Isolation and characterization of multipotent mesenchymal stromal cells from the gingiva and the periodontal ligament of the horse

<p>Abstract</p> <p>Background</p> <p>The equine periodontium provides tooth support and lifelong tooth eruption on a remarkable scale. These functions require continuous tissue remodeling. It is assumed that multipotent mesenchymal stromal cells (MSC) reside in the periodontal ligament (PDL) and play a crucial role in regulating physiological periodontal tissue regeneration. The aim of this study was to isolate and characterize equine periodontal MSC.</p> <p>Tissue samples were obtained from four healthy horses. Primary cell populations were har-vested and cultured from the gingiva, from three horizontal levels of the PDL (apical, midtooth and subgingival) and for comparison purposes from the subcutis (masseteric region). Colony-forming cells were grown on uncoated culture dishes and typical <it>in vitro </it>characteristics of non-human MSC, i.e. self-renewal capacity, population doubling time, expression of stemness markers and trilineage differentiation were analyzed.</p> <p>Results</p> <p>Colony-forming cell populations from all locations showed expression of the stemness markers CD90 and CD105. In vitro self-renewal capacity was demonstrated by colony-forming unit fibroblast (CFU-F) assays. CFU-efficiency was highest in cell populations from the apical and from the mid-tooth PDL. Population doubling time was highest in subcutaneous cells. All investigated cell populations possessed trilineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages.</p> <p>Conclusions</p> <p>Due to the demonstrated in vitro characteristics cells were referred to as equine subcutaneous MSC (eSc-MSC), equine gingival MSC (eG-MSC) and equine periodontal MSC (eP-MSC). According to different PDL levels, eP-MSC were further specified as eP-MSC from the apical PDL (eP-MSCap), eP-MSC from the mid-tooth PDL (eP-MSCm) and eP-MSC from the subgingival PDL (eP-MSCsg). Considering current concepts of cell-based regenerative therapies in horses, eP-MSC might be promising candidates for future clinical applications in equine orthopedic and periodontal diseases.</p

Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies

Background: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. Methods: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines’ representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. Results: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. Conclusion: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression

Analyse des zytotoxischen Effektes von geringen Konzentrationen beta-Amyloid über den Neurotrophin Rezeptor p75

Während ihrer Entwicklung benötigen Neuronen neurotrophe Faktoren wie den Nerven Wachstumsfaktor (NGF) um zu Differenzieren, synaptische Kontakte zu formen und ihr Überleben zu sichern. Die Effekte der neurotrophen Faktoren werden hierbei unter anderem über den p75 Rezeptor, der keine enzymatische Aktivität besitzt, vermittelt. Dennoch ist er in der Lage in Abhängigkeit des zellulären Umfeldes Differenzierung, Überleben oder den programmierten Zelltod auszulösen. Neben den Neurotrophinen bindet der p75 Rezeptor auch aggregiertes beta-Amyloid, welches der Hauptbestandteil der senilen Plaques ist, die sich im Verlauf der Alzheimerschen Krankheit im Gehirn ansammeln. Die vorliegenden Arbeit beschäftigt sich mit der Frage, welche Kinasen beim Start des p75 aktivierten Ras-MapK Signalweges beteiligt sind, und welche anderweitigen Effekte die Stimulation des Rezeptors mit geringen Konzentrationen beta-Amyloid auslöst

Loss of connexin43 (Cx43) in Sertoli cells leads to spatio-temporal alterations in occludin expression

Within the testis, Sertoli cell (SC) junctional complexes between somatic SC create a basal and apical polarity within the seminiferous epithelium, restrict movement of molecules between cells, and separate the seminiferous epithelium into a basal and adluminal compartment. This barrier consists of membrane integrated proteins known as tight, adherens, and gap junctions, which promote cell-cell contact along the blood-testis-barrier (BTB). Nevertheless, these junctions, which form the basis of the BTB are structures whose function and dynamic regulation is still poorly understood. Thus, in this study, through the use of immunohistochemistry (IHC), semi quantitative western blot (WB) analysis, and real-time-quantitative-PCR (qRT-PCR) we focused on the expression pattern of the main testicular tight junction protein, occludin, in SC. For this, the established transgenic SC specific connexin 43 (Cx43) knockout (SCCx43KO) mouse line was used; both knockout (KO) and wildtype (WT) males of different ages from juvenile to adult were compared. The object was to elucidate a possible role of Cx43 on the expression pattern and regulation of occludin. This conditional KO mouse line lacks the gap junction gene Gja1 (coding for Cx43) only in SC and reveals impaired spermatogenesis. The qRT-PCR indicates an increase in occludin mRNA in adult KO mice. These results correspond to the occludin protein synthesis of adult mice. Additionally, during puberty, occludin localization at the BTB barrier in KO mice is delayed. Our study demonstrates spatiotemporal alterations in occludin mRNA- and protein-expression, indicating that Cx43 might act as a regulator for BTB formation (and function)

Matrix metalloproteinase 2 (MMP-2) may be activated by binding of tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) to MMP-14 in bovine placentomes

Bu çalışma, 26-27 Şubat 2009 tarihlerinde Leipzig[Almanya]'da düzenlenen 42. Annual Conference of Physiology and Pathology of Reproduction/34th Mutual Conference on Veterinary and Human Reproductive Medicine'de bildiri olarak sunulmuştur

Establishment and functional characterization of a murine primary Sertoli cell line deficient of connexin43

The Sertoli cell (SC) specific connexin43 (Cx43) knockout (SCCx43KO) mouse line is ideal to gain insight into the mechanistic gap junction formation in SC and the seminiferous epithelium. A method for developing primary SC cultures from these mice was established, validated and successfully characterized via polymerase chain reaction, immunohistochemistry, immunofluorescence (IF), and Western blots (WB). It was evident that both knockout (KO) and wild-type (WT) primary cell cultures were similar in morphology. These highly pure SC cultures were subjected to cell proliferation assays indicating no notable proliferation in cultures of both genotypes. Measurements of cell monolayer integrity indicated significant increases in transepithelial electrical resistance and consequently in tight junction expression of the KO cultures. Using semi-quantitative WB and IF, tight junction protein claudin-11 was analyzed. These results support a role for Cx43 in regulating blood-testis barrier (BTB) function, composition, and dynamics in vitro. Thus, the SC deficient Cx43 cell cultures may provide a valuable in vitro tool for a better understanding of the mechanistic role of Cx43 in spermatogenesis and BTB assembly

Sternal bone marrow derived equine multipotent mesenchymal stromal cells (MSCs): investigations considering the sampling site and the use of different culture media

Aspiration of equine sternal bone marrow is required for the cultivation of bone marrow-derived multipotent mesenchymal stromal cells (BM-MSCs) for regenerative therapies. For bone marrow aspiration as well as for MSC cultivation, there is a need to optimize techniques and protocols to enhance MSC harvest at minimized culture times. In a comparative study bone marrow aspirates from sternebra 4 and 5 were collected at two different positions within the sternebrae, either from 10 mm or from 30 mm dorsal from the ventral margin of the sternebrae. Accuracy of the puncture depth was confirmed by ultrasonography and computed tomography. Isolated MSCs were cultivated using media supplemented with three alternative sera, i.e. fetal calf serum, standardized horse serum and autologous serum. Due to morphological characteristics (spherical shape, only thin layer of hyaline cartilage at the ventral site, reliable bone marrow aspiration from only 10 mm intraosseous depth), sternebra 5 appeared most suitable for bone marrow aspiration. Cultivation and expansion of BM-MSCs was most efficient using fetal calf serum