The PDF file includes:

• Section S1. Temporal and spatial concentration distribution within the cell culture chambers
• Section S2. Single 3T3 fibroblast cell culture and stimulation on chip
• Section S3. Culture and stimulation of human and mouse HSCs on chip
• Section S4. Culture and stimulation of NSC spheres on chip
• Section S5. Combinatorial and sequential experiments performed in 96-well plates
• Section S6. Extended discussion of high-throughput combinatorial and sequential input studies
• Section S7. Hes5 expression as a valid marker for NSC stemness
• Section S8. Immunostaining on chip and determining NSC phenotypes
• Section S9. Statistical analysis of combinatorial and sequential results
• Section S10. NSC single-cell tracking during combinatorial and sequential stimulation
• Fig. S1. Experimental characterization of concentration variations during medium exchange.
• Fig. S2. Assessment of the microfluidic system for dynamical cell culture and NF-κB signaling.
• Fig. S3. Culture and stimulation of human HSCs on chip.
• Fig. S4. Hes5 and Dcx expression regulating NSC cellular behavior.
• Fig. S5. Combinatorial and sequential stimulation of six ligands regulating NSC self-renewal and differentiation.
• Fig. S6. Correlation between Hes5 expression and NSC stemness.
• Fig. S7. Combinatorial and sequential inputs regulating NSC proliferation, Hes5, and Dcx expression.
• Fig. S8. Effect of various stimulation conditions on NSC cell fate subjected to statistical analysis.
• Legends for tables S1 and S2
• Legends for movies S1 to S5

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Other Supplementary Material for this manuscript includes the following:

• Table S1 (Microsoft Excel format). Microenvironment exposed to six single ligands and combinatorial and sequential ligand inputs (note: the order of the ligands in the table represents the order of ligands introduced into the microenvironments on daily bases).
• Table S2 (Microsoft Excel format). Statistical analysis results associated with sequential and combinatorial inputs of six ligands based on cell count measurements and Hes5 and Dcx expression level.
• Movie S1 (.mov format). COMSOL simulation and time-lapse video of fluid exchange in a unit chamber on the chip.
• Movie S2 (.avi format). Redistribution of GFP after medium exchange and all valves are closed.
• Movie S3 (.mov format). Retrieval of adherent cells (3T3, left) and suspension-cultured cells (Jurkat, right) from the chip.
• Movie S4 (.avi format). Stimulation of 3T3 cells by sinusoidal TNF-α inputs.
• Movie S5 (.avi format). Cell tracking videos of NSC spheres (top) and monolayer (bottom).