Microbioreactor arrays for full factorial screening of exogenous and paracrine factors in human embryonic stem cell differentiation

Titmarsh, Drew M., Hudson, James E., Hidalgo, Alejandro, Elefanty, Andrew G., Stanley, Edouard G., Wolvetang, Ernst J. and Cooper-White, Justin J. (2012) Microbioreactor arrays for full factorial screening of exogenous and paracrine factors in human embryonic stem cell differentiation. PLoS One, 7 12: e52405.1-e52405.12. doi:10.1371/journal.pone.0052405


Author Titmarsh, Drew M.
Hudson, James E.
Hidalgo, Alejandro
Elefanty, Andrew G.
Stanley, Edouard G.
Wolvetang, Ernst J.
Cooper-White, Justin J.
Title Microbioreactor arrays for full factorial screening of exogenous and paracrine factors in human embryonic stem cell differentiation
Journal name PLoS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2012-12-01
Year available 2012
Sub-type Article (original research)
DOI 10.1371/journal.pone.0052405
Open Access Status DOI
Volume 7
Issue 12
Start page e52405.1
End page e52405.12
Total pages 12
Place of publication San Francisco, CA, United States
Publisher Public Library of Science
Language eng
Subject 1100 Agricultural and Biological Sciences
1300 Biochemistry, Genetics and Molecular Biology
2700 Medicine
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-3 beta 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.
Formatted 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.
Keyword Multidisciplinary Sciences
Science & Technology - Other Topics
MULTIDISCIPLINARY SCIENCES
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Article number e52405

 
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