Mucin-Inspired Thermoresponsive Synthetic Hydrogels Induce Stasis in Human Pluripotent Stem Cells and Human Embryos

Abstract

Human pluripotent stem cells (hPSCs; both embryonic and induced pluripotent) rapidly proliferate in adherent culture to maintain their undifferentiated state. However, for mammals exhibiting delayed gestation (diapause), mucin-coated embryos can remain dormant for days or months in utero, with their constituent PSCs remaining pluripotent under these conditions. Here we report cellular stasis for both hPSC colonies and preimplantation embryos immersed in a wholly synthetic thermoresponsive gel comprising poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) [PGMA55-PHPMA135] diblock copolymer worms. This hydroxyl-rich mucin-mimicking nonadherent 3D gel maintained PSC viability and pluripotency in the quiescent G0 state without passaging for at least 14 days. Similarly, gel-coated human embryos remain in a state of suspended animation (diapause) for up to 8 days. The discovery of a cryptic cell arrest mechanism for both hPSCs and embryos suggests an important connection between the cellular mechanisms that evoke embryonic diapause and pluripotency. Moreover, such synthetic worm gels offer considerable utility for the short-term (weeks) storage of either pluripotent stem cells or human embryos without cryopreservation.

Figure 1

Physicochemical and biological characterization of mucin-mimicking PGMA₅₅-PHPMA₁₃₅ worm gels. (a) Schematic representation of the similar physical and chemical (hydroxyl-rich brush) structures of mucin gels and PGMA-PHPMA worm gels. (b) Temperature dependence of the storage and loss moduli (G′, G″) observed on cooling a 6% w/v worm gel reconstituted in various aqueous media from 37 to 2 °C at an applied strain of 1.0% and a fixed angular frequency of 1.0 rad s^–1. (c) Three protocols were evaluated with human dermal fibroblasts: protocol 1 (20% w/v copolymer gel prepared in PBS, diluted two-fold and dialyzed for 2 days against PBS), protocol 2 (same as protocol 1, followed by dialysis against PBS for 7 days), and protocol 3 (20% w/v copolymer gel prepared in PBS, dialyzed against deionized water for 7 days, followed by freeze-drying overnight and redispersion in DMEM cell culture medium). Cell viability was evaluated by direct-contact cell monolayers and also via an indirect assay using ThinCert inserts. Experiments were conducted in triplicate; **p < 0.01, *p < 0.05. (d) Five-day-old human blastocysts immersed in either 6% w/v worm gel or Matrigel incubated at 37 °C, 5% CO₂ and 5% O₂. After 4 days, embryos in Matrigel undergo fragmentation, whereas embryos immersed in worm gel remained intact but became compacted. Five-day-old embryos immersed within worm gel for 8 days (i.e., up to 13 days of development) stained positive for nuclear envelope statin (NES). Localization (red, nuclear envelope statin; blue, Hoechst 33342-nuclei; scale bar = 50 μm). n = 2 embryos per condition.

Figure 2

hESCs in PGMA₅₅-PHPMA₁₃₅ worm gel maintain viability and stem cell markers. (a) Proportion (%) of proliferative (hESC colonies recovered from reconstituted 6% w/v worm gels prepared using Nutristem medium for up to 21 days. (b) Syto 9 (live)/PI (dead) staining for typical cell colonies immersed in worm gel at 37 °C. Hoechst 33342 counter-stain. (c) FACS analysis of pluripotent (Tra-1-60, SSEA-4) and differentiation (SSEA-1) stem cell markers for hESCs recovered from worm gel after 21 days and then subjected to standard adherent cell culture for up to 14 days compared to control hESCs cultured in the absence of gel. Expression of pluripotent markers is equivalent or greater in cells stored in worm gel for 21 days prior to adherent culture and declines more slowly. (d) Immunofluorescent localization of Oct-4 and Nanog in hESCs after recovery from worm gel compared to a control. All experiments were performed in triplicate wells, with n = 3 independent experiments.

Figure 3

Changes in nuclear envelope statin (NES) and Ki-67 expression for hESCs immersed within a 6% w/v worm gel: (a) Overview of gelation/degelation process and associated changes in cell stasis markers. (b) Immunofluorescent localization of NES in hESC colonies. Control hESCs (no worm gel) did not display NES. After 2–6 h immersion, NES⁺ cells were only observed at the periphery of hESC colonies, but NES⁺ cells were distributed throughout the colony after 16–24 h. (c) Effect of degelation on NES expression. Cytosolic expression of NES after 16 h. Expression of NES⁺ was significantly reduced both 24 h after degelation and also after 5 days. (d) Immunofluorescent localization of Ki-67 in hESC colonies in gel. Control hESC (no gel) displayed Ki-67 expression, but this was considerably reduced after 16–24 h within the worm gel. All experiments were performed in triplicate, n = 3 independent experiments.