Fig. 1. Agarose microgel encapsulation of mouse ESCs supports naive pluripotency.

a Schematic illustration of the microfluidic cell encapsulation process. Tissue culture plastic (2D) mouse ESCs were single cell separated and microfluidically compartmentalized into agarose-in-oil microdroplets. Cell-laden agarose microgels (3D) were cultured in a static suspension culture in low adhesion tissue culture plates. b Phase contrast images of mESCs cultured on tissue culture plastic or encapsulated in microgels in naive pluripotent (2i/LIF) or metastable pluripotent (serum/LIF) conditions. Circularity of mESC colonies was measured using Fiji¹⁰⁷. The p-values were calculated using a two-tailed unpaired t-test with Welch’s correction; 3 independent experiments. Error bars indicate the mean and standard deviations. c Confocal immunofluorescence images of microgel-encapsulated mESCs stained for the general pluripotency marker OCT4 and the naive pluripotency marker KLF4 after being cultured for 48 h in 2i/LIF (naive pluripotent) or N2B27 (differentiation). KLF4 and OCT4 intensities (as shown on the right) were normalized to Hoechst. Error bars indicate the mean and standard deviations. n = 400 (2i/LIF), n = 285 (N2B27). The p-values were calculated using a two-tailed unpaired t-test with Welch’s correction; 3 independent experiments. d The Rex1::GFPd2 (RGd2) reporter system allows near real-time analysis of pluripotency (destabilized GFP with a half-life of 2 h). Homogenous expression (naive in 2i/LIF), heterogeneous expression (metastable in serum/LIF), and loss of expression upon exit from pluripotency (N2B27). e, f Flow cytometric analysis of RGd2 mESCs cultured on plastic compared to microgels. Negative control: wild-type mESCs. Error bars indicate the mean and standard deviations. The p-values were calculated using a two-tailed unpaired t-test with Welch’s correction. N = 6 (plastic 2i/LIF), N = 3 (microgel 2i/LIF), N = 16 (plastic serum/LIF), N = 6 (microgel serum/LIF). g Confocal immunofluorescence images of serum/LIF cultured (microgel and plastic) mESCs stained for KLF4. KLF4 intensities (as shown on the right) were normalized to Hoechst. Error bars indicate the mean and standard deviations. The p-values were calculated using a two-tailed unpaired t-test with Welch’s correction. n = 296 (plastic), n = 130 (microgel), 3 independent experiments. h Injection of microgel-cultured naive mESCs (nuclear H2B-tq reporter) into 8-cell stage embryos led to ~73% (N = 8/11) blastocysts chimeras 48 h after in vitro culture. Injected cells were identified via the H2B-tq reporter. Cells were stained for Lamin B1 (outlines all nuclei) and SOX2 (epiblast).