Mini shaker

Gastruloids were generated as previously described (Baillie-Johnson et al., 2015 ). Briefly, 300–700 mESCs were plated in 40 μL N2B27 in 96-well Clear Round Bottom Ultra-Low Attachment Microplates (7007, Corning). After 48 h, 150 μL of N2B27 containing 3 μM Chi were added to each well. After 72 h, medium was changed with N2B27. Starting from 96 h, the protocol was optimized as described in Figure S1A. At 96 h, gastruloids were transferred in Ultra-Low Attachment 24-well Plates (3473, Corning) in 100 μL of medium, plus 700 μL of fresh N2B27 containing 30ng ml⁻¹ bFGF (PMG0034, GIBCO), 5ng ml⁻¹ VEGF 165 (PHC9394, GIBCO) and 0.5mM L-ascorbic acid phosphate (013–12061, Wako) (N2B27+++) and cultured on an orbital shaker placed at 37°C, 5%CO₂ at 100rpm (VWR mini shaker). From 120 h onward, half medium was changed daily. Unless differently specified, N2B27+++ was applied from 96 to 144 h, while from 144 h to 168 h N2B27 was used for medium change. To generate Hcn4-GFP::Tbx1Cre-RFP gastruloids, 800–1200 cells were employed, due to initial difficulties in cell aggregation and extreme susceptibility to Chi treatment. For the same reason, Chi pulse for this line was done with 1 μM Chi, with the exact same modalities described for the other lines.

Hydrolysis reactions were performed with shaking by a Mini Shaker from VWR (Radnor, PA), which controlled both the stir rate (650 rpm) and the temperature (105 °C). ¹H-NMR spectroscopy was performed with 500 and 400 MHz spectrometers from Bruker (Billerica, MA). Samples were dried under high vacuum (<0.1 Torr) with a mechanical belt-drive oil pump from Welch (Niles, IL). Filtration used a suction pump from Gast Manufacturing (Benton Harbor, MI). Centrifugation was done with an IEC HN-SII centrifuge from Damon International Equipment (now, Thermo-Fischer, Waltham, MA).

PEGDA-co-A6ACA hydrogels were synthesized by reacting 1 M A6ACA and 4% PEGDA (M_n = 6 kDa) with 0.5% ammonium persulfate (APS) and 0.15% N,N,N′,N′-tetramethylethylenediamine (TEMED) in a glass mold with 1 mm spacer for 15 minutes at 25 °C.^{16 (link)} The resulting hydrogels were incubated in phosphate buffered saline (PBS) overnight with two changes of PBS. The equilibrium-swollen hydrogels were then cut into discs of 1 cm². To create bulk rigidity-matched biomineralized matrices, the above procedure was repeated except 2% PEGDA was used in the precursor solution.^{16 (link)} The hydrogel discs were biomineralized as described elsewhere.^{16 (link)} Briefly, hydrogel discs were incubated in DI water for 6 hours and then in modified simulated body fluid (m-SBF) for 6 hours. The hydrogel discs were then rinsed with DI water and incubated in a 40 mM Ca²⁺ and 24 mM HPO⁴³⁻ solution (pH = 5.2) at 25 °C for 45 minutes on a rotating shaker (VWR Mini-shaker) at 200 rpm. The discs were rinsed with DI water, incubated in m-SBF at 37 °C for 48 hours with a daily exchange of m-SBF followed by incubation in PBS for 6 hours. Both non-mineralized and mineralized hydrogels were sterilized through immersion in 70% ethanol (EtOH) for 6 hours, followed by washing in PBS for 4 days with three daily exchanges of PBS prior to cell culture.

The humic acid standards (Leonardite HA, Florida peat HA, and Suwannee River HA) were obtained from the International Humic Substances Society (Georgia, USA). More details on each of these HAs, including chemical composition, are available in the supplementary material, with further details available on the IHSS website (www.humicsubstances.org, accessed on Aug 8^th, 2015). The surfactants Triton X-100, cetylpyridinium chloride and sodium dodecyl sulfate were all purchased from Sigma Aldrich (Piscataway, NJ). Sodium chloride and sodium hydrogen carbonate for the saline solution were purchased from Sigma Aldrich. Sterile 18 MΩ deionized water was sourced from an apparatus by US filter. Artemia Franciscana was purchased from Brine Shrimp Direct (Ogdon, UT). Fisherbrand 100 × 15 mm petri dishes were purchased from Fisher Scientific (Somerville, NJ). A VWR mini shaker was used during the hatching assays. An AmScope SE305R-PZ stereoscopic microscope was utilized for observing and counting the Artemia.