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Title: Engineering 3D scaffolds with iPSCs towards regeneration of cardiac tissues
Authors: Krishnamoorthi, Muthu Kumar
Keywords: Engineering::Materials::Biomaterials
Issue Date: 26-Jun-2019
Source: Krishnamoorthi, M. K. (2019). Engineering 3D scaffolds with iPSCs towards regeneration of cardiac tissues. Doctoral thesis, Nanyang Technological University, Singapore.
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 highly bioactive, yet they are limited by their high batch-to-batch variability, and their poorly understood bioactivity mechanisms, particularly, in affecting stem cell fate. Conversely, commonly used synthetic materials, while offering good control over different parameters, generally lack the 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. The resulting 3D scaffolds will improve understanding of the cell material interactions and might produce a potential treatment for cardiac regeneration. One ideal group of natural biomaterials can be obtained by decellularization, yielding tissue specific bioactive, and cell supportive scaffolds of either solid, or liquid forms. Our lab has isolated a porcine cardiac extra cellular matrix (pcECM), in both solid and liquid forms, which preserves the 3D architecture of the heart ECM, while maintaining the bioactivity for cellular interaction. Nevertheless, the exact contribution of such pcECM to stem cell and tissue specific lineage commitment, and the possible mechanisms governing such bioactivity remain largely unknown. The complexity of the pcECM composition and 3D architecture hinder our ability to discriminate between different effectors and their resulting individual or combined effects. Hence, it is necessary to generate synthetic 3D biomimetic scaffolds with controllable architecture and bioactivity profiles that would enable the study of various components in a modular way. Electrospinning is an accessible and 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 improvised the liquid-collector of electrospinning to fabricate 3D fibrous scaffolds with high porosity. Though this ECM mimicking synthetic polymer scaffolds offer high reproducibility, they generally lack the bioactivity inherent to natural ECM biomaterials. Thus, different approaches have been used to confer bioactivity to synthetic materials, such as natural materials (e.g., short bioactive peptides) either on the surface or within the bulk, pre-culturing the scaffold with ECM producing cells (e.g., fibroblasts, and mesenchymal stem cells), and physical and/or chemical surface modifications. Our work aims to obtain 3D composite scaffolds (3DCS) with ECM mimicking synthetic ultra-structures, and tissue specific biochemical cues by fabricating 3D electrospun polymeric scaffolds and functionalizing them with liquidized pcECM. The 3DCS produced were studied in comparison to the natural pcECM highlighting the roles of architecture, biochemical composition, and various combinations thereof, in affecting the function and fate of seeded human multi and pluri-potent stem cells. hiPSCs were used in this study as an ideal model cell with clinical relevancy, given their possible autologous sourcing, and their ability to differentiate into all cardiac cell types, in particular, beating cardiomyocytes (hiPSC-CM). We hypothesized that, bioactive 3D scaffolds (pcECM and/or 3DCS) 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 the evaluated properties, and reproducibility of the 3D synthetic scaffolds, only 3D 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 also displayed cardiac relevant mechanical properties and did not elicit any immunogenicity in vitro. 3DCS also displayed the ability for cellular attachment and growth under static conditions when human mesenchymal stem cells (hMSCs) were used as model cells. hiPSC-CM seeded 3DCS maintained CM viability, beating functionality and phenotypic identity for two weeks as evaluated by protein expression. Moreover, the scaffolds’ microenvironment supported the 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.
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