Abstract
Concentration gradients of biochemical stimuli such as morphogens play a critical role in directing cell fate patterning across species and throughout development but are not commonly recapitulated in vitro. While in vitro biomolecule gradients have been generated using customized microfluidic platforms, broad implementation has been limited because these platforms introduce new variables to cell culture such as externally driven flow, culture in a specialized matrix, or extended time for in situ long range diffusion. Here we introduce a method that enables preforming and then transferring user-controlled gradients to cells in standard “open” cultures. Our gradient patterning devices are modular and decoupled from the culture substrate. We find that gradient generation and transfer are predictable by finite element modeling and that device and loading parameters can be used to tune the stimulus pattern. Furthermore, we demonstrate use of these devices to spatially define morphogen signal gradients and direct peri-gastrulation fate stratification of human pluripotent stem cells. This method for extrinsic application of biochemical signal gradients can thus be used to spatially influence cellular fate decisions in a user-controlled manner.
Highlights
Spatial and temporal patterns of biochemical signals play a central role in orchestrating the development of multicellular organisms
The model showed multiple dosing resulted in the desired higher final source-end concentrations with minimal change to sink-end concentrations compared to single dosing (Fig. 4C). Using this method to generate steep and stable morphogen gradients (Fig. 4A), we investigated whether parallel long-range BMP4 and CHIR99021 gradients could direct human pluripotent stem cells (hPSCs) differentiation patterning
We found that after exposure to BMP4/CHIR99021 gradients, hPSC cultures exhibited a gradient of T expression (Fig. 4E–E”), in agreement with our hypothesis
Summary
Spatial and temporal patterns of biochemical signals play a central role in orchestrating the development of multicellular organisms. Www.nature.com/scientificreports radial distribution of downstream signals and subsequent differentiation patterns across the hPSC colonies[15,16,17,18,19,20,21,22,23,24] While these studies provide informative in vitro models of self-driven peri-gastrulation fate patterning, they rely upon cell-directed signal patterning that occurs after homogenous application of soluble stimuli to the medium. Other groups have avoided issues associated with flow by patterning differentiation using morphogen gradients generated through source-to-sink diffusion in hydrogels[30,31,32] In these systems, cells are exposed to new matrices as well as to the morphogen itself while the gradient forms and stabilizes within the matrix (a time period that varies based on the biochemical cue’s molecular weight and matrix porosity). While these technologies have taken important steps forward towards creating user-defined gradients, they typically introduce new variables into hPSC cultures
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