Mutations in the COL1A1 and COL1A2 genes associated with osteogenesis imperfecta (OI) types I or III

Although over 85% of osteogenesis imperfecta (OI) cases are associated with mutations in the procollagen type I genes (COL1A1 or COL1A2), no hot spots for the mutations were associated with particular clinical phenotypes. Eight patients that were studied here, diagnosed with OI by clinical standards, are from the Polish population with no ethnic background indicated. Previously unpublished mutations were found in six out of those eight patients. Genotypes for polymorphisms (Sp1 - rs1800012 and PvuII - rs412777), linked to bone formation and metabolism were determined. Mutations were found in exons 2, 22, 50 and in introns 13 and 51 of the COL1A1 gene. In COL1A2, one mutation was identified in exon 22. Deletion type mutations in COL1A1 that resulted in OI type I had no effect on collagen type I secretion, nor on its intracellular accumulation. Also, a single base substitution in I13 (c.904-9 G>T) was associated with the OI type I. The OI type III was associated with a single base change in I51 of COL1A1, possibly causing an exon skipping. Also, a missense mutation in COL1A2 changing Gly→Cys in the central part of the triple helical domain of the collagen type I molecule caused OI type III. It affected secretion of the heterotrimeric form of procollagen type I. However, no intracellular accumulation of procollagen chains could be detected. Mutation in COL1A2 affected its incorporation into procollagen type I. The results obtained shall help in genetic counseling of OI patients and provide a rational support for making informed, life important decisions by them and their families

The comparison of multipotential for differentiation of progenitor mesenchymal-like stem cells obtained from livers of young and old rats.

The presence of stem cells differentiating to hepatocytes and cholangiocytes has been previously reported in livers of young rats. Here, we have isolated, cultured, and characterized mesenchymal stem cells (MSCs) from livers of young and old rats and tested their multipotential for differentiation. The mesenchymal stem cells in liver sections were identified by the presence of markers, respectively for primary stem cells Thy-1 and CD34, for differentiation to early cholangiocytes GST and CK19, and for differentiation to hepatocytes GSTalpha and CK18. Ki67 was detected as the cell proliferation marker. Cells isolated from livers of either age group were tested in a culture for their viability following storage and were characterized for the presence of most of the markers detected in cells in situ. The results revealed age-dependent changes in the number of recovered primary MSCs. In both age groups we have observed cells changing under differentiating conditions to liver cell lineages, such as cholangiocytes and hepatocytes, as well as to non-liver cells such as adipocytes, astrocytes, neuroblasts, and osteoblasts. Our data revealed that from the livers of rats 20 months and older the primary MSCs could be isolated and expanded; however, they were significantly fewer, even though their differentiation multipotential was preserved. The mechanism involved in the differentiation of liver MSCs seemed to depend on a constellation of signals in Notch signalling pathways. Thus, our results support the idea of potential use of liver as a source of MSCs, not only for liver reconstruction but also for cell therapy in general

Characteristic of cells isolated from human Abdominal Aortic Aneurysm samples cultured in vitro

Abstract Background: This study aimed to standardize cell culture methods for major cell types isolated from three layers of human AAA. We also aimed to determine cell types in each layer of each AAA segment and compare them with cell types in layers of control, unchanged segments. Material and methods We divided AAAs into three segments along the AAA and control segments flanking the aneurysm. Isolated cells following expansion were analyzed by flow cytometry, immunochemistry and microscopic methods. Fluorochrome-conjugated antibodies were used to detect the three major cell types (endothelial cells, smooth muscle cells, and fibroblasts) in each layer of every AAA segment. Results: Culture of cells from the three AAA segments was successfully established in 21% of patients. In all of the layers, only a small proportion of cells showed layer- specific markers of cell types. The majority of cells from every layer were positive for CD90, which is considered specific marker of fibroblasts in the aorta. Conclusions: We describe methodology for isolation of cells, their culture conditions, and phenotypic characterization for AAA. The wall of AAA loses its specific types of cells in all of the layers compared with the normal abdominal aortic wall

