Brain-stiffness-mimicking tilapia collagen gel promotes the induction of dorsal cortical neurons from human pluripotent stem cells

The mechanical properties of the extracellular microenvironment, including its stiffness, play a crucial role in stem cell fate determination. Although previous studies have demonstrated that the developing brain exhibits spatiotemporal diversity in stiffness, it remains unclear how stiffness regulates stem cell fate towards specific neural lineages. Here, we established a culture substrate that reproduces the stiffness of brain tissue using tilapia collagen for in vitro reconstitution assays. By adding crosslinkers, we obtained gels that are similar in stiffness to living brain tissue (150–1500 Pa). We further examined the capability of the gels serving as a substrate for stem cell culture and the effect of stiffness on neural lineage differentiation using human iPS cells. Surprisingly, exposure to gels with a stiffness of approximately 1500 Pa during the early period of neural induction promoted the production of dorsal cortical neurons. These findings suggest that brain-stiffness-mimicking gel has the potential to determine the terminal neural subtype. Taken together, the crosslinked tilapia collagen gel is expected to be useful in various reconstitution assays that can be used to explore the role of stiffness in neurogenesis and neural functions. The enhanced production of dorsal cortical neurons may also provide considerable advantages for neural regenerative applications.


Peptide sequence of collagen derived from tilapia skin
Three bands were manually cut after SDS-PAGE ( Supplementary Fig. S1Bb). Proteins included in each band were subsequently digested by treatment with Trypsin. MALDI-TOF/MS analysis was performed using a Microflex LRF 20 (Bruker) (GENOMINE Inc., Pohang, Korea). The obtained peptide sequences are listed in Supplementary Table S1. Each peptide sequence was aligned using Clustal Omega (version 1.2.4).

Scanning electron microscopy (SEM) of collagen gels
The surface structure of gels was observed using an SEM (SU8220, Hitachi). The fixation and drying procedures were as follows. The gels were fixed with 2.5% glutaraldehyde (25% stock, 18427, Ted Pella) in 0.1 M phosphate buffer (PB) at 4˚C overnight. After 2 washes in 0.1 M PB for 15 min, the gels were post-fixed with 1% OsO 4 (2% aqueous stock, 18466, Ted Pella) at 4˚C for 1 h, then washed twice in 0.1 M PB for 15 min. Subsequently, the gels were dehydrated by sequential immersion in 50, 60, 70, 80, 90 and 95% ethanol for 10 min at 4˚C. The gels were then dehydrated by immersion in 100% ethanol for 15 min twice at 4˚C. Before freezing the gels, the ethanol was replaced with t-butyl alcohol by incubating the gels in t-butyl alcohol twice for 20 min at room temperature. Finally, the gels were soaked in t-butyl alcohol, placed at 4˚C overnight, and 4 freeze-dried using a vacuum freeze dryer (IlShin Valves). Samples were affixed to the stage with carbon tape, and vapor deposition with platinum (E-1030, Hitachi) was performed prior to observation.

Quantitative analysis of ligand distribution
To investigate the distribution of vitronectin on the culture substrate, gels (SOFT and HARD) and coverslips were coated with Vitronectin XF as described in the Methods. The coated culture substrates were subsequently fixed with 4% PFA. Vitronectin was detected by anti-vitronectin antibody and visualized by Alexa488 similar to immunocytochemistry. The surface images of the culture substrate were subsequently acquired using a confocal microscope (Nikon A1R-MP). To obtain a higher magnified view, a 60x oil objective lens with 5x digital zoom was used. Acquired images were analyzed by ImageJ. The background was initially subtracted, and the vitronectin-positive region was then extracted using the threshold and converted to the binary image. Finally, the extracted area was measured. Statistical analysis was performed using Prism version 4.0 (GraphPad Software). The antibody information is described in Supplementary Table   S5.

Immunocytochemistry and microscopy of cultured cells
The cells were fixed in medium containing 4% PFA at room temperature for 10 min. After washing with PBS 3 times for 5 min each, the cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min. The cells were then washed with PBS 3 times for 5 min each and blocked with 2% BSA in PBS containing 0.1% Triton X-100 at room temperature for 1 h. The cells were incubated with primary antibodies at 4˚C overnight and then with secondary antibodies at room temperature for 2 h.
Counterstaining was performed using DAPI (Sigma-Aldrich). The information of antibodies is described in Supplementary Table S5.  Supplementary Table S6 and are derived from previously published sequences. Relative 6 fold changes in mRNA expression were calculated using the 2 -∆∆Ct method: 2 -∆∆Ct in which Ct=Ct target -Ct normalizer , Ct=Ct-Ct reference . GAPDH, a housekeeping gene, was used to normalize gene expression. In the experiment shown in Fig. 6C to E, the value of Ct on Day 0 was used as a reference in each condition. In the experiments shown in Fig. 7B and C, the value of

Embryoid body (EB) formation
Human iPS cells were expanded on gels or plastic dishes coated with Vitronectin in StemFit medium. After 1 week, the hiPSC colonies were detached by treatment with Dispase I (0.5 Unit/ml) (BD Biosciences). Harvested cells were transferred to a petri dish in DMEM/F12/GlutaMAX-I (Invitrogen) that contained 20% knockout serum replacement (Invitrogen) (20% KSR EB medium), 0.1 mM of Non-Essential Amino Acids (Invitrogen), 0.1 mM of 2-mercaptoethanol (Invitrogen) and penicillin/streptomycin (Invitrogen). After 4 days of culture in the floating condition, the EBs 7 were transferred to a 0.1% gelatin-coated dish and cultured in the same medium for an additional 10 days. The medium was changed on Day 4, 7 and 12. EBs were collected for RNA extraction with DNase I treatment on Day 14. cDNA was synthesized as described above. To confirm the undifferentiated state, RNA was extracted from expanded hiPSCs on the harvested day. RT-PCR was performed to confirm the gene expression of three germ layer markers. The primer sequences for RT-PCR are listed in Supplementary Table S7.      Figure S3 A B