Effect of Fibrin on MSC Proliferation and Gene Expression of Phenotypic Markers
We first examined whether fibrin supports MSC proliferation by quantifying cell numbers at various time points. In agreement with published literature, MSCs cultured on fibrin-coated dishes increased in number with time (Suppl. Fig. 1
]. We next determined whether fibrin-coated substrates could modulate MSC phenotype across a range of cell lineages, including vascul ogenic, myogenic, neurogenic, chondrogenic, osteogenic, and adipogenic lineages. Based on qPCR analysis after 5 days of culture on fibrin-coated substrates, we showed that fibrin differentially regulated phenotypic markers of multiple lineages ( and ). Gene expression was markedly induced on the fibrin-coated substrate for smooth muscle markers. In particular, smooth muscle calponin (CNN1) demonstrated a 2-fold increase (0.67±0.20 fibrin vs 0.30±0.10 control), smooth muscle α-actin (ACTA2) increased by 1.5-fold (2.47±0.94 fibrin vs 1.62±0.52 control), and transgelin (SM22) showed a modest increase by 1.3-fold (1.45±0.28 fibrin vs 1.09±0.18 control) (). Endothelial marker vascular endothelial growth factor receptor 2 (VEGFR2/FLK1) was upregulated by 40-fold (1.17±0.53 fibrin vs 0.02± 0.01 control) and vascular endothelial cadherin (VECAD) increased by 3-fold (0.0032±0.0014 fibrin vs 0.00092± 0.0056 control) (). In stark contrast, cardiac markers troponin-I and GATA-4 were not detectable in any treatment groups (data not shown).
Mesenchymal stem cell (MSC) phenotype on fibrin-coated surfaces
Besides vascular lineages, we also surveyed phenotypic markers representing myogenic, chondrogenic, osteogenic, neurogenic, and adipogenic lineages (). Myogenic factor 5 (MYF5), an early muscular transcriptional factor, was significantly upregulated by 16-fold (1.39±0.53 fibrin vs 0.08±0.05 control) on fibrin-coated substrates (). Similarly, skeletal myosin heavy chain (MYH13), a mature skeletal muscle marker, was significantly induced by 34-fold (24.68±15.69 fibrin vs 0.72±0.87 control) (). Among neurogenic markers, the dopaminergic neuronal marker TH was upregulated by 14-fold (0.99±0.38 fibrin vs 0.05±0.07 control) (), and astrocyte phenotypic marker glial fibrillary acidic protein (GFAP) was upregulated by 29-fold (0.048±0.026 fibrin vs 0.0016±0.0012 control) (). In addition, chondrogenic extracellular matrix collagen IIα1 (COL2A1) was also significantly induced by the fibrin-coated substrate by 24-fold (1.48±0.51 fibrin vs 0.06±0.03 control) (). On the other hand, osteogenic lineage marker alkaline phosphatase (ALPL) and adipogenic marker peroxisome proliferator-activated receptor γ (PPARG) did not show significant differential gene expression on fibrin-coated substrates (). These results suggested that fibrin could modulate phenotypic expression of MSCs towards numerous lineages, including vascular (SM22, CNN1, FLK1, VECAD), neural (TH, GFAP), muscular (MYF5, MYH13), and chondrogenic (COL2A1) lineages after 5 days of incubation.
To confirm the differential gene expression results, we carried out immunoblotting assay after 5 days of incubation for a subset of markers. In agreement with the gene expression patterns, the relative expression of early lineage-specific markers such as FLK1, ACTA2, and MYF5 were significantly induced on the fibrin substrate, when normalized to total actin abundance (). Fibrin-coated substrates promoted an increased expression of FLK1 by over 400-fold, ACTA2 by 0.5-fold, and MYF5 by 8-fold (). On the other hand, we could not detect the expression of mature markers such as skeletal muscle myosin and GFAP (data not shown). These results suggest that fibrin could mediate phenotypic changes in MSCs at the gene and, to some degree, the protein levels.
