Interpreted gene expression of human dermal fibroblasts after adipo-, chondro- and osteogenic phenotype shifts

Autologous cell-based therapies promise important developments for reconstructive surgery. In vitro expansion as well as differentiation strategies could provide a substantial benefit to cellular therapies. Human dermal fibroblasts, considered ubiquitous connective tissue cells, can be coaxed towards different cellular fates, are readily available and may altogether be a suitable cell source for tissue engineering strategies. Global gene expression analysis was performed to investigate the changes of the fibroblast phenotype after four-week inductions toward adipocytic, osteoblastic and chondrocytic lineages. Differential gene regulation, interpreted through Gene Set Enrichment Analysis, highlight important similarities and differences of induced fibroblasts compared to control cultures of human fibroblasts, adipocytes, osteoblasts and articular chondrocytes. Fibroblasts show an inherent degree of phenotype plasticity that can be controlled to obtain cells supportive of multiple tissue types.     

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture

Summary In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.     

Demineralized bone promotes chondrocyte or osteoblast differentiation of human marrow stromal cells cultured in collagen sponges

Demineralized bone implants have been used for many types of craniomaxillofacial, orthopedic, periodontal, and hand reconstruction procedures. In previous studies, we showed that demineralized bone powder (DBP) induces chondrogenesis of human dermal fibroblasts in a DBP/collagen sponge system that optimized interactions between particles of DBP and target cells in cell culture. In this study, we test the hypothesis that DBP promotes chondrogenesis or osteogenesis of human marrow stromal cells (hMSCs) in 3-D collagen sponge culture, depending upon the culture conditions. We first confirmed that hMSCs have chondrogenic potential when treated with TGF-, either in 2-D monolayer cultures or in 3-D porous collagen sponges. Second, we found that DBP markedly enhanced chondrogenesis in hMSCs in 3-D sponges, as assessed by metachromasia and expression of chondrocyte-specific genes AGGRECAN, COL II, and COL X. Human dermal fibroblasts (hDFs) were used to define mechanisms of chondroinduction because unlike hMSCs they have no inherent chondrogenic potential. In situ hybridization revealed that hDFs vicinal to DBPs express chondrocyte-specific genes AGGRECAN or COL II. Macroarray analysis showed that DBP activates TGF-/BMP signaling pathway genes in hDFs. Finally, DBP induced hMSCs to express the osteoblast phenotype when cultured with osteogenic supplements. These studies show how culture conditions can influence the differentiation pathway that human marrow stromal cells follow when stimulated by DBP. These results support the potential to engineer cartilage or bone in vitro by using human bone marrow stromal cells and DBP/collagen scaffolds.     

Effect of strain on human dermal fibroblasts in a three-dimensional collagen sponge

Our aim was to design a simple compression system and investigate the influence of mechanical stress on skin-like structures. Many mechanical compression studies have employed intricate culture systems, so the relationship between extracellular matrix material and the response of skin cells to mechanical stress remains unknown. Our approach uses only glass vials, 6-well plates and standard laboratory equipment. We examined the influence of mechanical stress on human skin fibroblasts embedded within a collagen sponge. The results show that mechanical compression increases MMP-1 and MMP-2 release by the cells into the the cell culture. Our results suggest that pressure on the skin may affect extracellular matrix degradation through some as yet unidentified pathways and that IL-6 mRNA expression may be involved in this effect. Using our approach, the effects of static mechanical stress on protein expression by cells in the culture medium and in sponges can be easily examined, and therefore this system will be useful for further analyses of skin responses to mechanical stress.     

Up-regulated type I collagen expression by the inhibition of Rac1 signaling pathway in human dermal fibroblasts

Tissue remodeling is known to play important roles in wound healing. Although Rac1 is reported to be one of the key signaling molecules in cutaneous wound healing process, the exact mechanisms of Rac1-mediated tissue remodeling is still unknown. This study investigated the role of Rac1 in the regulation of extracellular matrix in cultured human dermal fibroblasts obtained by skin biopsy from three healthy donors. Protein levels of type I collagen in cultured human fibroblasts were increased by the treatment with Rac1 inhibitor NSC23766 in a dose-dependent manner. However, the mRNA levels of α2(I) collagen was not altered by the inhibitor. On the other hand, by the addition of inhibitor, half-lives of type I collagen protein were increased and MMP1 levels were reduced. These data suggest that blockade of Rac1 signaling results in accumulation of type I collagen due to decreased collagenase activity. This study also suggests that controlling Rac1 signaling is a new therapeutic approach to chronic/untreatable ulcer.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Persistent effect of TGF-beta 1 on extracellular matrix gene expression in human dermal fibroblasts

TGF-beta, a potent inducer of the extracellular matrix, is also known to stimulate its own synthesis. In this report we have analyzed long term effects of TGF-beta 1 on its own expression and on the expression of extracellular matrix genes. We demonstrated that 24 hours of incubation of human dermal fibroblasts with TGF-beta 1 (1 ng/ml) under serum free conditions resulted in an elevated expression of TGF-beta 1, collagen alpha 2(I) and fibronectin mRNAs that persisted at least 96 hours after removal of TGF-beta 1. These data suggest the possibility of persistent in vivo activation of target cells following exposure to TGF-beta 1.     

