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Lim M.L.,Advanced Center for Translational Regenerative Medicine | Jungebluth P.,Advanced Center for Translational Regenerative Medicine | Sjoqvist S.,Advanced Center for Translational Regenerative Medicine | Nikdin H.,Karolinska Institutet | And 4 more authors.
Stem Cells Translational Medicine | Year: 2013

Pluripotent cells such as human embryonic stem cells and human induced pluripotent stem cells are useful in the field of regenerative medicine because they can proliferate indefinitely and differentiate into all cell types. However, a limiting factor for maintaining and propagating stem cells is the need for inactivated fibroblasts as a growth matrix, since these may potentially cause cross-contamination. In this study, we aimed to maintain stem cells on the extracellular matrix (ECM) of either nonirradiated or γ{phonetic}-irradiated fibroblasts. It has been demonstrated that the ECM contains factors and proteins vital for the adhesion, proliferation, and differentiation of pluripotent cells. In order to preserve the ECM, the cell layers of the fibroblasts were decellularized by treatment with 0.05% sodium dodecyl sulfate (SDS), which resulted in an absence of DNA as compared with conventional feeder culture. However, SDS treatment did not cause a detectable change in the ECM architecture and integrity. Furthermore, immunohistochemistry demonstrated that expressions of major ECM proteins, such as fibronectin, collagen, and laminin, remained unaltered. The human pluripotent cells cultured on this decellularized matrix maintained gene expression of the pluripotency markers NANOG and OCT4 and had the potency to differentiate to three germ layers. The in vitro culture system shown here has an excellent potential since the main allogeneic components (i.e.,DNAof the feeder cells) are removed. It is also a technically easy, fast, safe, and cheap method for maintaining a refined feeder-free stem cell culture for further cell differentiation studies. © AlphaMed Press 2013.

Jungebluth P.,Advanced Center for Translational Regenerative Medicine | Haag J.C.,Advanced Center for Translational Regenerative Medicine | Sjoqvist S.,Advanced Center for Translational Regenerative Medicine | Gustafsson Y.,Advanced Center for Translational Regenerative Medicine | And 9 more authors.
Nature Protocols | Year: 2014

Tissue-engineered tracheal transplants have been successfully performed clinically. However, before becoming a routine clinical procedure, further preclinical studies are necessary to determine the underlying mechanisms of in situ tissue regeneration. Here we describe a protocol using a tissue engineering strategy and orthotopic transplantation of either natural decellularized donor tracheae or artificial electrospun nanofiber scaffolds into a rat model. The protocol includes details regarding how to assess the scaffolds' biomechanical properties and cell viability before implantation. It is a reliable and reproducible model that can be used to investigate the crucial aspects and pathways of in situ tracheal tissue restoration and regeneration. The model can be established in <6 months, and it may also provide a means to investigate cell-surface interactions, cell differentiation and stem cell fate. © 2014 Nature America, Inc. All rights reserved.

Sjoqvist S.,Advanced Center for Translational Regenerative Medicine | Jungebluth P.,Karolinska University Hospital | Ling Lim M.,Karolinska Institutet | Haag J.C.,University of Rome Tor Vergata | And 20 more authors.
Nature Communications | Year: 2014

A tissue-engineered oesophageal scaffold could be very useful for the treatment of pediatric and adult patients with benign or malignant diseases such as carcinomas, trauma or congenital malformations. Here we decellularize rat oesophagi inside a perfusion bioreactor to create biocompatible biological rat scaffolds that mimic native architecture, resist mechanical stress and induce angiogenesis. Seeded allogeneic mesenchymal stromal cells spontaneously differentiate (proven by gene-, protein and functional evaluations) into epithelial-And muscle-like cells. The reseeded scaffolds are used to orthotopically replace the entire cervical oesophagus in immunocompetent rats. All animals survive the 14-day study period, with patent and functional grafts, and gain significantly more weight than sham-operated animals. Explanted grafts show regeneration of all the major cell and tissue components of the oesophagus including functional epithelium, muscle fibres, nerves and vasculature. We consider the presented tissue-engineered oesophageal scaffolds a significant step towards the clinical application of bioengineered oesophagi. © 2014 Macmillan Publishers Limited. All rights reserved.

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