Supporting Information for: Mucin-inspired thermoresponsive synthetic hydrogels induce stasis in human pluripotent stem cells and human embryos

Transcription

1 Supporting Information for: Mucin-inspired thermoresponsive synthetic hydrogels induce stasis in human pluripotent stem cells and human embryos Irene Canton*, Nicholas J. Warren, Aman Chahal, Katherine Amps, Andrew Wood, Richard Weightman, Eugenia Wang, Harry Moore* and Steven P. Armes* S1

2 Figure S1. Variation in storage and loss moduli (G, G ) with applied strain at 37 C (at a fixed angular frequency of 1 rad s -1 ) and variation in storage and loss moduli (G, G ) with angular frequency (at a fixed applied strain of 1 %) for a 6% w/v PGMA 55 -PHPMA 135 worm gel reconstituted in various cell culture media. S2

3 Figure S2. Effect of different commercial media on hesc colony size over a 7-day gelation period. hesc colonies were dispersed mechanically in an aqueous 6% w/v PGMA 55 - PHPMA 135 worm gel comprising Nutristem cell culture medium (NS) or Embryoid Body medium (EB) and stored for 7 days at 37 o C in a humidified incubator. Optical micrographs of the same colonies were recorded at day 0 (gelation day) and at day 7 in the gel. Note that there is no discernible evidence for colony expansion. S3

4 Figure S3. Gelation and recovery of hes cell suspensions using a 6% PGMA 55 -PHPMA 135 worm gel reconstituted with 3i medium. Cell monolayers were washed with PBS and dissociated using TrypLE Select. Suspensions of 1 x 10 5 hescs were gelled in 6% PGMA 55 - PHPMA 135 worm gel reconstituted with 3i medium and stored at 37 C in a humidified incubator (5% CO 2 /95% air). Optical micrographs were recorded for the same gelled-3i medium well over time (a) at time 0, (b) time 24h and (c) time 7 days. Cells were recovered via degelation at 4 C and resuspended in liquid 3i medium and cultured on CELLstart TM - coated six-well plates for 48 h. Recovered cells were examined by optical microscopy and compared to a control group (not shown) with 1 x 10 5 hescs grown for 48 h after dissociation. Recovered cells after 24 h gelation in 3i medium (d) were able to grow into colonies of similar size to those of the control group. No viable colonies were observed upon degelation after 7 days (e). S4

5 Figure S4. Live/dead imaging of hesc colonies immersed within PGMA 55 -PHPMA 135 worm gels for 21 days. Representative fluorescence micrographs recorded after live/dead staining using Syto9 and propidium iodide (PI) dyes for cell colonies immersed in a worm gel for 21 days at 37 o C. The cell-permeable dye, Syto-9, indicates cell membrane integrity (live cells), whereas the cell-impermeable dye, PI, stains dead cells only. Hoechst was used as a cell nuclei counter-stain. The three micrographs represent a typical colony, the best colony and the worst colony with respect to colony viability within a given experiment. S5

6 Figure S5. Effect of environment on hesc colony morphology. The micrographs represent (a) a typical flat hesc colony growing in Nutristem + CELLstart TM after fixation and (b) the morphology of a domed hesc colony recovered after 7 days immersion in a 6% w/v PGMA 55 -PHPMA 135 worm gel, followed by recovery for 24 h in Nutristem + CELLstart TM and immediate fixation. S6

7 Figure S6. Differentiation of human embryonic stem cell colonies (MShef 4) recovered from worm gel after 14 days and passaged to standard adherent culture conditions. (A) Differentiating hesc colonies after 7 days; white arrow indicates remnants of colony; inset: undifferentiated hesc colonies immediately after recovery. (B) differentiated cells after 10 days; black arrow indicates bone-like progenitor cells; white arrow indicates trophoblast syncytial cells. (C) differentiated neural epithelial cells after 10 days with axon projections. (D) rapid differentiation of endothelial-like cells after 10 days. Bar = 100 m. S7

