Dataset Information

Comparative Decellularization and Recellularization of Wild-Type and Alpha 1,3 Galactosyltransferase Knockout Pig Lungs: A Model for Ex Vivo Xenogeneic Lung Bioengineering and Transplantation.

ABSTRACT:

Background

A novel potential approach for lung transplantation could be to utilize xenogeneic decellularized pig lung scaffolds that are recellularized with human lung cells. However, pig tissues express several immunogenic proteins, notably galactosylated cell surface glycoproteins resulting from alpha 1,3 galactosyltransferase (?-gal) activity, that could conceivably prevent effective use. Use of lungs from ?-gal knock out (?-gal KO) pigs presents a potential alternative and thus comparative de- and recellularization of wild-type and ?-gal KO pig lungs was assessed.

Methods

Decellularized lungs were compared by histologic, immunohistochemical, and mass spectrometric techniques. Recellularization was assessed following compartmental inoculation of human lung bronchial epithelial cells, human lung fibroblasts, human bone marrow-derived mesenchymal stromal cells (all via airway inoculation), and human pulmonary vascular endothelial cells (CBF) (vascular inoculation).

Results

No obvious differences in histologic structure was observed but an approximate 25% difference in retention of residual proteins was determined between decellularized wild-type and ?-gal KO pig lungs, including retention of ?-galactosylated epitopes in acellular wild-type pig lungs. However, robust initial recellularization and subsequent growth and proliferation was observed for all cell types with no obvious differences between cells seeded into wild-type versus ?-gal KO lungs.

Conclusion

These proof of concept studies demonstrate that decellularized wild-type and ?-gal KO pig lungs can be comparably decellularized and comparably support initial growth of human lung cells, despite some differences in retained proteins. ?-Gal KO pig lungs are a suitable platform for further studies of xenogeneic lung regeneration.

SUBMITTER: Platz J

PROVIDER: S-EPMC4991572 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Publications

Comparative Decellularization and Recellularization of Wild-Type and Alpha 1,3 Galactosyltransferase Knockout Pig Lungs: A Model for Ex Vivo Xenogeneic Lung Bioengineering and Transplantation.

Platz Joseph J Bonenfant Nicholas R NR Uhl Franziska E FE Coffey Amy L AL McKnight Tristan T Parsons Charles C Sokocevic Dino D Borg Zachary D ZD Lam Ying-Wai YW Deng Bin B Fields Julia G JG DeSarno Michael M Loi Roberto R Hoffman Andrew M AM Bianchi John J Dacken Brian B Petersen Thomas T Wagner Darcy E DE Weiss Daniel J DJ

Tissue engineering. Part C, Methods 20160714 8

<h4>Background</h4>A novel potential approach for lung transplantation could be to utilize xenogeneic decellularized pig lung scaffolds that are recellularized with human lung cells. However, pig tissues express several immunogenic proteins, notably galactosylated cell surface glycoproteins resulting from alpha 1,3 galactosyltransferase (α-gal) activity, that could conceivably prevent effective use. Use of lungs from α-gal knock out (α-gal KO) pigs presents a potential alternative and thus compa ...[more]

PMID: 27310581

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Project description:OBJECTIVE:To evaluate the pancreatic differentiation potential of ?-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media. METHODS:The BM-MSCs have been differentiated into pancreatic ?-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media - advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated. RESULTS:The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic ?-like cells in the N2B27-based media than in the ADMEM-based media. CONCLUSION:5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic ?-like cells.

Dataset Information

Comparative Decellularization and Recellularization of Wild-Type and Alpha 1,3 Galactosyltransferase Knockout Pig Lungs: A Model for Ex Vivo Xenogeneic Lung Bioengineering and Transplantation.

Background

Methods

Results

Conclusion

Publications

Comparative Decellularization and Recellularization of Wild-Type and Alpha 1,3 Galactosyltransferase Knockout Pig Lungs: A Model for Ex Vivo Xenogeneic Lung Bioengineering and Transplantation.

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