Mechanically stable fibrin scaffolds promote viability and induce neurite outgrowth in neural aggregates derived from human induced pluripotent stem cells

Abstract

Recent work demonstrated that 3D fibrin scaffolds function as an effective substrate for engineering tissues from pluripotent stem cells. However, the rapid degradation rate of fibrin remains a major limitation when differentiating human pluripotent stem cells for tissue engineering applications. The addition of crosslinking agents, such as genipin, during the polymerization process increases scaffold stability while decreasing the degradation rate of fibrin. Genipin crosslinking alters the physical characteristics of the fibrin scaffolds, which influences the behaviour of the differentiating cells seeded inside. It also possesses neuritogenic and neuroprotective properties, making it particularly attractive for engineering neural tissue from pluripotent stem cells. Here we show that genipin enhances neuronal differentiation of neural progenitors derived from human induced pluripotent stem cells (hiPSCs) in 2D culture and genipin concentration influences the morphological and mechanical properties of 3D fibrin scaffolds. These mechanically stable genipin-crosslinked fibrin scaffolds support hiPSC-derived neural aggregates and induce neurite outgrowth while remaining intact for 2 weeks as opposed to 5 days for unmodified fibrin scaffolds.

3D Bioprinting Human Induced Pluripotent Stem Cell-Derived Neural Tissues Using a Novel Lab-on-a-Printer Technology

“…hiPSC-derived NPCs were obtained as previously described from the hiPSC line, 1-DL-01, from WiCell [16,28]. Experiments using hiPSC-derived NPCs were conducted with the approval of the University of Victoria's Human Ethics Committee under protocol number: 12-187.…”

“…Each treatment was placed on a 15 mL conical tube and centrifuged at 300 rpm for 5 min. Preparation of the cell suspension was performed as previously reported following the Guava ViaCount ® protocol for cell viability determinations [16]. The supernatant was removed, and the pellet was resuspended in 1 mL of phosphate buffered solution (PBS) (10010023, Thermo Fisher, Waltham, MA, USA).…”

“…Being a naturally-derived material, fibrin has a decreased mechanical stability and a slow polymerization process [8,15]. The polymerization of fibrin starts with the addition of the enzyme, thrombin [16]. Alginate, which polymerizes quickly, can be mixed with fibrin as a second component and make the bioink suitable for bioprinting [8].…”

“…Alginate, which polymerizes quickly, can be mixed with fibrin as a second component and make the bioink suitable for bioprinting [8]. To address the challenge of the lack of mechanical stability of fibrin scaffolds, small molecules, such as genipin, can enhance the structural properties of the scaffold and promote neural outgrowth [16]. The cationic polysaccharide, chitosan, is the third component of the novel bioink used to provide mechanical strength to the scaffolds by ionically interacting with alginate and covalently crosslinking with genipin [17].…”

Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions

“…The results of this work showed that the partially differentiated iPSC‐RPE can directly adhere to the fibrin gel, form a monolayer and express phenotype specific marker proteins . Similarly, work from Willerths' group has demonstrated culture of iPSC‐derived neural aggregates on fibrin hydrogels can be used to generate a mixed population of dorsal and ventral spinal neurons . These examples highlight the ability to differentiate iPSC‐derived progenitor‐type populations to terminally differentiated cells using fibrin hydrogels.…”