Keratocyte phenotype mediates proteoglycan structure: a role for fibroblasts in corneal fibrosis

In pathological corneas, accumulation of fibrotic extracellular matrix is characterized by proteoglycans with altered glycosaminoglycans that contribute to the reduced transparency of scarred tissue. During wound healing, keratocytes in the corneal stroma transdifferentiate into fibroblasts and myofibroblasts. In this study, molecular markers were developed to identify keratocyte, fibroblast, and myofibroblast phenotypes in primary cultures of corneal stromal cells and the structure of glycosaminoglycans secreted by these cells was characterized. Quiescent primary keratocytes expressed abundant protein and mRNA for keratocan and aldehyde dehydrogenase class 3 and secreted proteoglycans containing macromolecular keratan sulfate. Expression of these marker compounds was reduced in fibroblasts and also in transforming growth factor-beta-induced myofibroblasts, which expressed high levels of alpha-smooth muscle actin, biglycan, and the extra domain A (EDA or EIIIA) form of cellular fibronectin. Collagen types I and III mRNAs were elevated in both fibroblasts and in myofibroblasts. Expression of these molecular markers clearly distinguishes the phenotypic states of stromal cells in vitro. Glycosaminoglycans secreted by fibroblasts and myofibroblasts were qualitatively similar to and differed from those of keratocytes. Chondroitin/dermatan sulfate abundance, chain length, and sulfation were increased as keratocytes became fibroblasts and myofibroblasts. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated increased N-acetylgalactosamine sulfation at both 4- and 6-carbons. Hyaluronan, absent in keratocytes, was secreted by fibroblasts and myofibroblasts. Keratan sulfate biosynthesis, chain length, and sulfation were significantly reduced in both fibroblasts and myofibroblasts. The qualitatively similar expression of glycosaminoglycans shared by fibroblasts and myofibroblasts suggests a role for fibroblasts in deposition of non-transparent fibrotic tissue in pathological corneas.     

Differentiation of Human Embryonic Stem Cells into Cells with Corneal Keratocyte Phenotype

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.     

Enhanced cell accumulation and collagen processing by keratocytes cultured under agarose and in media containing IGF-I,TGF-β or PDGF

We previously showed an agarose overlay on keratocytes cultured in media containing pharmacological levels of insulin enhanced collagen processing and collagen fibril formation. In this study, we compared collagen processing by keratocytes cultured in media containing physiological levels of IGF-I, TGF-β, FGF-2, and PDGF in standard and in agarose overlay cultures. Pepsin digestion/SDS PAGE was used to determine the levels of procollagen secreted into the media and the collagen content of the ECM associated with the cell layer. Distribution of collagen type I and fibronectin in the ECM of the agarose cultures was determined by immunoflorescence. Collagen fibril and keratocyte morphology was evaluated by electron microscopy. The agarose overlay significantly enhanced the cell number in the IGF-I, TGF-β and PDGF treated cultures by 2–3 fold. The overlay also significantly enhanced the processing of procollagen to collagen fibrils from 29% in standard cultures to 63–68% in agarose cultures for the IGF-I and PDGF cultures, and from 66% in standard culture to 85% in agarose culture for the TGF-β cultures. Cell accumulation and collagen processing was not enhanced by agarose overlay of the FGF-2 treated cultures. Collagen type I and fibronectin were more uniformly distributed and the collagen fibrils smaller in the ECM of the TGF-β treated cultures. Keratocytes in the FGF-2 treated cultures were in close cell contact with few collagen fibrils while IGF-I, TGF-β, and PDGF cultures had an extensive ECM with abundant collagen fibrils. The results of this study indicate that the agarose overlay enhances collagen fibril assembly and cell accumulation by keratocytes when both collagen synthesis and cell proliferation are stimulated.     

Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.     

Phenotypic characterization of culture expanded rabbit limbal corneal keratocytes

The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits’ corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.     

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.     

Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Bone marrow mesenchymal stem cells can differentiate and assume corneal keratocyte phenotype

It remains elusive as to what bone marrow (BM) cell types infiltrate into injured and/or diseased tissues and subsequently differentiate to assume the phenotype of residential cells, for example, neurons, cardiac myocytes, keratocytes, etc., to repair damaged tissue. Here, we examined the possibility of whether BM cell invasion via circulation into uninjured and injured corneas could assume a keratocyte phenotype, using chimeric mice generated by transplantation of enhanced green fluorescent protein (EGFP)(+) BM cells into keratocan null (Kera(-/-)) and lumican null (Lum(-/-)) mice. EGFP(+) BM cells assumed dendritic cell morphology, but failed to synthesize corneal-specific keratan sulfate proteoglycans, that is KS-lumican and KS-keratocan. In contrast, some EGFP(+) BM cells introduced by intrastromal transplantation assumed keratocyte phenotypes. Furthermore, BM cells were isolated from Kera-Cre/ZEG mice, a double transgenic mouse line in which cells expressing keratocan become EGFP(+) due to the synthesis of Cre driven by keratocan promoter. Three days after corneal and conjunctival transplantations of such BM cells into Kera(-/-) mice, green keratocan positive cells were found in the cornea, but not in conjunctiva. It is worthy to note that transplanted BM cells were rejected in 4 weeks. MSC isolated from BM were used to examine if BM mesenchymal stem cells (BM-MSC) could assume keratocyte phenotype. When BM-MSC were intrastromal-transplanted into Kera(-/-) mice, they survived in the cornea without any immune and inflammatory responses and expressed keratocan in Kera(-/-) mice. These observations suggest that corneal intrastromal transplantation of BM-MSC may be an effective treatment regimen for corneal diseases involving dysfunction of keratocytes.     

Embryogenic suspension cultures of Pinus nigra Arn.: growth parameters and maturation ability

Suspension cultures have been established from embryogenic tissues of Pinus nigra initiated from immature zygotic embryos. The growth of tissues in liquid medium has been influenced by initial tissue weight used for the establishment of the cultures as well as by genotype. In most of the cases initial tissue weight 0.5 g was insufficient and the cultures showed poor growth and later degeneration. Higher amount of initial tissues (1 or 2.5 g) was more efficient for the establishment and proliferation of somatic embryos in liquid medium. The growth of suspension cultures was also cell line dependent. Somatic embryo maturation in liquid medium was very limited and no plantlet regeneration occurred. Cotyledonary somatic embryos developed and produced emblings when the suspension was plated on filter paper discs and cultured on solid maturation medium. Based on our experiments we can state that the embryogenic tissues are able to grow and proliferate in liquid medium but somatic embryo maturation and plantlet regeneration occur only on solid medium.     

Chitosan-functionalized silk fibroin 3D scaffold for keratocyte culture

The goal of this study was to evaluate the potential suitability of an artificial membrane composed of silk fibroin (SF) functionalized by different ratios of chitosan (CS) as a substrate for the stroma of the cornea. Keratocytes were cultured on translucent membranes made of SF and CS with different ratios. The biophysical properties of the silk fibroin and chitosan (SF/CS) membrane were examined. The SF/CS showed tensile strengths that increased as the CS concentration increased, but the physical and mechanical properties of chitosan-functionalized silk fibroin scaffolds weakened significantly compared with those of native corneas. The resulting cell scaffolds were evaluated using western blot in addition to light and electron microscopy. The cell attachment and proliferation on the scaffold were similar to those on a plastic plate. Keratocytes cultured in serum on SF/CS exhibited stellate morphology along with a marked increase in the expression of keratocan compared with identical cultures on tissue culture plastics. The biocompatibility was tested by transplanting the acellular membrane into rabbit corneal stromal pockets. There was no inflammatory complication detected at any time point on the macroscopic level. Taken together, these results indicate that SF/CS holds promise as a substrate for corneal reconstruction.     

Stimulation of embryonic mouse limb bud mesenchymal cell growth by peptide growth factors

The possible role of peptide growth factors in mammalian intrauterine cell growth has been investigated using primary cultures of undifferentiated mesenchymal cells from 11-day mouse embryo limb buds. When grown as monolayer cultures, proliferation is greatly favored by high cell densities. In medium containing 0.2% serum, purified epidermal growth factor (EGF), fibroblast growth factor (FGF), multiplication stimulating activity (MSA), insulin, and somatomedin-C (Sm-C) do not increase cell growth, but a 30-40,000 molecular weight component of mouse fetal liver conditioned medium is stimulatory. On the other hand, when limb bud cells are grown as high density or micromass cultures, a method which better approximates in vivo growth conditions, all of the purified growth factors tested stimulate cell growth significantly. These growth factors have additive effects when used in combination, the best stimulation being observed with liver medium (10% v/v), EGF (10 ng/ml), FGF (200 ng/ml), and either insulin (1 microgram/ml) or Sm-C (20 ng/ml). We conclude that the response of limb bud cells to growth stimulation is influenced by the manner in which the cells are cultured and that at least four different growth factors are required for optimal in vitro proliferation. One of these, the active component of liver medium, appears to be a previously uncharacterized growth factor.     