Effect of Physical and Mechanical Properties of Fibrin on MSC Gene Expression
To understand the mechanism of fibrin-induced phenotypic expression, we questioned whether the differential gene expression was related to the physical or mechanical properties of fibrin, namely its pore size and elastic modulus. To address this, we modulated the pore size and elastic modulus by changing the composition of the fibrinogen and thrombin components. We prepared several formulations of fibrin by diluting fibrinogen 10-fold (F10) and/or thrombin by 10-fold (T10) or 50-fold (T50) and found that the pore size of fibrin could be significantly altered. As shown by the H&E-stained cross sections (), the equivalent pore diameter was significantly higher in the T10F10 formulation (112.1±15.41 μm), when compared to that of the standard formulation (83.53±6.81 μm), T10 (86.89±14.70 μm), and T50 (81.29±16.48 μm) formulations (P<0.05). In addition to significantly modulating the fibrin pore size, diluting the thrombin and fibrinogen components also altered its mechanical properties. As shown in , the Young’s modulus for the standard FG formulation was 26.96 ± 8.14 kPa, which was significantly higher than the T10 (9.45±3.10 kPa), T50 (5.29±1.08 kPa), and T10F10 (5.74±1.64 kPa) formulations (P<0.05).
Effects of physical and mechanical properties of fibrin on gene expression changes in MSCs
After characterizing the physical and mechanical properties of different fibrin formulations, we next studied gene expression of MSCs on the 4 formulations of fibrin. To minimize the possible changes in physical and mechanical properties during in vitro studies due to protease digestion or other factors, we chose an early time point of 2 days for the assessment of gene expression changes. Within the range of mechanical properties and pore sizes examined, we did not detect statistically significant differences in gene expression of phenotypic markers after 2 days of culture (). This data suggests that these biomechanical and biophysical factors did not play a significant role in modulating MSC behavior after 2 days of culture.
Comparison of MSC Gene Expression on Various ECMs
In addition to assessing the effect of the physical and mechanical properties of fibrin on MSC gene expression, we sought to determine whether the modulation of gene expression changes was specific to the chemical composition of fibrin. To address this, we compared MSC gene expression on fibrin or on other ECMs, namely laminin, fibronectin, and collagen I. To further elucidate the structural effect of substrates on gene expression, collagen I was prepared as either a monomeric coating or polymerized thin gel. A 2-day time point was selected to minimize the possibility of biochemical and biophysical changes in ECM caused by cells during in vitro studies. The MSCs on cultured on laminin, fibronectin, and collagen I matrices appeared to have an elongated morphology as on the non-coated control substrate (). On the other hand, the cells on fibrin-coated substrates appeared to form tube-like structures that were consistent with early phenotypic changes towards vascular cell phenotype.
MSC morphology on substrates coated with laminin, fibronectin, fibrin, collagen I (coll I) monomer, or coll I polymer matrices after 2 days of culture
Comparative gene expression analysis of MSCs on the various ECM substrates revealed a number of differentially expressed phenotypic markers. The mature smooth muscle marker, CNN1 was shown to be significantly upregulated only on the fibronectin substrate by 1.5-fold (1.7 ±0.51 fibronectin vs 1.20±0.18 control) (). Another smooth muscle marker, ACTA2, was induced on both fibronectin- and fibrin-coated substrates by 1.5-fold and 1.1-fold, when compared to the control sample, whereas collagen I polymer induced a 1-fold downregulation in gene expression respectively (1.78±0.51 fibronectin vs 1.27±0.11 fibrin vs 0.17±0.02 collagen polymer vs 1.20±0.18 control) (). In addition to smooth muscle phenotypic markers, endothelial precursor marker FLK1 was upregulated on both fibrin and collagen I polymer substrates by 55-fold and 395-fold, respectively (0.07±0.06 fibrin vs 0.53±0.28 collagen polymer vs 0.0013±0.0019 control) (), although the mature endothelial marker, VECAD, was differentially expressed only on collagen I polymer (0.037±0.007 collagen polymer vs 0.004±0.001 control) substrate (). The gene expression of CNN1 and VECAD was not significantly different on fibrin and control surfaces at day 2, suggesting that these two gene were induced at later time points (e.g., day 5; ).
