Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells
Background Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC)...
| Main Authors: | , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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Public Library of Science
2011
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| Online Access: | http://hdl.handle.net/1721.1/66169 https://orcid.org/0000-0001-8898-2296 |
| _version_ | 1826201276867674112 |
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| author | Blagovic, Katarina Kim, Lily Y. Voldman, Joel |
| author2 | Whitaker College of Health Sciences and Technology |
| author_facet | Whitaker College of Health Sciences and Technology Blagovic, Katarina Kim, Lily Y. Voldman, Joel |
| author_sort | Blagovic, Katarina |
| collection | MIT |
| description | Background
Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands.
Methodology/Principal Findings
We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs.
Conclusions/Significance
Our results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems. |
| first_indexed | 2024-09-23T11:49:28Z |
| format | Article |
| id | mit-1721.1/66169 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:49:28Z |
| publishDate | 2011 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | mit-1721.1/661692022-09-27T22:08:58Z Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells Blagovic, Katarina Kim, Lily Y. Voldman, Joel Whitaker College of Health Sciences and Technology Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science Massachusetts Institute of Technology. Research Laboratory of Electronics Voldman, Joel Voldman, Joel Blagovic, Katarina Kim, Lily Y. Background Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands. Methodology/Principal Findings We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs. Conclusions/Significance Our results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems. National Institutes of Health (U.S.) (NIH grant No. EB007278) Swiss National Science Foundation Swiss National Science Foundation 2011-10-03T20:40:03Z 2011-10-03T20:40:03Z 2011-08 2010-11 Article http://purl.org/eprint/type/JournalArticle 1932-6203 http://hdl.handle.net/1721.1/66169 Blagovic, Katarina, Lily Y. Kim, and Joel Voldman. “Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells.” Ed. Wei-Chun Chin. PLoS ONE 6 (2011): e22892. https://orcid.org/0000-0001-8898-2296 en_US http://dx.doi.org/10.1371/journal.pone.0022892 PLoS ONE Creative Commons Attribution http://creativecommons.org/licenses/by/2.5/ application/pdf Public Library of Science PLoS |
| spellingShingle | Blagovic, Katarina Kim, Lily Y. Voldman, Joel Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells |
| title | Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells |
| title_full | Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells |
| title_fullStr | Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells |
| title_full_unstemmed | Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells |
| title_short | Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells |
| title_sort | microfluidic perfusion for regulating diffusible signaling in stem cells |
| url | http://hdl.handle.net/1721.1/66169 https://orcid.org/0000-0001-8898-2296 |
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