Human pluripotent stem cells: towards the definition of new engineering approaches to target heart and kidney disease
[eng] Human pluripotent stem cells (hPSCs) offer a significant advantage in recapitulating key features related to tissue differentiation, morphogenesis and, conversely, human diseases. Taking advantage of their inherent ability to differentiate into the three germ layers, and their unlimited self-r...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/209017 |
| Acceso en línea: | https://hdl.handle.net/2445/209017 http://hdl.handle.net/10803/690399 |
| Access Level: | acceso abierto |
| Palabra clave: | Medicina regenerativa Bioenginyeria Cèl·lules mare Cor Cultiu de teixits Regenerative medicine Bioengineering Stem cells Heart Tissue culture |
| Sumario: | [eng] Human pluripotent stem cells (hPSCs) offer a significant advantage in recapitulating key features related to tissue differentiation, morphogenesis and, conversely, human diseases. Taking advantage of their inherent ability to differentiate into the three germ layers, and their unlimited self-renewal capacity, the field has been able to establish novel methodologies to generate three dimensional (3D) self-organized organ-like structures, known as organoids. Moreover, the discovery and development of CRISPR/Cas9 technology has revolutionized our ability to introduce permanent or transient changes in the genome of living organisms and cells. In this context, the host laboratory has previously developed a cellular platform, called iCRISPR2 (iC2), which utilizes an inducible Cas9 under the endogenous TET/ON promoter to enable the efficient genome editing of hPSCs. The iC2 system allows the generation of reporter, knock-out and knock-in hPSC lines. In the present thesis, we have established new tools and methodologies to generate hPSC-derived cardiac models both in two- dimensional (2D) and three-dimensional (3D) culture conditions. To this aim, by exploiting the iC2 platform, we have first generated and characterized hPSC cardiac reporter lines mirroring the endogenous expression of MYH6, MYL2, and SIRPA. Following a well-established cardiac differentiation procedure developed previously in the host laboratory, we have demonstrated the efficient generation of beating cardiac- like cells from these reporter lines in 2D culture conditions. Leveraging this new tool, we have defined a novel approach to generate 3D cardiac-like organoids in a robust and efficient manner that exhibit relevant cardiac differentiation and functional characteristics. Building on the newly defined cardiac-like organoids, we have directed our attention toward establishing a new cardiac in vitro model that can emulate important aspects of diabetic cardiomyopathy. For this purpose, we have investigated the impact of exposing hPSC-derived cardiac-like organoids to a diabetic-like insult that was previously reported by the host laboratory to induce early hallmarks of diabetic-like disease in hPSC-derived kidney organoids. Cardiac-like organoids exposed to diabetic-like culture conditions compared to normoglycemia were interrogated at the morphological, transcriptional, and functional levels to assess on the potential phenotypic alterations associated to early hallmarks of diabetic cardiopathy. Furthermore, in the last part of the present thesis, we have focused our efforts on combining hPSC-derived organoid technology with bioengineering to develop new approaches to enhance the differentiation and functional maturation of hPSC-derived organoids. To this end, we have established novel procedures based on organ decellularization technology to fabricate biomaterials mimicking the biochemical composition of the native organ. We have derived biomimetic hydrogels from decellularized extracellular matrices (dECM) obtained from kidney and heart that have been applied as new cell culture biomaterials during the differentiation of hPSC- derived kidney and cardiac organoid models. All in all, by harnessing the power of organoid technology and pioneering the development of novel biomaterials, we have successfully established advanced hPSC-derived biomodels serving as valuable tools for conducting promising investigations for future advancements in regenerative medicine applications. In view of the current advances in the field of hPSC-derived organoids, genome engineering and bioengineering, in the present thesis, we further discuss the major achievements and ongoing challenges in the development of cardiac and kidney organoids as human in vitro models to study organ development and disease. |
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