Development of Organ-Specific Progenitor Cell Cultures as Efficacy Test Platforms for Electron-Spun Fibre Meshes in Regenerative Medicine

Applications

Master Thesis by Vijayalakshmi Rajendran Supervisor: Prof. May Griffith

vijra393@student.liu.se

BackgroundThe nervous and cardiovascular system plays the most complex and vital role in all organisms. The main goal of regenerative medicine is to repair or recreate the damaged tissues using stem cells to restore the vital function of the targeted organ. Organ specific progenitor cells with non-toxic, biodegradable synthetic polymers gives an effective reparative therapy. The effect of PCL materials and surface modified (PEDOT coated) PCL materials of different topology with neural progenitor cells as test platforms are evaluated for cytotoxicity and neuron differentiation. To establish an effective proliferation and differentiation system, the stem cells from mice heart are isolated and characterized as cardiac stem cells by Fluorescence activated cell sorting through specific antigen expression and differentiated to cardiomyocytes.Further, nerve and cardiac tissue rejuvenation on biomaterials would serve as a regenerative therapy for numerous neurodegenerative disorders and cardiovascular disorders respectively.ObjectivesTo develop reproducible nerve and cardiac stem/progenitor cell cultures as test platforms for biomaterials to be used in nerve and cardiac tissue regeneration.To test the effect of biomaterials composition and topology on neural progenitor proliferation and differentiation.

AcknowledgementI extend my heartful and sincere gratitude towards Prof May Griffith, Dr. Naresh Polisetti, Abeni Wickham, Prof. Johan Edqvist, Prof.Uno Wennergren for their remarkable efforts in guiding me throughout my thesis period.

Conclusion The regeneration of nerve tissue using neuroblastoma derived cell line as potential test platform for PEDOT coated PCL and pure PCL electrospun meshes was accomplished. The effect of the PEDOT coated PCL and pure PCL with different topology was evaluated for cytotoxicity and differentiation.The reproducible cardiac stem/progenitor cells were characterized and differentiated into cardiomyocytes. Further, the characterized CSCs should be used as efficacy test platforms for PEDOT/PCL fibers for effective cardiac regeneration. Hence, the organ specific stem cell cultures could be used as efficacy test platforms for biomaterials in regenerative medicine applications

Materials and methods Neural Regeneration on sythetic polymersCell seeding on Materials: NDCs maintained in DMEM containing 10% FCS, 1% PEST were seeded 5000 cells/sample of synthetic substrateCell Viability Assay: NDCs on materials were subjected to Viability test using calcein and ethidium homodimer 1. Neural Induction of NDC: KSFM basal medium containing 0.1µM dexamethasone, 50µg*/ml ascorbic acid, 0.5%DMSO, 20µM cAMP and 0.5µg/ml NGF for 7 daysICC: Differentiation of NDCs into neurons was confirmed by the expression of neuronal markers, β-Tubulin-III and Neurofilament – Heavy chain .Statistics: Simple unpaired T-test for Cytotoxic assay and neurite length measurement ; Two samples, T-test between percents to determine % of neurites formed on PEDOT/PCL fibers at p≤ 0.05 level.Differentiation of CSCs into cardiomyocytes Cardiac stem cells isolated from C57BL/6 mouse were maintained in DMEM/HamsF12 media containing 10%FBS, 1%PEST,1X ITS, 0.5% DMSO , 20ng/ml EGF.FACS- Charectarization of CSCs by surface marker expression such as Sca1,c-kit, CD34, GATA4, CD44,CD29, APC cocktail lineage and CD31.ICC- Expression of GATA4 and ISL1Differentiation of CSCs- 5’azacytidine for 3 days and in DMEM/HamsF12 with 1X ITS, 0.5% DMSO , 20ng/ml EGF for 21 days and confirmed by GATA 4, Troponin 1, ANP and Actinin expression.

Viability test for NDCs seeded on PEDOT coated PCL electrospun microfibres. Green fluorescence

represents live cells.

Aligned-10µm Random-10µm

Neural differentiation of NDCs expressing green fluorescence neural markers in 10X resolution

PCL;Random 10µm PCL;Aligned 10µmPEDOT;random 10µm PEDOT;aligned 10µm

Differentiation of CSCs to cardiomyocytes

GATA-4 Troponin I ANP Actinin

Sca1 GATA 4

Characterization of CSCs by FACS analysis-:green curve- Marker expression, black curve- isotype control

Results & Discussion

CD31