IL-4 induces differentiation of human embryonic stem cells into fibrogenic fibroblast-like cells

Abstract

Background: Fibroblast heterogeneity is recognized, and fibroblasts from diseased tissues, including those of asthmatic subjects, have functional phenotypes that differ from normal tissue. However, progenitor-progeny relationships and the factors that control fibroblast differentiation are poorly defined.

Objective: We sought to determine whether IL-4 could alter the functional phenotype of fibroblasts during their differentiation from stem/progenitor cells.

Methods: Using a 3-dimensional collagen gel system, we obtained embryoid bodies derived from human embryonic stem cells and recovered spindle-shaped cells consistent with fibroblasts that had differentiated in the presence or absence of IL-4.

Results: IL-4-induced fibroblast-like cells were more active in contraction of collagen gels, migration, and production of fibronectin than control (without IL-4) cells. IL-4-induced cells demonstrated less expression of miR-155, which modulated contraction, migration, and fibronectin production. These differences persisted in culture without further addition of IL-4, suggesting the differentiated phenotype might be a permanent alteration.

Conclusion: The current study demonstrates that IL-4 induces differentiation of stem/precursor cells into fibroblast-like cells that demonstrate a more fibrogenic phenotype, which is due to reduced expression of miR-155. These findings provide a novel mechanism for the persistent abnormalities in IL-4-related diseases and a novel target to regulate tissue remodeling by fibroblasts.

Figure 1. Representative immunocytochemistry of differentiated fibroblast-like cells

(A) Vimentin in control cells. (B) Vimentin in IL-4 induced cells. (C) Cytokeratin in control cells. (D) Cytokeratin in IL-4 induced cells. (E) Desmin in control cells. (F) Desmin in IL-4 induced cells. (G) α-SMA in control cells. (H) α-SMA in IL-4 induced cells.

Figure 1. Representative immunocytochemistry of differentiated fibroblast-like cells

(A) Vimentin in control cells. (B) Vimentin in IL-4 induced cells. (C) Cytokeratin in control cells. (D) Cytokeratin in IL-4 induced cells. (E) Desmin in control cells. (F) Desmin in IL-4 induced cells. (G) α-SMA in control cells. (H) α-SMA in IL-4 induced cells.

Figure 2

α-SMA expression in undifferentiated hESCs, EBs and differentiated fibroblast-like cells. (A) Representative western blots for α-SMA. β-actin expression is shown as a loading control. (B) Densitometric analysis of α-SMA expression expressed relative amount to β-actin. Three different batches of control and IL-4 induced cells (indicated by #1, #2 and #3 in Panel A), each prepared from hECSs on separate occasions, were each evaluated three times. Values are mean ± SD. * p < 0.05, ** p < 0.01.

Figure 2

α-SMA expression in undifferentiated hESCs, EBs and differentiated fibroblast-like cells. (A) Representative western blots for α-SMA. β-actin expression is shown as a loading control. (B) Densitometric analysis of α-SMA expression expressed relative amount to β-actin. Three different batches of control and IL-4 induced cells (indicated by #1, #2 and #3 in Panel A), each prepared from hECSs on separate occasions, were each evaluated three times. Values are mean ± SD. * p < 0.05, ** p < 0.01.

Figure 3. Physiological differences between control and IL-4 induced fibroblast-like cells derived from hESCs

(A) Collagen gel contraction. The size of gels was measured on day 3 and shown as percentage of initial area. (B) Chemotaxis towards fibronectin. Chemotaxis was assessed by counting the number of migrated cells in five high-power fields (HPF). (C) Migration of fibroblast-like cells in the wound closure assay. The cell migration is expressed as a percentage of the cell occupied area in the initial wound area. (D) Cell proliferation. Cells were plated at a density of 2 × 10⁴ cells/well in and cultured. Cell numbers were determined on days 3 and 5 using an electronic cell counter. For all experiments, three different batches of control and IL-4 induced cells were evaluated on three separate occasions, each performed in triplicate. Values are mean ± SD. * p < 0.05, ** p < 0.01.

Figure 3. Physiological differences between control and IL-4 induced fibroblast-like cells derived from hESCs

(A) Collagen gel contraction. The size of gels was measured on day 3 and shown as percentage of initial area. (B) Chemotaxis towards fibronectin. Chemotaxis was assessed by counting the number of migrated cells in five high-power fields (HPF). (C) Migration of fibroblast-like cells in the wound closure assay. The cell migration is expressed as a percentage of the cell occupied area in the initial wound area. (D) Cell proliferation. Cells were plated at a density of 2 × 10⁴ cells/well in and cultured. Cell numbers were determined on days 3 and 5 using an electronic cell counter. For all experiments, three different batches of control and IL-4 induced cells were evaluated on three separate occasions, each performed in triplicate. Values are mean ± SD. * p < 0.05, ** p < 0.01.

