Bioengineered Scaffold Microenvironment Promotes Assembly and Differentiation of Stem Cell-Derived Human Organoids

Abstract

Human pluripotent stem cells (hPSCs) differentiated into complex three-dimensional (3D) structures, referred to as ‘organoids’ due to their organ-like properties, offer ideal platforms to study human development, disease and regeneration. However, studying organ morphogenesis has been hindered by the lack of appropriate culture systems that can spatially enable cellular interactions that are needed for organ formation. Many organoid cultures rely on decellularized extracellular matrices as supportive scaffolds, which are often poorly chemically defined and allow only limited tunability and reproducibility. By contrast, engineered synthetic matrices can be tuned and optimized to mimic the embryo environment in order to enhance development and maturation of organoid cultures. Herein, this dissertation primarily focuses on testing the hypothesis that using synthetic microporous polymer matrices can guide key interactions guiding stem-cell decisions for the reproducible generation and control of organoid cultures. One study showed microporous synthetic biomaterials can guide the assembly of pancreatic progenitors into insulin-producing clusters that further developed into islet organoids. Immunofluorescent analysis showed the scaffold culture facilitated cell-cell interactions by significantly increasing protein expression of ECAD compared to suspension (42 ± 5% vs 21 ± 4% of total cell population, n=4; P< 0.01). PLG scaffold cultures supported cell-mediated matrix deposition of extracellular matrix (ECM) proteins associated with the basement membrane of islet cells as well. Furthermore, hPSC-derived β-cells cultured on the PLG scaffold showed an enhanced insulin secretion index compared to the suspension culture control (1.3 ± 0.2 vs 0.43 ± 0.06, n=3, P< 0.01), indicating the development of functional β-cells. By modifying the stage that cells were seeded on scaffolds from pancreatic progenitor to pancreatic endoderm, islet organoids showed increased amounts of insulin secreted per cell. In addition, seeding scaffolds with dense clusters instead of a single suspension minimized cell manipulation during the differentiation, which was shown to be influential to the development of the islet organoids. An engineered insulin reporter further identified how mechanistic changes in vitro influenced function within individual cells by measuring insulin storage and secretion through non-invasive imaging. Human lung organoids (HLOs) were also evaluated for in vivo maturation on biomaterial scaffolds, where HLOs were shown improved tissue structure and cellular differentiation. We sought to examine the contribution of polymer degradation to the number of airway-like structures with the hypothesis that faster degradation would permit more HLOs fusing in adjacent pores, thus, support larger airway-like structures. Investigative studies demonstrated slower degrading 85:15 PLG HLOs had significantly smaller airway diameter than the faster degrading 75:25 PLG (224 µm vs. 333 µm, P< 0.05) confirming that scaffold pore interconnectivity and polymer degradation contributed to in vivo maturation. Polymer biomaterials were also developed to modulate local tissue and systemic inflammation through local delivery of human interleukin 4 (hIL-4)-expressing lentivirus. Microporous scaffold culture strategies improve organoid complexity and exert fine control over the system using engineering solutions, thus, allowing the community to build more realistic organoid tools. Taken together, the microporous scaffold culture has the feasibility to translate organoid culture to the clinic as a biomanufacturing platform.