Increased levels of a particular phosphatidylcholine species in senescent human dermal fibroblasts in vitro

Plasma membranes are essential components of living cells, and phospholipids are major components of cellular membranes. Here, we used liquid chromatography/mass spectrometry to investigate changes in the membrane phospholipid content that occur in association with aging. Our results indicate that the levels of a particular species of phosphatidylcholine comprised of stearic acid and arachidonic acid increased with age. To determine the reason for the increased levels of this particular phosphatidylcholine, we examined the effect of highly unsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, on cellular aging. Applied arachidonic acid was incorporated into phosphatidylcholine molecules, but neither arachidonic acid nor other related unsaturated fatty acids had any effect. We conclude that increased levels of this distinctive phosphatidylcholine are a result of in vitro senescence.     

A novel method to prepare size-regulated spheroids composed of human dermal fibroblasts

A simple method to prepare size-regulated spheroids has been successfully developed by combining a temperature responsive polymer, poly-N-isopropyl-acrylamide (PNIPAAm), conjugated with collagen and ultraviolet (UV) irradiation with photomasks. The coating layer composed of PNIPAAm conjugated with collagen functions as a cell substratum at 37 degrees C, then when lowering the temperature of culture medium the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature LCST; about 30 degrees C, but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. On the other hand, UV effectively immobilized collagen in the coating layer because UV generates crosslinkages in collagen molecules. Crosslinkages were quantitatively introduced by controlling the energy of UV-irradiation thus the ability of human dermal fibroblasts to attach to and detach from the surface was tightly controlled. When the collagen content in the coating layer was 9 mug/cm(2) (collagen ratio, 4.5%), UV-irradiation energy of 2000 J/m(2) was suitable to obtain 100% of the attachability and detachability. However, the cells did not attach to the nonirradiated surface at this collagen content because insufficient collagen was immobilized. Using photomakes to apply UV-irradiation, it was possible to obtain cell-adhesive areas(irradiated areas) and nonadhesive areas (nonirradiated areas) on the same surface. Consequently, spheroids of any size and in any number from one dish were prepared. The viability of cells in spheroids 350 mum in diameter was maintained at a high level for 28 days; however, viability of spheroids 800 mum in diameter rapidly decreased for 2 days. The size was very important to maintain the viability. This novel method is useful to develop size-regulated spheroids for different applications; for example, in toxicology tests. (c) 1994 John Wiley & Sons, Inc.     

Influence of serum on adult and fetal dermal fibroblast migration adhesion, and collagen expression

Summary The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration. The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although there was a greater faction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining. Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences in adult and fetal wound healing.     

Effect of growth arrest on the doubling potential of human fibroblasts in vitro: A possible influence of the donor

Summary Population doublings versus time in culture were compared in human postnatal skin fibroblasts from normal donors, a cancer patient, and from donors suffering from Cockayne syndrome, Ataxia telangiectasia, and Fanconi’s anemia (FA). Confluent cultures were maintained in a nonproliferating state for 14 to 27 d in 0.5% serum medium. The results show that the ability of cells to resume division after a resting stage can be influenced by pathologic conditions. In arrested FA cell populations an increase of the population doublings and of the calendar time were observed. It is possible that in some cell populations the resting stage favors the expression of growth potentialities related to an instability of the cells. This work was supported by contract CRL 802030 from the I.N.S.E.R.M. and by Euratom.     

A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts

BACKGROUND INFORMATION: Different in vitro models, based on co-culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions. In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co-culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin-1 and ED-A-fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored. RESULTS: The comparison of the HDMEC-NHDF co-culture with the respective mono-cultures revealed that HDMECs showed a lower proliferation activity when co-cultured, but their number was stable throughout a period of 48 h. NHDFs in co-culture were slightly slower at proliferating than in the mono-culture. The MF population was stable for 48 h in the co-culture, as well as in NHDF mono-culture. Co-cultures and HDMEC mono-cultures were characterized by a slower migration rate than NHDF mono-cultures. Hypoxia decreased both cell proliferation and migration in the mono-cultures, as well as in the co-cultures, indicating the general suitability of the assay. Exclusively, in co-cultures well-defined cell clusters comprising HDMECs and MFs formed at the edges of the in vitro wounds. CONCLUSIONS: On the basis of these results, the CCSWMA developed using co-cultures, including HDMECs, NHDFs and MFs, proved to be an effective tool to directly visualize cellular interaction. Therefore, it will serve in the future to evaluate the influence of wound-healing-related factors in vitro, as shown for hypoxia in the present study.     

