Microbioreactor Arrays for Full Factorial Screening of Exogenous and Paracrine Factors in Human Embryonic Stem Cell Differentiation

Abstract

Timed exposure of pluripotent stem cell cultures to exogenous molecules is widely used to drive differentiation towards desired cell lineages. However, screening differentiation conditions in conventional static cultures can become impractical in large parameter spaces, and is intrinsically limited by poor spatiotemporal control of the microenvironment that also makes it impossible to determine whether exogenous factors act directly or through paracrine-dependent mechanisms. We detail here the development of a continuous flow microbioreactor array platform that combines full-factorial multiplexing of input factors with progressive accumulation of paracrine factors through serially-connected culture chambers, and further, the use of this system to explore the combinatorial parameter space of both exogenous and paracrine factors involved in human embryonic stem cell (hESC) differentiation to a MIXL1-GFP+ primitive streak-like population. We show that well known inducers of primitive streak (BMP, Activin and Wnt signals) do not simply act directly on hESC to induce MIXL1 expression, but that this requires accumulation of surplus, endogenous factors; and, that conditioned medium or FGF-2 supplementation is able to offset this. Our approach further reveals the presence of a paracrine, negative feedback loop to the MIXL1-GFP+ population, which can be overcome with GSK-3β inhibitors (BIO or CHIR99021), implicating secreted Wnt inhibitory signals such as DKKs and sFRPs as candidate effectors. Importantly, modulating paracrine effects identified in microbioreactor arrays by supplementing FGF-2 and CHIR in conventional static culture vessels resulted in improved differentiation outcomes. We therefore demonstrate that this microbioreactor array platform uniquely enables the identification and decoding of complex soluble factor signalling hierarchies, and that this not only challenges prevailing strategies for extrinsic control of hESC differentiation, but also is translatable to conventional culture systems.

Engineering stem cell niches in bioreactors

“…Continuous flow microbioreactor arrays revealed the effect of paracrine-dependent mechanism during human ESC differentiation into early mesoderm [15] . The negative feedback loop to the mesoderm progenitors could be overcome by adding the glycogen synthase kinase (GSK)-3β inhibitor CHIR99021 and the positive induction signals were enhanced by supplementing FGF-2.…”

“…The critical culture parameters that have been widely studied in a bioreactor system for engineering stem cell niches include oxygen tension, 3-D scaffolds (synthetic or decellularized), physical forces such as hydrodynamic forces, mechanical strain, flow shear and electrical stimulation (Figure 1). Recently, microfluidic devices and microbioreactors have been designed and used for high throughput regulation of microenvironmental factors to better understand stem cell niches and for drug screening [14,15] . Paracrine/autocrine factors can also be revealed using microfluidic devices and microbioreactors to better control stem cell differentiation.…”

High-throughput screening approaches and combinatorial development of biomaterials using microfluidics

“…In such cases, other techniques, including 3D printing [14][15][16], sequential overlapping layers [17][18][19], porous or impermeable membranes [20][21][22] or valves [23] may help to increase multiplicity of variables with higher level of freedom in interconnectivity. Highly sophisticated operations and effective use of the hierarchical structuring of microfluidic compartments can also be achieved in centrifugal microfluidics [24].…”

Advances in microfluidic platforms for analyzing and regulating human pluripotent stem cells

“…Titmarsh et al . [35] developed a continuously-connected flow array that applied exogenous soluble cues and circulated endogenous paracrine factors. Using this device, they determined that Wnt signaling-induced primitive streak differentiation in hESCs required accumulation of endogenous factors or FGF-2 supplementation.…”