Full factorial screening of human embryonic stem cell maintenance with multiplexed microbioreactor arrays

Use of human pluripotent stem cells (hPSCs) in regenerative medicine applications relies on control of cell fate decisions by exogenous factors. This control can be hindered by the use of undefined culture components, poorly understood autocrine/paracrine effects, spatiotemporal variations in microenvironmental composition inherent to static culture formats, and signal cross-talk between multiple factors. We recently described microbioreactor arrays that provide a full factorial spectrum of exogenous factors, and allow gradual accumulation of paracrine factors through serial culture chambers. We combined these with defined biochemical conditions, and in situ reporter gene- and immunofluorescence-based readouts to create an hPSC screening platform with enhanced data throughput and microenvironmental control. HES3-EOS-C(3+)-EiP reporter hESCs were screened against FGF-2, TGF-β1, and retinoic acid in a modified mTeSR-1 medium background. Differential pluripotency marker expression reflected mTeSR-1's maintenance capacity, and differentiation in response to removal of maintenance factors or addition of retinoic acid. Interestingly, pluripotency marker expression was downregulated progressively through serial chambers. Since downstream chambers are exposed to greater levels of paracrine factors under continuous flow, this effect is thought to result from secreted factors that negatively influence pluripotency. The microbioreactor array platform decodes factor interplay, and has a broad application in deciphering microenvironmental control of cell fate.