Figure 3. Physiological differences between control and IL-4 induced fibroblast-like cells derived from hESCs

(A) Collagen gel contraction. The size of gels was measured on day 3 and shown as percentage of initial area. (B) Chemotaxis towards fibronectin. Chemotaxis was assessed by counting the number of migrated cells in five high-power fields (HPF). (C) Migration of fibroblast-like cells in the wound closure assay. The cell migration is expressed as a percentage of the cell occupied area in the initial wound area. (D) Cell proliferation. Cells were plated at a density of 2 × 10⁴ cells/well in and cultured. Cell numbers were determined on days 3 and 5 using an electronic cell counter. For all experiments, three different batches of control and IL-4 induced cells were evaluated on three separate occasions, each performed in triplicate. Values are mean ± SD. * p < 0.05, ** p < 0.01.

Figure 3. Physiological differences between control and IL-4 induced fibroblast-like cells derived from hESCs

(A) Collagen gel contraction. The size of gels was measured on day 3 and shown as percentage of initial area. (B) Chemotaxis towards fibronectin. Chemotaxis was assessed by counting the number of migrated cells in five high-power fields (HPF). (C) Migration of fibroblast-like cells in the wound closure assay. The cell migration is expressed as a percentage of the cell occupied area in the initial wound area. (D) Cell proliferation. Cells were plated at a density of 2 × 10⁴ cells/well in and cultured. Cell numbers were determined on days 3 and 5 using an electronic cell counter. For all experiments, three different batches of control and IL-4 induced cells were evaluated on three separate occasions, each performed in triplicate. Values are mean ± SD. * p < 0.05, ** p < 0.01.

Figure 4. Production of biomarkers from control and IL-4 induced fibroblast-like cells derived from hESCs

(A) Fibronectin production. (B) PGE₂ production. (C) TGF-β1 production. For the measurement of TGF-β1, only media without TGF-β1 were used. Three different batches of control and IL-4 induced cells were evaluated on three separate occasions, each performed in triplicate. Values are mean ± SD. * p < 0.01.

Figure 4. Production of biomarkers from control and IL-4 induced fibroblast-like cells derived from hESCs

(A) Fibronectin production. (B) PGE₂ production. (C) TGF-β1 production. For the measurement of TGF-β1, only media without TGF-β1 were used. Three different batches of control and IL-4 induced cells were evaluated on three separate occasions, each performed in triplicate. Values are mean ± SD. * p < 0.01.

Figure 4. Production of biomarkers from control and IL-4 induced fibroblast-like cells derived from hESCs

(A) Fibronectin production. (B) PGE₂ production. (C) TGF-β1 production. For the measurement of TGF-β1, only media without TGF-β1 were used. Three different batches of control and IL-4 induced cells were evaluated on three separate occasions, each performed in triplicate. Values are mean ± SD. * p < 0.01.

Figure 5. MiR-155 expression in undifferentiated hESCs, EBs and differentiated fibroblast-like cells

Cell layers of differentiated cells were extracted after 48 h treatment with or without TGF-β1 (100 pM). MiR-155 expression by real time PCR is normalized to the amount of rRNA and expressed as 2−ΔΔC_T values. * p < 0.01, compared with other groups.

Figure 6. MiR-155 regulates the differentiated fibroblast-like cell phenotype

(A) Collagen gel contraction on day 3. (B) Chemotaxis towards fibronectin. (C) Cell migration by wound closure assay. (D) Fibronectin production. Three different batches of control and IL-4 induced cells were evaluated on three separate occasions in all experiments. Values are mean ± SD. * p < 0.05, ** p < 0.01.

Figure 6. MiR-155 regulates the differentiated fibroblast-like cell phenotype

(A) Collagen gel contraction on day 3. (B) Chemotaxis towards fibronectin. (C) Cell migration by wound closure assay. (D) Fibronectin production. Three different batches of control and IL-4 induced cells were evaluated on three separate occasions in all experiments. Values are mean ± SD. * p < 0.05, ** p < 0.01.

Figure 6. MiR-155 regulates the differentiated fibroblast-like cell phenotype

(A) Collagen gel contraction on day 3. (B) Chemotaxis towards fibronectin. (C) Cell migration by wound closure assay. (D) Fibronectin production. Three different batches of control and IL-4 induced cells were evaluated on three separate occasions in all experiments. Values are mean ± SD. * p < 0.05, ** p < 0.01.

Figure 6. MiR-155 regulates the differentiated fibroblast-like cell phenotype

(A) Collagen gel contraction on day 3. (B) Chemotaxis towards fibronectin. (C) Cell migration by wound closure assay. (D) Fibronectin production. Three different batches of control and IL-4 induced cells were evaluated on three separate occasions in all experiments. Values are mean ± SD. * p < 0.05, ** p < 0.01.