Iron salts and transferrin are specifically required for cell division of cultured 3T6 cells.

3T6 Swiss mouse fibroblasts can be plated in medium without serum. Prostaglandin F₂^∞, fibroblastic growth factor, epidermal growth factor and insulin stimulate DNA synthesis in medium containing vitamin B₁₂. A combination of these factors, however, does not stimulate cell division under our conditions. Iron salts and transferrin or low concentrations of serum are required to be concurrently present with the growth factors before cell division is observed.     

An adhesion-dependent switch between mechanisms that determine motile cell shape

Keratocytes are fast-moving cells in which adhesion dynamics are tightly coupled to the actin polymerization motor that drives migration, resulting in highly coordinated cell movement. We have found that modifying the adhesive properties of the underlying substrate has a dramatic effect on keratocyte morphology. Cells crawling at intermediate adhesion strengths resembled stereotypical keratocytes, characterized by a broad, fan-shaped lamellipodium, clearly defined leading and trailing edges, and persistent rates of protrusion and retraction. Cells at low adhesion strength were small and round with highly variable protrusion and retraction rates, and cells at high adhesion strength were large and asymmetrical and, strikingly, exhibited traveling waves of protrusion. To elucidate the mechanisms by which adhesion strength determines cell behavior, we examined the organization of adhesions, myosin II, and the actin network in keratocytes migrating on substrates with different adhesion strengths. On the whole, our results are consistent with a quantitative physical model in which keratocyte shape and migratory behavior emerge from the self-organization of actin, adhesions, and myosin, and quantitative changes in either adhesion strength or myosin contraction can switch keratocytes among qualitatively distinct migration regimes.     

Factors controlling embryonic heart cell proliferation in serum-free synthetic media

Summary Embryonic chick cardiac cell cultures, plated on collagen-coated dishes, containing serum-free synthetic media proliferate actively. The basic medium contained Ham's F12 nutrient mixture, fetuin, ascorbic acid, and bovine serum albumin. This medium was supplemented with various combinations of factors; endothelial cell growth supplement (ECGS), epidermal growth factor (EGF), insulin (I), transferrin (T), selenium (S), hydrocortisone, and thyroxine or supplemented alone. Basic medium supplemented with ECGS alone contributes to the highest final cell density among all other factors used in various combinations or alone. The final cell density of the control culture with 2% fetal bovine serum was higher than those of all experimental cultures and an additional control culture grown in the basic medium. Combinations of factors without ECGS do not promote significant cell proliferation. Thyroxine is required to induce optimal differentiation and contractility of cardiac myocytes in vitro. Fibronectin and laminin did not show any more influence than collagen did on the growth and maintenance of cardiac myocytes in serum-free media. The proportion of cardiac muscle cells in ECGS-containing media was higher than those in other experimental media and control media with the exception of ECGS and ITS-containing medium that showed lower proportion of cardiac myocytes than that of serum-containing medium on Days 3 and 5. The profiles of incorporation of [³H]thymidine into DNA of heart cells in experimental and control cultures showed a peak in incorporation values within the first week of culture and subsequently declined. Autoradiography studies revealed that cardiac myocytes in culture supplemented with ECGS alone attained a peak in labeling index on Day 1 with approximately 62% labeled cells. Subsequently, the labeling indices declined. Cardiac myocytes grown in media without ECGS showed significantly lower labeling indices than those in ECGS-containing media. This study has demonstrated the influence of ECGS, EGF and ITS in promoting the growth of cardiac myocytes and also in contributing to the maintenance of contractile cardiac myocytes in serum-free, long-term culture. The influence of ECGS on heart cell proliferation is considered to be superior to that of EGF and ITS. This study was supported in part by a grant HL-25482 from the National Heart Lung and Blood Institute and a grant from the American Heart Association of Michigan.     

CELL—CELL CONTACTS ACCELERATE CELL COLONY GROWTH IN SPARSE CULTURES OF CHICK EMBRYO FIBROBLASTS

There is no cell proliferation in very sparcely plated chick embryo cell cultures. Substituting conditioned medium or adding of ethanol-fixed homologous cells to the cultures accelerates cell colony growth. The mechanism for the mitogenic action of fixed cells is considered to be the contact stimulation of cell proliferation, and addition of extra cells to sparse culture is believed to mimic the cell micro-environment existing in subconfluent cultures. The role of diverse cell—cell contacts in cultured cell growth regulation is discussed. The procedure used (addition of ethanol-fixed cells) may improve normal cell cloning techniques.