8 Figure S7. Effect of original hesc colony size on colony recovery after immersion in a 6% w/v worm gel for 14 days at 37 o C, followed by isolation via degelation. hesc colonies were dispersed mechanically in an aqueous PGMA 55 -PHPMA 135 worm gel comprising Nutristem cell culture medium and stored for 14 days at 37 o C. Colonies were then isolated via thermally-triggered degelation and subsequently plated on Cellstart and Nutristem liquid medium. (a)-(e) Optical micrographs of various hesc colonies recorded 5 days after culture. Small colonies (< 100 m) either exhibited (a) minimal growth over time and differentiation or (b) failed to thrive after degelation. (c) Enhanced attachment and growth was observed for colonies greater than 100 m in size and (d) optimal growth was obtained for m colonies, which also exhibited the characteristic ES cell morphology. (e) Differentiated neuronal cells observed at the edge of a relatively large (>> 200 m) colony in a high-density culture. The traced line denotes the original colony size after initial degelation. S8

9 Figure S8. Ki-67 immunolabeling of hesc colonies immersed within a 6% w/v worm gel compared to after degelation. hesc colonies were positively stained for Ki-67 growing normally in a liquid medium on a CELLstart TM matrix (control). Colonies were then mechanically removed and immersed in PGMA 55 -PHPMA 135 worm gels containing Nutristem medium at 37 o C for 7 days, 14 days or 21 days, then de-gelled, fixed and immunolabeled for Ki-67. While immersed within the worm gel, no Ki-67 protein was detected for any of the cell colonies. Colonies were also isolated via degelation after 7 days, 14 days or 21 days, re-plated onto wells coated with CELLstart TM and grown in Nutristem medium for 5 days prior to conducting further immunolabeling experiments for Ki-67. In all cases, Ki-67 expression was confirmed via positive staining (Cy3, red) with a nuclei counterstain (Hoechst 33342, blue). Experiments were performed in triplicate wells, with n = 3 independent experiments. S9

10 Figure S9. hpscs proliferate in a PNIPAAm-PEG based thermo-responsive gel. hpscs were gelled in Mebiol Gel following manufacturer s instructions and cell proliferation was observed for 14 days. (a) Optical micrographs show growth of the gelled hpsc colonies into embryoid bodies. (b) Immunofluorescence analysis revealed that colonies gelled in Mebiol Gel remained Ki-67 (+) and NES (-) indicating that cells were actively cycling. S10

11 Figure S10. Adapted hpscs in PGMA 55 -PHPMA 135 worm gels. hpscs H7S6 that have been adapted on feeder cells and have developed genetic modifications similar to those seen in teratomas were able to grow on PGMA-PHPMA worm gels. The colonies expanded and pushed out of the original gelling site invading the surrounding gel, much like tumours. S11

12 Figure S11. Effect of immersing hpscs in PGMA-PHPMA worm gels on their attachment and metabolic activity under various culture conditions.(a) Schematic cartoon summarizing the sequential experimental protocol for the various culture conditions employed for the hpscs. (b) When hpscs are provided with a suitable 2D substrate for attachment (e.g. Cellstart), a normalized cell viability (MTT) assay confirmed that they could tolerate direct contact (d.c.) with the worm gel without any adverse effect on their metabolic activity. (c) hpscs do not attach to the surface of the worm gel, suggesting minimal interaction, but the same hpscs remain attached in d.c. with worm gel when Cellstart substrate was added to the wells. In summary, these 2D experiments demonstrate that PGMA-PHPMA worm gels exhibit minimal interaction with hpscs, but are biocompatible under the experimental conditions used to induce stasis. S12

13 Table 1. Analysis* of the proliferative status of hesc colonies immersed in a 6% w/v PGMA 55 -PHPMA 135 worm gel: % Amount of DNA. Gelation time % Normalized %SEM (Days) amount of DNA ± ± ± ±7.8 Micrographs ** t=0 t=7 t=14 t=21 *Gelled hesc colonies were kept for 0, 1, 14 or 21 days at 37 o C in a humidified incubator. Colonies were harvested via degelation at the relevant time points and total DNA was harvested and quantified, as reported in materials and methods. Data were normalized to the initial DNA amount (taken to be 100%) obtained from colonies seeded at day 0. Data represent average values of n = 3 experiments conducted in duplicate wells. **Microscopy images indicated very little change in colony size over time, which is consistent with these observations (Scale bar= 200 m). S13