Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease

Progressive photoreceptor degeneration resulting from genetic and other factors is a leading and largely untreatable cause of blindness worldwide. The object of this study was to find a cell type that is effective in slowing the progress of such degeneration in an animal model of human retinal disease, is safe, and could be generated in sufficient numbers for clinical application. We have compared efficacy of four human-derived cell types in preserving photoreceptor integrity and visual functions after injection into the subretinal space of the Royal College of Surgeons rat early in the progress of degeneration. Umbilical tissue-derived cells, placenta-derived cells, and mesenchymal stem cells were studied; dermal fibroblasts served as cell controls. At various ages up to 100 days, electroretinogram responses, spatial acuity, and luminance threshold were measured. Both umbilical-derived and mesenchymal cells significantly reduced the degree of functional deterioration in each test. The effect of placental cells was not much better than controls. Umbilical tissue-derived cells gave large areas of photoreceptor rescue; mesenchymal stem cells gave only localized rescue. Fibroblasts gave sham levels of rescue. Donor cells were confined to the subretinal space. There was no evidence of cell differentiation into neurons, of tumor formation or other untoward pathology. Since the umbilical tissue-derived cells demonstrated the best photoreceptor rescue and, unlike mesenchymal stem cells, were capable of sustained population doublings without karyotypic changes, it is proposed that they may provide utility as a cell source for the treatment of retinal degenerative diseases such as retinitis pigmentosa.     

Insulin-like growth factor (IGF) binding proteins and their mRNAs in connective tissues of fasted guinea-pigs

Fasting (with vitamin C-supplementation) and vitamin C-deficiency in guinea-pigs are associated with decreased collagen gene expression in connective tissues. Recently we presented evidence that circulating insulin-like growth factor binding protein (IGFBP)-1 and-2 that are induced during both nutritional deficiencies may be responsible for this inhibition by interfering with IGF-I action. The present objective was to determine whether circulating IGFBPs are accumulated in bone, skin and cartilage during fasting, which would support an endocrine role for them. IGFBP-1 mRNA was not detected in any of the connective tissues. The protein, as measured by ligand blotting, was not present in tissues of normal animals but accumulated early during fasting in all of the tissues. Bone and cartilage from normal animals contained IGFBP-2 and its mRNA, but only in bone did their levels increase during fasting. IGFBP-3 mRNA was not detected in connective tissues from normal or fasted guinea-pigs. Little or no IGFBP-3 was detected in normal tissue extracts, but protein accumulated during fasting and presumably was derived from the circulation. IGF-I and-II mRNAs were expressed in bone and cartilage but in skin, only IGF-II mRNA was detected. Affinity cross-linking revealed that in skin, IGFBP-3 contained relatively few unoccupied IGF-I binding sites compared to IGFBP-1 while in bone and cartilage, only IGFBP-1 contained unoccupied binding sites. IGFBP-1, acting by endocrine action, is probably the major factor responsible for inhibition of IGF-I-dependent collagen gene expression during fasting.     

Development of a three-dimensional transmigration assay for testing cell--polymer interactions for tissue engineering applications.

The ability of synthetic or natural scaffolds to support invasion of cells from surrounding tissue is a key parameter for tissue engineering (TE). In this study, the migration of fibroblasts, chondrocytes, and osteoblasts into biodegradable polymer scaffolds was evaluated using a novel, three-dimensional (3-D) transmigration assay. This assay is based on a cell-populated contracted collagen lattice with a biodegradable polymer scaffold implanted at the center of the collagen gel. Cell migration into the scaffolds was assessed both quantitatively and qualitatively following various time lengths in culture using image analysis. Chondrocytes, incorporated within the collagen lattice, migrated into polymer scaffolds, when cultured both statically or in a rotating bioreactor. However, the bioreactor cultures resulted in a significantly greater cell invasion as compared to static cultures. There was a cell density-dependent osteoblast migration from collagen lattice into polymer scaffold, when tested in the transmigration assay. In addition, polymer scaffolds, treated with or without recombinant human platelet-derived growth factor (rh-PDGF-BB) were evaluated for fibroblast migration. The presence of rh-PDGF-BB resulted in significantly greater fibroblast invasion as compared to untreated scaffolds. Our studies suggest that the transmigration model provides a rapid system for testing cell invasion of potential scaffolds for tissue engineering applications.     

