Use of genes and cells in regenerative medicine

Abstract

Regenerative medicine is a discipline that aims to achieve regeneration of cells, tissue or organs in order to restore or establish normal functions. There are several strategies that can be used to achieve this goal. Many of the strategies are based on use of genes or cells to regenerate organ functions. The present thesis aim to investigate different gene and cell based methods for the use in regenerative medicine.

In paper I a novel peptide conjugate is described for the use of gene transfer in vitro and in vivo. It shows that serum-resistant nanoparticles are formed upon co-incubation of Stearyl- TP10 with plasmid DNA and that these nanoparticles efficiently transfect cells ubiquitously with minimal toxicity. It is also shown that stearyl-TP10/plasmid nanoparticles enable efficient dose-dependent gene delivery in vivo when being administered intramuscularly or intradermally without any associated observed toxicity or induction of immune response. In vivo transfection was highly dependent on a specific charge ratio; this stands in contrast to the in vitro response. Altogether these results show that stearyl-TP10 is an attractive non viral, peptide-based mediator of plasmid delivery, that is effective both in vitro and in vivo. This peptide based vector could be adapted for delivery of other nucleic acids.

Paper II investigates natural conditions that allow clonal survival and self-renewal of human embryonic stem (ES) cells. Only two laminins, LN-511 and LN-521, could support long-term self-renewal of the cells in a completely defined and xeno-free environment but, unlike LN- 511, LN 521 permits survival of individualized human ES or induced pluripotent stem (iPS) cells plated at certain densities. The paper also shows that the use of LN-521 and E-cadherin together as a culture matrix supports both derivation and clonal survival of hES cells. The paper demonstrates that a single cell obtained by biopsy from an 8-cell human in vitro fertilized (IVF) embryo can give rise to new human ES cell lines under completely chemically defined and xeno-free conditions. The methods developed here may have significance for research and clinical applications of human ES cells or other cell types.

Paper III describes the treatment with messenchymal stromal cells (MSC) in two patients with severe refractory ARDS on extracorporeal membrane oxygenation. (ECMO) Although no conclusions could be drawn on effectiveness of MSC treatment in ARDS, clinical improvement was shown after MSC treatment allowing discontinuation of ECMO support and, subsequently, a progressive decrease in the need for mechanical ventilation. Apart from monitoring the clinical progress the in vivo actions of the MSCs on lung and systemic inflammation are correlated to vitro potency assays, including effects on inflammatory and immune modulatory cells. Also proteomic assessments of the MSCs and extra cellular vesicles (EV) released by the MSCs are described. This paper outlines a case report and is now planned to be expanded in to a phase 1 clinical trial with MSC treatment to ARDS patients ECMO support.