טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKrishnamoorthi Muthu Kumar
SubjectEngineering 3D Scaffolds with iPSCs towards Regeneration of
Cardiac Tissues
DepartmentDepartment of Biotechnology and Food Engineering
Supervisors Professor Marcelle Machluf
Professor Tan Lay Poh


Abstract

This work focuses on engineering 3 dimensional (3D) fibrous hybrid scaffolds with human induced pluripotent stem cells (hiPSC) for cardiac tissue engineering (CTE). Natural materials are bioactive, yet are limited by their variability, and poorly understood bioactivity mechanisms. Conversely, synthetic materials, while offering good reproducibility, generally lacks suitable bioactivity for cellular interactions. In this work, we propose comparing and combining natural and synthetic scaffolds to enjoy the advantages of both platforms while circumventing their inherent limitations.

One ideal group of natural biomaterials can be obtained by decellularization. Our lab has isolated a porcine cardiac extra cellular matrix (pcECM), in both solid and liquid forms, which preserves the architecture and bioactivity of the heart ECM. Nevertheless, the exact contribution of such pcECM to stem cell lineage commitment, and the possible mechanisms governing such bioactivity remain largely unknown. Hence, it is necessary to generate 3D biomimetic scaffolds with controllable architecture and bioactivity profiles that would enable the study of various components in a modular way.

Electrospinning is an inexpensive means to fabricate fibrous matrices but the fundamental limitation with traditional electrospinning is that the scaffold produced is usually two dimensional (2D) dense mats rather than 3D porous structures. Here, we used liquid-collector for electrospinning to fabricate 3D fibrous scaffolds with high porosity. Though this ECM mimicking synthetic scaffolds offer high reproducibility, they generally lack the bioactivity inherent to natural ECM biomaterials.

Our work aims to obtain 3D composite scaffolds (3DCS) with ECM mimicking synthetic architecture, and tissue specific biochemical cues by fabricating 3D electrospun polymeric scaffolds and functionalizing them with liquidized pcECM. hiPSCs were used as an ideal model cell, given their possible autologous sourcing, and their ability to differentiate into all cardiac cell types, particularly, beating cardiomyocytes (hiPSC-CM). We hypothesized that, bioactive 3D scaffolds that maintain a balance and cooperation between architectural and biochemical signals, are needed to initialize differentiation of hiPSC towards cardiac lineages.

Our results show that the pcECM can be mimicked by wet electrospinning of poly lactide-co-glycolide (PLGA), and poly lactide-co-ε-caprolactone (PLCL). However, based on evaluated properties, and reproducibility of the 3D synthetic scaffolds, only PLGA exhibited adequate profile and was therefore used for further studies. After modification with pcECM gel, the 3DCS displayed similarities with pcECM in terms of morphology, chemistry, biochemical composition. The 3DCS displayed cardiac relevant mechanical properties and did not elicit immunogenicity in vitro. 3DCS also displayed the ability for cellular attachment and growth when human mesenchymal stem cells (hMSCs) were used. hiPSC-CM seeded 3DCS maintained CM viability, beating functionality, phenotypic identity and calcium handling ability. Finally, hiPSCs seeded on these scaffolds, differentiated into cardiac lineage cells spontaneously without the addition of any external factors or molecules, asserting the role and importance of a tissue specific biochemical microenvironment for cardiac applications. Taken together, our results here contribute to the understanding of how the biology and architecture of the pcECM can affect and determine the fate of the seeded hiPSCs. This knowledge is relevant not only for basic research but also for possible CTE applications.