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- Creators: Stabenfeldt, Sarah
- Creators: Harrington Bioengineering Program
- Status: Published
I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics.
Protein and gene circuit level synthetic bioengineering can require years to develop a single target. Phage assisted continuous evolution (PACE) is a powerful new tool for rapidly engineering new genes and proteins, but the method requires an automated cell culture system, making it inaccessible to non industrial research programs. Complex protein functions, like specific binding, require similarly dynamic PACE selection that can be alternatively induced or suppressed, with heat labile chemicals like tetracycline. Selection conditions must be controlled continuously over days, with adjustments made every few minutes. To make PACE experiments accessible to the broader community, we designed dedicated cell culture hardware and integrated optogenetically controlled plasmids. The low cost and open source platform allows a user to conduct PACE with continuous monitoring and precise control of evolution using light.