Control of matrix stiffness promotes endodermal lineage specification by regulating SMAD2/3 via lncRNA LINC00458

Abstract

During endoderm formation, cell identity and tissue morphogenesis are tightly controlled by cell-intrinsic and cell-extrinsic factors such as biochemical and physical inputs. While the effects of biochemical factors are well studied, the physical cues that regulate cell division and differentiation are poorly understood. RNA sequencing analysis demonstrated increases of endoderm-specific gene expression in hPSCs cultured on soft substrate (Young's modulus, 3 ± 0.45 kPa) in comparison with hard substrate (Young's modulus, 165 ± 6.39 kPa). Further analyses revealed that multiple long noncoding RNAs (lncRNAs) were up-regulated on soft substrate; among them, LINC00458 was identified as a stiffness-dependent lncRNA specifically required for hPSC differentiation toward an early endodermal lineage. Gain- and loss-of-function experiments confirmed that LINC00458 is functionally required for hPSC endodermal lineage specification induced by soft substrates. Our study provides evidence that mechanical cues regulate the expression of LINC00458 and induce differentiation of hPSC into hepatic lineage progenitors.

Fig. 1. Soft substrate up-regulates endoderm-specific genes in hPSCs.

(A) Experimental setup for the examination of the effect of different substrate stiffnesses on hPSCs. (B) Morphology of hPSCs cultured on different substrate stiffnesses; arrows indicate typical petal and cobblestone-shaped cells. (C) hPSCs were stained for actin filaments on soft and hard substrates (top). Angular plots corresponding to soft and hard substrates are shown in the bottom compiled from 15 individual cells. (D) Hierarchical clustering analysis of mRNA expression in hPSCs cultured on soft and/or hard substrates and TCPS. (E) Volcano plot representative of the entire gene expression dataset (hPSCs cultured on soft versus hard substrates for 3 days). Gene groups are shown by different colors (light red, differentially expressed; blue, ectoderm; dark red, endoderm; black, housekeeping genes; green, mesoderm; yellow, mesoendoderm), and dots represent individual genes. Insignificant genes are in gray. DEG, differentially expressed genes. (F) Gene ontology (GO) terms highlighting the categories of up-regulated genes in the cells on soft substrates in comparison to those on hard substrates. FC, fold change.

Fig. 2. Soft substrate induces endodermal lineage commitment.

(A) A schematic drawing of the differentiation of hPSCs into liver and pancreatic cells. Meso, mesoderm; AFG, anterior foregut; PFG, posterior foregut; MHG, midgut/hindgut. Day 3 mRNA expression of hPSC genes involved in (B) APS/PPS, (C) DE, and (D) mesoderm differentiations. The results are presented as means ± SD of triplicates. One-way analysis of variance (ANOVA; n = 3 independent experiments). (E) Immunofluorescent staining and (F) quantification of SOX17 and NANOG in hPSCs grown on substrates with different stiffnesses. The results are presented as means ± SD of triplicates. One-way ANOVA (n = 3 independent experiments). Different letters indicate significant differences, and the same letters indicate no significant difference.

Fig. 3. LINC01356 and LINC00458 are associated with soft matrix–induced and cell type–specific expression signatures.

(A) Schematic plan to compare lncRNAs between soft and hard substrates. (B) Top 20 differentially regulated lncRNAs in hESCs cultured on soft or hard substrates. (C) LINC01356 and LINC00458 expressions in different human cells by qRT-PCR. hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; THP-1; monocytic leukemia cell; hADSC, human adipose–derived stem cell. The results are presented as means ± SD of triplicates. One-way ANOVA (n = 3 independent experiments). Different letters indicate significant differences, and the same letters indicate no significant difference. (D) Expression levels of LINC01356 and LINC00458 were determined by qRT-PCR after 3 days of culture on the indicated substrate stiffness. The results are presented as means ± SD of triplicates. One-way ANOVA (n = 3 independent experiments). Different letters indicate significant differences, and the same letters indicate no significant difference. (E) Induction of LINC01356 and LINC00458 expressions over 3 days. Cells were cultured on soft and hard substrates. RNA was isolated at the indicated time points and assessed by qRT-PCR. The results are presented as means ± SD of triplicates. ***P < 0.001, **P < 0.005, *P < 0.05, two-sided Student’s t test. (F) Total RNA was isolated from nuclear and cytoplasmic fractions and analyzed by qRT-PCR. MALAT1, metastasis-associated lung adenocarcinoma transcript 1.

Fig. 4. LINC00458 is necessary for soft substrate–induced endoderm commitment.

(A) hPSCs were cultured on TCPS and transfected with LNA GapmeRs targeting LINC00458 to determine knockdown efficiency. (B) hPSCs were cultured on soft substrate with LNA GapmeRs targeting LINC00458, and the expression of endodermal genes SOX17, FOXA2, EOMES, and GATA6 was analyzed by qRT-PCR. (C) Immunoblotting analysis of the FOXA2 and SOX17 expressions in hPSCs cultured on soft substrates with LNA GapmeRs targeting LINC00458. (D and E) Immunofluorescence detection of FOXA2 and SOX17 expressions in hPSCs cultured on hard substrates with LINC00458 transfection. The results are presented as means ± SD of triplicates. **P < 0.005, *P < 0.05, two-sided Student’s t test.

Fig. 5. LINC00458 interacts with SMAD3, a core component of the endoderm-specific transcription factor.

(A) Immunoblot of SMAD2/3 (SMAD2 (Ser^465/467)/SMAD3 (Ser^423/425) phosphorylation in hPSCs cultured on soft and/or hard substrates and TCPS. (B) qRT-PCR detection of LINC01356 and LINC00458 pulled down by SMAD2/3- or SMAD1-specific antibodies, as compared to that with immunoglobulin G (IgG), based on the RNA-binding protein immunoprecipitation (RIP) assay. SMAD2/3_S and SMAD2/3_H, soft and hard group samples, respectively, with anti-SMAD2/3 antibody; SMAD1_S and SMAD1_H, soft and hard group samples, respectively, with anti-SMAD1 antibody. (C) Immunoblot detection of the levels of SMAD2/3 in hPSCs on soft substrates transfected with SMAD2 or SMAD3 siRNAs. (D) qRT-PCR detection of LINC00458 and MALAT1 in RIP complex in hPSCs with SMAD2 or SMAD3 siRNAs. (E) A working model of soft substrate induces LINC00458 to interact with SMAD2/3, which promotes endoderm specification. The results are presented as means ± SD of triplicates. **P < 0.005, *P < 0.05, one-way ANOVA (n = 3 independent experiments). NS, not significant.