Interleukin-1 alpha and beta induce interleukin-1 beta gene expression in human dermal fibroblasts

The ability of the two forms of interleukin-1, IL-1 alpha and IL-1 beta, to induce IL-1 beta gene expression in human skin fibroblasts was studied in vitro, using Northern blot hybridization. Both recombinant IL-1 alpha and IL-1 beta caused a dramatic increase in IL-1 beta mRNA levels, IL-1 alpha being more efficient than IL-1 beta. Blockage of the prostaglandin synthesis by indomethacin reduced the basal level of IL-1 beta mRNA in control cultures and decreased also the stimulatory effect exerted by both IL-1s on IL-1 beta gene expression. These data suggest that IL-1 and prostaglandin (mainly PGE2) may act synergistically to stimulate IL-1 gene expression in dermal fibroblasts, contributing as a local amplifier system to the alterations of connective tissue in inflammatory processes.     

IGF-1 modulation of rat cardiac fibroblast behavior and gene expression is age-dependent

The collagenous extracellular matrix (ECM) forms a stress-tolerant network that is essential for proper function of the vertebrate heart. Profound changes have been detected in the interstitial ECM concurrent with developmental and disease processes of the heart. These alterations in either the organization or accumulation of ECM components markedly affect myocardial function. Studies have shown that a number of biochemical factors, including angiotensin II, transforming growth factor-β, and insulin-like growth factors, modulate collagen expression by heart fibroblasts, however, few studies have examined the differential effects of these factors on fibroblasts from animals of different physiological backgrounds. The present studies were carried out to determine whether cardiac fibroblasts isolated from different aged animals (fetal, neonatal, and adult) have diverse responses to insulin-like growth factor-1 (IGF-1). Fibroblasts isolated from fetal, neonatal, and adult rat hearts were treated with IGF-1, and several downstream responses were measured, including collagen gel contraction, adhesion to ECM, and expression of interstitial collagen and integrins. IGF-1 affected these parameters to different degrees, depending on the age of the animal from which the fibroblasts were isolated. These experiments indicate that IGF-1 is a potent modulator of fibroblast behavior in general; however, significant differences are apparent in the responsiveness of cells to this growth factor depending on the age of the animal of origin. Future experiments will be directed at determining how the in vivo chemical and biomechanical environment affects the response of heart fibroblasts to growth factors such as IGF-1.     

Effects of pulsed electrical stimulation on growth factor gene expression and proliferation in human dermal fibroblasts

Molecular and Cellular Biochemistry - Human dermal fibroblast proliferation plays an important role in skin wound healing, and electrical stimulation (ES) promotes skin wound healing. Although the...     

Persistent biologic actions of insulin on cultured fetal human fibroblasts

Summary Monolayer cultures of fetal human fibroblasts, preincubated in serum-free culture medium overnight (about 18 hr), were incubated with insulin (0.1 to 100 mU per ml), then washed and incubated in an insulin-free medium. The effect of insulin on glucose utilization, uridine incorporation into RNA and leucine incorporation into protein was maintained after removal of insulin and washing. For both glucose utilization and uridine incorporation into RNA, this effect was demonstrated at physiologic levels of insulin (0.1 mU per ml). When anti-insulin serum was added to the cultures after the cell preincubated with insulin were washed, this effect was greatly attenuated. This lasting effect of insulin was probably not due to nonspecifically bound insulin becoming available to the cells. Binding of¹²⁵I-monoiodoinsulin was examined in monolayer cultures of fetal human fibroblasts. When unlabeled insulin was present at about 1 mU per ml concentration, 50% displacement of monoiodoinsulin occured. When fibroblasts were incubated with monoiodoinsulin and then removed from the radioactive medium, initial dissociation of the bound hormone occurred rapidly but then reached a plateau. This prolonged insulin effect appears to result from persistent binding of insulin to its receptor. Supported in part by PHS Grants AM-02456, AM-05020 and AM-15312, by the Kroc Foundation and by the Diabetes Center (AM-17047). Supported in part by Research Career Development Award AM-47142 from NIAMDD.     

Apoptosis in v-myc-transfected MSU-1.1 fibroblasts is induced by cell-matrix contact and differs from that of normal dermal fibroblasts

In order to characterize a fibroblast cell line representing normal human skin fibroblasts in three-dimensional cultures, we compared the fibroblast line MSU-1.1, derived from human foreskin and immortalized by v-myc, to primary human dermal fibroblasts (NDF). Our results demonstrate that in contrast to NDF, all MSU-1.1 fibroblasts die within 3-4 d when cultured within three-dimensional contractile collagen matrices. Also, in contrast to NDF. MSU-1.1 cells die markedly in anchored collagen gels as well. Death is due to apoptosis and is attenuated by addition of antibodies against collagen-recognizing receptors alpha1beta1 and alpha2beta1. Apoptosis of NDF in collagen lattices was repressed by an inhibitor of caspase-1, which was ineffective on apoptosis of MSU-1.1. Further, apoptosis by MSU-1.l fibroblasts was also observed in anchored, i.e., restrained collagen lattices, an environment that supports proliferation of NDF.