Polycationic star polymers with hyperbranched cores for gene delivery

Core-shell type stars synthesized via atom transfer radical polymerization were used for the delivery of nucleic acids. The interior of the stars consisted of hyperbranched poly(arylene oxindole), while the arms were composed of poly(N,N-dimethylaminoethyl methacrylate). The length of the star arms varied in degree of polymerization (DP) from 14 to 98. The hydrodynamic radius of the structures measured in water indicated the presence of small aggregates, while isolated stars ranging in size from 14 to 29 nm were seen in organic solvent. The phase transition temperatures of the stars in water, measured in basic conditions, were shifted to lower values with increasing DP of the arms. Stable polyplexes of stars with plasmid DNA were formed. Their size varied from 300 nm to 400 nm, depending upon the DP of arms. The zeta potential of the polyplexes was positive, which facilitated their cellular uptake. The DP of the arms influenced the transfection efficiency of HT-1080 cells, demonstrating that stars are promising candidates for synthetic gene vectors. © 2014 Elsevier Ltd. All rights reserved.publisher: Elsevier articletitle: Polycationic star polymers with hyperbranched cores for gene delivery journaltitle: Polymer articlelink: http://dx.doi.org/10.1016/j.polymer.2014.07.013 content_type: article copyright: Copyright © 2014 Elsevier Ltd. All rights reserved.status: publishe

Preparation and Characterization of Nanofibrous Polymer Scaffolds for Cartilage Tissue Engineering

Polymer substrates obtained from poly(lactic acid) (PLA) nanofibres modified with carbon nanotubes (CNTs) and gelatin (GEL) for cartilage tissue engineering are studied. The work presents the results of physical, mechanical, and biological assessment. The hybrid structure of PLA and gelatine nanofibres, carbon nanotubes- (CNTs-) modified PLA nanofibres, and pure PLA-based nanofibres was manufactured in the form of fibrous membranes. The fibrous samples with different microstructures were obtained by electrospinning method. Microstructure, physical and mechanical properties of samples made from pure PLA nanofibres, CNTs-, and gelatin-modified PLA-nanofibres were studied. The scaffolds were also tested in vitro in cell culture of human chondrocytes collected from patients. To assess the influence of the nanofibrous scaffolds upon chondrocytes, tests for cytotoxicity and genotoxicity were performed. The work reveals that the nanofibrous structures studied were neither genotoxic nor cytotoxic, and their microstructure, physical and mechanical properties create promising scaffolds for potential use in cartilage repairing

Nonviral Plasmid DNA Carriers Based on N,N'-Dimethylaminoethyl Methacrylate and Di(ethylene glycol) Methyl Ether Methacrylate Star Copolymers

Star polymers with random and block copolymer arms made of cationic N,N'-dimethylaminoethyl methacrylate (DMAEMA) and nonionic di(ethylene glycol) methyl ether methacrylate (DEGMA) were synthesized via atom transfer radical polymerization (ATRP) and used for the delivery of plasmid DNA in gene therapy. All stars were able to form polyplexes with plasmid DNA. The structure and size of the polyplexes were precisely determined using light scattering and cryo-TEM microscopy. The hydrodynamic radius of a complex of DNA with star was dependent on the architecture of the star arms, the DEGMA content and the number of amino groups in the star compared to the number of phosphate groups of the nucleic acid (N/P ratio). The smallest polyplexes (Rh90°∼50 nm) with positive zeta potentials (∼15 mV) were formed of stars with N/P=6. The introduction of DEGMA into the star structure caused a decrease of polyplex cytotoxicity in comparison to DMAEMA homopolymer stars. The overall transfection efficiency using HT-1080 cells showed that the studied systems are prospective gene delivery agents. The most promising results were obtained for stars with random copolymer arms of high DEGMA content.status: publishe

Mutations in COL1A1 and COL1A2 Genes Associated with Osteogenesis Imperfecta (OI) Types I or III.

Although over 85% of osteogenesis imperfecta (OI) cases are associated with mutations in the procollagen type I genes (COL1A1 or COL1A2), no hot spots for the mutations were associated with particular clinical phenotypes. Eight patients that were studied here, diagnosed with OI by clinical standards, are from the Polish population with no ethnic background indicated. Previously unpublished mutations were found in six out of those eight patients. Genotypes for polymorphisms (Sp1 - rs1800012 and PvuII - rs412777), linked to bone formation and metabolism were determined. Mutations were found in exons 2, 22, 50 and in introns 13 and 51 of the COL1A1 gene. In COL1A2, one mutation was identified in exon 22. Deletion type mutations in COL1A1 that resulted in OI type I had no effect on collagen type I secretion, nor on its intracellular accumulation. Also, a single base substitution in I13 (c.904-9 G>T) was associated with the OI type I. The OI type III was associated with a single base change in I51 of COL1A1, possibly causing an exon skipping. Also, a missense mutation in COL1A2 changing Gly→Cys in the central part of the triple helical domain of the collagen type I molecule caused OI type III. It affected secretion of the heterotrimeric form of procollagen type I. However, no intracellular accumulation of procollagen chains could be detected. Mutation in COL1A2 affected its incorporation into procollagen type I. The results obtained shall help in genetic counseling of OI patients and provide a rational support for making informed, life important decisions by them and their families