Characterization of a macrophage-based system for studying the activiation of latent TGF-β

TGF- has been implicated in scarring and tissue fibrosis. Most cells secrete TGF- as a high molecular weight, latent complex that must be processed to a lower molecular weight, biologically active form. A number of molecules are involved in this activation step including the mannose 6-phosphate/insulin-like growth factor- II receptor, tissue transglutaminase, thrombospondin, plasmin, and others. Here we describe a rapid macrophage-based system for TGF- 1 activation, which could be used for screening potential anti- fibrotic agents. The system employs transformed mouse peritoneal macrophages treated with lipopolysaccharide as a cell line capable of activating latent TGF-. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor andtransglutaminase. The activation of latent TGF- in this system can be prevented by the addition of mannose-6-phosphate but not mannose-1-phosphate. In addition, transglutaminase inhibitors, antibodies to thrombospondin, insulin-like growth factor- II in the presence of its binding protein IGFBP-2, but not IGFBP-1, suppressed the activation of TGF-. Anti-inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF- activation by a mechanism, that may involve downregulation of transglutaminase expression. In summary, this new, rapid and reproducible system allows testing molecules for their ability to inhibit TGF- activation, thus providing a screening method for potential anti-scarring molecules.     

Differential regulation of collagen gene expression in granulation tissue and nonrepair connective tissues in vitamin C-deficient guinea pigs

Poor wound healing during vitamin C deficiency is thought to be due to decreased hydroxylation of proline residues in collagen. In non-repair connective tissues of guinea pigs, however, procollagen gene expression is not decreased until weight loss occurs during the third and fourth weeks of scurvy (phase II) with only a moderate decrease in proline hydroxylation. Decreased procollagen gene expression is related to the induction of insulin-like growth factor binding proteins 1 and 2 that inhibit insulin-like growth factor-I action. We examined wound healing and granulation tissue formation during phase I of vitamin C deficiency. Synthetic sponges were implanted on day 7 of vitamin C deficiency and analyzed at 6 and 10 days after surgery, when there was no weight loss or induction of insulin-like growth factor binding proteins. Healing of incisions was almost complete at 10 days after surgery in normal controls but not in scorbutic animals. The area around the incision and implant exhibited excessive angiogenesis and hemorrhaging of vessels in the scorbutic animals at 6 and 10 days after surgery. At 10 days after surgery, collagen synthesis in the implants of scorbutic guinea pigs was 36% lower than control values, with a normal extent of proline hydroxylation. Concentrations of messenger RNAs for types I and III procollagens were slightly increased by scurvy at 6 days after surgery but were decreased by 26% and 40%, respectively, at 10 days. Fibronectin mRNA levels were unaffected by scurvy at both time points. Our results suggest that poor wound healing in phase I of scurvy may be related to defective interstitial procollagen gene expression and defective blood vessel formation, but it does not involve inhibition of proline hydroxylation or induction of insulin-like growth factor binding proteins. mRNA for insulin-like growth factor-II, transforming growth factor-beta(1), and transforming growth factor-beta(2) were significantly expressed in implants, but their patterns of expression did not correlate with changes in procollagen gene expression.     

Differential expression and regulation of extracellular matrix-associated genes in fetal and neonatal fibroblasts

Adults and neonates heal wounds by a repair process associated with scarring in contrast to scar-free wound healing in the fetus. In the present study, human dermal fetal fibroblasts, representing the scarless phenotype, and neonatal human dermal fibroblasts, representing scar-forming phenotype, were examined for potential differences that might influence the wound healing process. Fetal fibroblasts secreted four- to tenfold more latent transforming growth factor-beta1 depending on the cell strains compared. Fetal fibroblasts also produced higher levels of collagen protein and mRNA for most types of collagen (particularly type III) as compared to neonatal cells. Interestingly, mRNA for type V collagen was significantly reduced in fetal cells. Neonatal fibroblasts expressed significantly higher levels of latent transforming growth factor-beta1 binding protein mRNA, in contrast to almost undetectable levels in fetal fibroblasts. By ligand blot analysis, the levels of insulin-like growth factor binding protein-3, a reported mediator of transforming growth factor-beta1 activity, was eightfold higher in neonatal versus fetal fibroblasts. Approximately 20 other mRNAs for various cytokines, matrix molecules and receptors were examined and found to be similar between the two cell types. The phenotypic differences described in this article may represent potentially important mechanisms to explain the differences in the quality of wound repair observed in fetal versus adult/neonatal tissues.