Soft Matrix Combined With BMPR Inhibition Regulates Neurogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells

Abstract

Stem cells constantly encounter as well as respond to a variety of signals in their microenvironment. Although the role of biochemical factors has always been emphasized, the significance of biophysical signals has not been studied until recently. Additionally, biophysical elements, like extracellular matrix (ECM) stiffness, can regulate functions of stem cells. In this study, we demonstrated that soft matrix with 1-10 kPa can induce neural differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs). Importantly, we used a combination of soft matrix and bone morphogenetic protein receptor (BMPR) inhibition to promote neurogenic differentiation of hUC-MSCs. Furthermore, BMPR/SMADs occurs in crosstalk with the integrinβ1 downstream signaling pathway. In addition, BMPR inhibition plays a positive role in maintaining the undifferentiated state of hUC-MSCs on the hydrogel substrate. The results provide further evidence for the molecular mechanisms via which stem cells convert mechanical inputs into fateful decisions.

Keywords: bone morphogenetic protein receptor; hydrogel; mesenchymal stem cells; neural differentiation; stiffness.

FIGURE 1

Matrix stiffness regulates cell morphology and neural differentiation ofhuman umbilicalcordmesenchymal stem cells (hUC-MSCs). (A) A summary of substrate stiffness construction. Matrixes of different stiffness were prepared by mixing the same concentration of acrylamide (AAm) with different concentrations of bis-acrylamide (Bis-AAm; 0.05, 0.3, and 0.7%). Sulfo-SANPAH (hexanoate) was cross-linked via ultraviolet irradiation and then coated with fibronectin for cell adhesion. (B) Matrix stiffness affects the morphological characteristics of hUC-MSCs. Morphological characteristics of hUC-MSCs were observed under a scanning electron microscope at days 1 and 7. TCP group: cells on Tissue Culture Plate. Scale bar = 20 μm. n = 3 independent wells. (C) The cell aspect ratio was calculated using NIH Image J. The aspect ratio of the cell is the ratio of the major to minor axes (n = 3, *p < 0.05,and **p < 0.01). (D) The cell area was calculated using NIH Image J (n = 3, *p < 0.05,and **p < 0.01). (E) qRT-PCR analyses were performed to detect the expression of neuronal-specific markers Nestin and βIII-tubulin in hUC-MSCs at day 1 and day 7 (Results are presented as mean ± SEM, n = 5 independent experiments, *p < 0.05, and **p < 0.01). (F) Western blotting analysis of Nestin and βIII-tubulin at day 7 (Results are presented as mean ± SEM, n = 3, *p < 0.05, and **p < 0.01). (G) qRT-PCR analysis of stem cells self-renewal markers SOX2 and OCT4 at day 1 (Results are presented as mean ± SEM, n = 5 independent experiments, *p < 0.05, **p < 0.01, and ***p < 0.001).

FIGURE 2

BMPR is involved in stiffness-mediated neural differentiation of hUC-MSCs. (A) qRT-PCR detection of BMPR subtypes expression at day 1 and day 7 (Results are presented as mean ± SEM, n = 5 independent experiments, *p < 0.05, and **p < 0.01). (B) Expression of BMPR subtypes detected using western blotting, and statistical analysis diagram (Results are presented as mean ± SEM, n = 3, *p < 0.05, and **p < 0.01). (C) The best concentration of BMPR inhibitor was determined using western blotting, the best concentration of inhibitor was 2 μM (n = 3 independent experiments). (D) The cells were collected at day 7 for detection of BMPR expression after stiffness groups adding inhibitor using RT-qPCR (Results are presented as mean ± SEM, n = 5 independent experiments, *p < 0.05, and **p < 0.01). (E) Expression of p-SMAD was detected using western blotting before and after BMPR inhibition, and statistical analysis diagram (Results are presented as mean ± SEM, n = 3, *p < 0.05, and **p < 0.01). (F) qRT-PCR analysis of Nestin and βIII-tubulin expression after BMPR inhibition for 24 h. Statistical analysis was performed using one-way ANOVA. Statistical significance was defined as p < 0.05. Results are presented as mean ± SEM (n = 5 independent experiments, p < 0.05). (G) qRT-PCR analysis of SOX2 and OCT4 expression after BMPR inhibition for 24 h. Statistical analysis was performed using one-way ANOVA. Statistical significance was defined as p < 0.05. Results are presented as mean ± SEM (n = 5 independent experiments, p < 0.05, and ***p < 0.001).

FIGURE 3

The downstream molecules of integrinβ1 were detected after BMPR inhibition. (A) qRT-PCR analysis of integrinβ1 after BMPR inhibition for 24 h. Results are presented as mean ± SEM (n = 5 independent experiments, *p < 0.05, and **p < 0.01). “+”represents the group contained inhibitor. (B) Staining for integrinβ1 in hUC-MSCs, which were treated on 1–10 kPa before and after BMPR inhibition for 24 h, and then observed under a confocal microscope (Olympus FV 1200; n = 3 independent experiments, scale bars, 10 μm). (C) Western blotting detection and statistical analysis diagram showing proteins associated with integrinβ1 including AKT, GSK-3β, and FAK on TCP, 1–10 kPa, and 62–68 kPa before adding inhibitor (Results are presented as mean ± SEM, n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001). (D) Western blotting detection of expression of AKT, GSK-3β, and FAK on TCP and 1–10 kPa after adding inhibitor (Results are presented as mean ± SEM, n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001). “+” represents the group contained inhibitor.