Site-matched papillary and reticular human dermal fibroblasts differ in their release of specific growth factors/cytokines and in their interaction with keratinocytes.

The interfollicular dermis of adult human skin is partitioned into histologically and physiologically distinct papillary and reticular zones. Each of these zones contains a unique population of fibroblasts that differ in respect to their proliferation kinetics, rates at which they contract type I collagen gels, and in their relative production of decorin and versican. Here, site-matched papillary and reticular dermal fibroblasts couples were compared to determine whether each population interacted with keratinocytes in an equivalent or different manner. Papillary and reticular fibroblasts grown in monolayer culture differed significantly from each other in their release of keratinocyte growth factor (KGF) and granulocyte-macrophage colony stimulating factor (GM-CSF) into culture medium. Some matched fibroblast couples also differed in their constitutive release of interleukin-6 (IL-6). Papillary fibroblasts produced a higher ratio of GM-CSF to KGF than did corresponding reticular fibroblasts. Interactions between site-matched papillary and reticular couples were also assayed in a three-dimensional culture system where fibroblasts and keratinocytes were randomly mixed, incorporated into type I collagen gels, and allowed to sort. Keratinocytes formed distinctive cellular masses in which the keratinocytes were organized such that the exterior most layer of cells exhibited characteristics of basal keratinocytes and the interior most cells exhibited characteristics of terminally differentiated keratinocytes. In the presence of papillary dermal fibroblasts, keratinocyte masses were highly symmetrical and cells expressed all levels of differentiation markers. In contrast, keratinocyte masses that formed in the presence of reticular fibroblasts tended to have irregular shapes, and terminal differentiation was suppressed. Furthermore, basement membrane formation was retarded in the presence of reticular cells. These studies indicate that site-matched papillary and reticular dermal fibroblasts qualitatively differ in their support of epidermal cells, with papillary cells interacting more effectively than corresponding reticular cells.

Dilution of human mesenchymal stem cells with dermal fibroblasts and the effects on in vitro and in vivo osteochondrogenesis.

The stromal elements of human bone marrow include cells, referred to as mesenchymal stem cells (MSCs), that have the potential to differentiate into bone, cartilage, fat, and hematopoietic-supportive stromal tissue. MSCs have been isolated and maintained in culture, and in vivo and in vitro assays have been used to show that these cultured cells possess osteochondral potential. Human mesenchymal stem cells (hMSCs) were combined in a range of proportions with human dermal fibroblasts (hDFs), shown to be devoid of osteochondral potential, and tested in these assays. Results suggest that hMSCs may be intentionally "contaminated" with 25-50% hDFs and still elicit a positive response in alkaline phosphatase and calcium in vitro osteogenic assays, form cartilage in pellet culture conditions, and produce bone when loaded into porous hydroxyapatite-tricalcium phosphate ceramic cubes and then implanted subcutaneously into immunocompromised mice. Although hMSCs can be purified and culture-expanded as a homogeneous subset of marrow cells, the dilution results reported here are encouraging for the prospective use of these cells in clinical applications, where repair grafts that contain 100% hMSCs almost surely will become infiltrated with host connective tissue and vasculature, which will dilute the initial concentration of hMSCs.

A monoclonal antibody which recognizes a glycosaminoglycan epitope in both dermatan sulfate and chondroitin sulfate proteoglycans of human skin.

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.

Versican in human fetal skin development.

The extracellular matrix of human fetal skin differs substantially from that of adult skin. Fetal skin contains sparse amounts of fibrillar collagen enmeshed in a highly hydrated amorphous matrix composed of hyaluronan and sulfated proteoglycans. Both fetal and adult skin contain two major interstitial proteoglycans that are extracted by chaotrophic agents and detergents. These are the large chondroitin sulfate proteoglycan versican and the small dermatan sulfate proteoglycan decorin. For this study, proteoglycans extracted from fetal and adult skin were compared on Western blots to determine the relative amounts of versican. Decorin present in the same samples provided an internal standard for these studies. Fetal skin differed from adult skin in that it contained a significantly higher proportion of versican than did adult skin. Immunohistochemical studies compared early-fetal with mid-fetal skin and found that versican was a significant component of the interstitial extracellular matrix at both of these stages of skin development. However, by the mid-fetal period, interstitial versican became restricted to the upper half of the dermis, although versican also continued to be highly expressed around hair follicles, glands, and vasculature in the lower half of the dermis. Fetal skin extracts differed from an adult skin extract by the presence of a 66-kDa protein immunologically related to versican and by the absence of a 17-kDa core protein of a proteoglycan related to decorin. Both of these molecular species may represent degradation products of their respective proteoglycans. Monoclonal antibodies which detect epitopes in native chondroitin sulfate glycosaminoglycan chains recognized versican extracted from fetal skin. However, the tissue distribution of these antigens did not entirely conform to that for versican core protein, suggesting that versican in different regions of the skin may be substituted with glycosaminoglycan chains with different microchemistries. The results of these studies indicate that human fetal skin is structurally different from adult skin in terms of both the distribution and the composition of the large, aggregating chondroitin sulfate proteoglycan versican.

Monoclonal antibody against adult marrow-derived mesenchymal stem cells recognizes developing vasculature in embryonic human skin.