Effect of ECM on MSC differentiation markers after 2 days
In regards to myogenic phenotypic markers, MYF5 was significantly induced on the collagen I polymer substrate by 40-fold, when compared to the control (0.88±0.62 collagen polymer vs 0.02±0.03), and MYH13 was induced on both the fibrin and collagen I polymer (70-fold and 120-fold, respectively) substrates (0.53±0.43 fibrin vs 0.94±0.23 collagen polymer vs 0.01±0.01 control) (). In regards to neural markers, fibrin induced a 67-fold upregulation of TH and 173-fold increase in GFAP, whereas collagen polymer I induced these same genes by 173-fold and 526-fold, respectively (0.28±0.18 fibrin vs 0.53±0.21 collagen polymer vs 0.0041±0.0029 control for TH; 0.20±0.15 fibrin vs 0.59±0.28 collagen polymer vs 0.0011±0.0013 control for GFAP) (). In addition, chondrogenic marker COL2A1 was upregulated on both fibrin and collagen I polymer by 98-fold and 226-fold, respectively (0.33±0.36 fibrin vs 0.77±0.30 collagen polymer vs 0.0034± 0.0033 control) (), and osteogenic marker BMP2 was 40-fold higher on the collagen I polymer substrate when compared to the control substrate (5.72±2.14 collagen polymer vs 0.14±0.01 control) ().
Interestingly, the effects on collagen I thin gels were not reproduced on collagen I monomeric coating, suggesting that the structural aspect of the substrate may also be an important factor in matrix regulation of cell phenotype. Together, these findings reveal that some phenotypic markers could be differentially expressed on both fibrin-coated substrates as well as others such as fibronectin and collagen I polymer, suggesting that more than one ECM composition may be capable of modulating MSC phenotype.
Combined Effects of Fibrin with Growth Factors on MSC Expression
In addition to assessing the effect of fibrin on MSC phenotype, the combined effect of fibrin and growth factors was examined by culturing MSCs on fibrin-coated substrates for 12 days in the presence of growth factors known to stimulate muscular, vascular, or neural lineages, namely VEGF, TGFβ, IGF-1, PDGF, and NGF. Morphologically, the cells in the presence of TGFβ on the non-coated substrate appeared to have a flattened cellular shape and pronounced formation of stress fibers, whereas no obvious differences were observed in the remaining growth factor-treated groups (). In the presence of fibrin, the cells had an elongated morphology for all growth factor-treated groups. After 12 days of incubation, fibrin was macroscopically visible on the dish.
MSC morphology when cultured on fibrin and/or growth factors for 12 days
At day 12, in the absence of growth factors, only GFAP gene was still induced by fibrin matrix, suggesting that the effects of fibrin are time-dependent and the further differentiation of MSCs needs other biochemical factors (). In some cases, the addition of growth factors to fibrin could further modulate differential gene expression changes after 12 days. Whereas fibrin appeared to have no significant effect in modulating TGFβ-induced upregulation of CNN1 () or TGFβ-mediated inhibition of VECAD expression (), the early myogenic transcriptional factor, MYF5, was significantly induced in the fibrin+NGF (0.31±0.33) group when compared to fibrin (0.05±0.07) or NGF (0.0013±0.0010) alone (). Similarly, MYH13 resulted with a significantly higher mRNA abundance in the fibrin+NGF (0.06±0.05) group when compared to fibrin (0.05±0.12) or to NGF (0.0015±0.0026) alone (). In the area of neural phenotype, the expression of GFAP was significantly upregulated in the presence of fibrin by VEGF, PDGF, IGF-1, or NGF, when compared to growth factor treatment alone (). Since VEGF, PDGF, and IGF-1 are not commonly associated with neural phenotype, this data suggests that fibrin may be involved in growth factor-stimulated pathways that enhance neural lineage phenotype. Finally, BMP2 gene expression was not modulated by the presence of fibrin, but could be significantly induced in the presence of PDGF alone, when compared to the control group (). These results suggest that the combined delivery of fibrin with growth factors play diverse roles in upregulating, downregulation, or having no effect on MSC gene expression.
Combined effects of fibrin and growth factors on MSC phenotypic markers after 12 days
To determine whether fibrin and/or growth factors could induce the post-transcriptional expression of mature lineage-specific markers, we carried out immunofluorescence staining on samples cultured for 12 days using antibodies against VECAD, smooth muscle calponin, and skeletal muscle myosin. Contrary to transcriptional analysis, no treatment group could produce detectable immunofluorescence staining of the mature phenotypic markers, suggesting that fibrin could modulate cell phenotype on the transcriptional level but could not induce MSCs to become maturely differentiated cells (data not shown).