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Reprogramming of human PBMCs into iPSCs for the In-vitro manufacture of engineered vascular grafts based on biodegradable synthetic polymers


Generali, Melanie. Reprogramming of human PBMCs into iPSCs for the In-vitro manufacture of engineered vascular grafts based on biodegradable synthetic polymers. 2017, University of Zurich, Faculty of Science.

Abstract

Cardiovascular diseases (CVD) remains to be the leading cause of morbidity and mortality worldwide.
Replacement of affected vascular tissues has been widely used to treat CVD such as coronary heart
disease, aortic aneurysm and peripheral vascular disease. However, successful treatment of CVD is
often limited by the lack of suitable autologous replacement tissue. Therefore, tissue engineering (TE)
represents a promising solution to replace diseased vessels. TE aims at the development of constructs
that integrate with the patient’s native tissue to restore physiologic function. The success of any TE
approach is dependent on three main factors: (i) the cell source, (ii) the scaffold matrix, and (iii) the
ambient biochemical and physical factors. During the last years several different starter materials and
cell sources have been investigated.
On the one hand, biodegradable scaffold matrixes form the basis of any in vitro tissue engineering
approach by acting as a temporary matrix for cell proliferation and extracellular matrix deposition until
the scaffold is replaced by neo-tissue. The present study systematically compares three frequently
used polymers for the in vitro engineering of extracellular matrix based on poly-glycolic acid (PGA)
under static as well as dynamic conditions. Ultra-structural analysis was used to examine the polymer
structure. For tissue engineering (TE) three human fibroblast cell lines were seeded on either PGApoly-
4-hydroxybutyrate (P4HB), PGA-poly-lactic acid (PLA) or PGA-poly–caprolactone (PCL) patches.
Later, these patches were analyzed qualitatively and quantitatively. We found that PGA-P4HB and
PGA-PLA scaffolds enhance tissue formation significantly higher than PGA-PCL scaffolds. Polymer
remnants were visualized by polarization microscopy. In addition, biomechanical properties of the
tissue engineered patches were determined in comparison to native tissue. This study may allow
future studies to specifically select certain polymer starter matrices aiming at specific tissue properties
of the bioengineered constructs in vitro.
On the other hand, an ideal cell source for human therapeutic and disease modeling applications
should be easily accessible and possess unlimited differentiation and expansion potential. Human
induced pluripotent stem cells (hiPSCs) derived from peripheral blood mononuclear cells (PBMCs)
represent a promising source given their ease of harvest combined with their pluripotent nature.
Therefore, hiPSCs were generated based on PBMCs and differentiated into smooth muscle cells (SMCs)
as well as endothelial cells (ECs). These cells were seeded onto PGA-P4HB starter matrices and cultured
under static or dynamic conditions to induce tissue formation in vitro. Resulting tissue-engineered
vascular grafts (TEVGs) showed abundant amounts of extracellular matrix, containing an αSMApositive
layer in the interstitium and a thin luminal layer of vWF-positive cells approximating native
vessels. These results pave the way for developing autologous PBMC-derived hiPSC-based vascular
constructs for therapeutic applications or disease modelling.

Abstract

Cardiovascular diseases (CVD) remains to be the leading cause of morbidity and mortality worldwide.
Replacement of affected vascular tissues has been widely used to treat CVD such as coronary heart
disease, aortic aneurysm and peripheral vascular disease. However, successful treatment of CVD is
often limited by the lack of suitable autologous replacement tissue. Therefore, tissue engineering (TE)
represents a promising solution to replace diseased vessels. TE aims at the development of constructs
that integrate with the patient’s native tissue to restore physiologic function. The success of any TE
approach is dependent on three main factors: (i) the cell source, (ii) the scaffold matrix, and (iii) the
ambient biochemical and physical factors. During the last years several different starter materials and
cell sources have been investigated.
On the one hand, biodegradable scaffold matrixes form the basis of any in vitro tissue engineering
approach by acting as a temporary matrix for cell proliferation and extracellular matrix deposition until
the scaffold is replaced by neo-tissue. The present study systematically compares three frequently
used polymers for the in vitro engineering of extracellular matrix based on poly-glycolic acid (PGA)
under static as well as dynamic conditions. Ultra-structural analysis was used to examine the polymer
structure. For tissue engineering (TE) three human fibroblast cell lines were seeded on either PGApoly-
4-hydroxybutyrate (P4HB), PGA-poly-lactic acid (PLA) or PGA-poly–caprolactone (PCL) patches.
Later, these patches were analyzed qualitatively and quantitatively. We found that PGA-P4HB and
PGA-PLA scaffolds enhance tissue formation significantly higher than PGA-PCL scaffolds. Polymer
remnants were visualized by polarization microscopy. In addition, biomechanical properties of the
tissue engineered patches were determined in comparison to native tissue. This study may allow
future studies to specifically select certain polymer starter matrices aiming at specific tissue properties
of the bioengineered constructs in vitro.
On the other hand, an ideal cell source for human therapeutic and disease modeling applications
should be easily accessible and possess unlimited differentiation and expansion potential. Human
induced pluripotent stem cells (hiPSCs) derived from peripheral blood mononuclear cells (PBMCs)
represent a promising source given their ease of harvest combined with their pluripotent nature.
Therefore, hiPSCs were generated based on PBMCs and differentiated into smooth muscle cells (SMCs)
as well as endothelial cells (ECs). These cells were seeded onto PGA-P4HB starter matrices and cultured
under static or dynamic conditions to induce tissue formation in vitro. Resulting tissue-engineered
vascular grafts (TEVGs) showed abundant amounts of extracellular matrix, containing an αSMApositive
layer in the interstitium and a thin luminal layer of vWF-positive cells approximating native
vessels. These results pave the way for developing autologous PBMC-derived hiPSC-based vascular
constructs for therapeutic applications or disease modelling.

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Additional indexing

Item Type:Dissertation (monographical)
Referees:Weber Benedikt, Cinelli Paolo, Verrey François, von Eckardstein Arnold, Hoerstrup Simon P, Gantenbein Benjamin
Communities & Collections:04 Faculty of Medicine > Institute for Regenerative Medicine (IREM)
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2017
Deposited On:20 Oct 2017 13:36
Last Modified:19 Mar 2018 08:50
Number of Pages:119
Additional Information:Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich
OA Status:Green

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