We have described previously a monoclonal antibody (SH2) that specifically recognizes undifferentiated mesenchymal progenitor cells isolated from adult human bone marrow. These cells, which we operationally refer to as mesenchymal stem cells, have the capacity to differentiate and form distinct mesenchymal tissues such as bone and cartilage when the isolated cells are placed in the appropriate in vivo or in vitro environment. We report here the partial biochemical characterization of the antigen recognized by the SH2 antibody. Metabolically radiolabelled adult marrow-derived mesenchymal stem cells in culture were extracted and immunoprecipitated with the SH2 antibody. The purified antigen migrated as a single band of 90 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed under reducing conditions. The SH2-immunoprecipitated protein exhibited a molecular weight band shift after removal of N-linked oligosaccharides. We investigated the expression of the SH2 antigen, along with the endothelial markers factor VIII-related antigen and Ulex europaeus I (UEA-I) lectin during specific developmental periods in human dermal embryogenesis and in the postnatal period through aged adults. Frozen sections of human embryonic, fetal, or postnatal skin ranging from 8 weeks estimated gestational age (EGA) through 84 years of age were immunostained or double immunolabelled with antibodies SH2, UEA-I, or factor VIII-related antigen followed by second antibodies with fluorescent markers. Positive cell surface reactivity with the SH2 antibody was seen in cells in the vascular plane in the earliest specimens (day 55 EGA) corresponding to the late cellular dermis period. During the period of the cellular to fibrous transition, in which the initiation of appendage development occurs, most SH2-reactive cells colocalized with vasculature markers UEA-I and factor VIII-related antigen, although there was a subset of cells recognized by SH2 antibody that did not colocalize with the endothelial markers. In contrast to the endothelial markers UEA-I and factor VIII-related antigen, in which the number of immunopositive cells became more prominent with age and maturation of the dermis, the frequency of cells that contained the SH2-reactive antigen diminished with age. The SH2 reactivity evident in embryonic, fetal, and early postnatal periods was not observed in human skin specimens taken from adults greater than 30 years old. These observations support the hypothesis that the SH2 antigen is a cell surface marker of developing microvasculature and may play a role in dermal embryogenesis and angiogenesis.

Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha.

We previously reported the purification, culture-expansion, and osteogenic differentiation potential of mesenchymal progenitor cells (MPCs) derived from human bone marrow. As a first step to establishing the phenotypic characteristics of MPCs, we reported on the identification of unique cell surface proteins which were detected with monoclonal antibodies. In this study, the phenotypic characterization of human marrow-derived MPCs is further established through the identification of a cytokine expression profile under standardized growth medium conditions and in the presence of regulators of the osteogenic and stromal cell lineages, dexamethasone and interleukin-1 alpha (IL-1 alpha), respectively. Constitutively expressed cytokines in this growth phase include G-CSF, SCF, LIF, M-CSF, IL-6, and IL-11, while GM-CSF, IL-3, TGF-beta 2 and OSM were not detected in the growth medium. Exposure of cells in growth medium to dexamethasone resulted in a decrease in the expression of LIF, IL-6, and IL-11. These cytokines have been reported to exert influence on the differentiation of cells derived from the bone marrow stroma through target cell receptors that utilize gp130-associated signal transduction pathways. Dexamethasone had no effect on the other cytokines expressed under growth medium conditions and was not observed to increase the expression of any of the cytokines measured in this study. In contrast, IL-1 alpha increased the expression of G-CSF, M-CSF, LIF, IL-6 and IL-11 and induced the expression of GM-CSF. IL-1 alpha had no effect on SCF expression and was not observed to decrease the production of any of the cytokines assayed. These data indicate that MPCs exhibit a distinct cytokine expression profile. We interpret this cytokine profile to suggest that MPCs serve specific supportive functions in the microenvironment of bone marrow. MPCs provide inductive and regulatory information which are consistent with the ability to support hematopoiesis, and also supply autocrine, paracrine, and juxtacrine factors that influence the cells of the marrow microenvironment itself. In addition, the cytokine profiles expressed by MPCs, in response to dexamethasone and IL-1 alpha, identify specific cytokines whose levels of expression change as MPCs differentiate or modulate their phenotype during osteogenic or stromagenic lineage entrance/progression.

Construction of a bilayered dermal equivalent containing human papillary and reticular dermal fibroblasts: use of fluorescent vital dyes.

Bilayered dermal equivalents were constructed by seeding human papillary and reticular dermal fibroblasts into separate layers of type I collagen and allowing these layers to gel into a single entity. That these bilayered gels had fused was established through histologic examination and from the fact that these gels, when detached, contracted as a single unit. Papillary and reticular dermal fibroblasts remained in their respective layers as established by differentially labeling these dermal cells with fluorescent vital dyes l,l'-dioctacecyl-3,3,3',3'-tetramethylindocabocyanine perchlorate, DiIC(18)(3) (Dil), and 3,3'-dioctadecyloxacarbocyanine perchlorate, DiOC(18)(3) (DiO). The labeling with these vital dyes did not interfere with the ability of the cells to proliferate or to contract floating type I collagen gels. Thus, these bilayered gels can provide the means of creating dermal equivalents that contain a variety of different dermal cell types to assess their relative abilities, either alone or in various combinations, to support keratinocyte proliferation and differentiation and to contribute, eventually, to the formation of a multilayered skin equivalent.

Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies.

Human bone marrow has been shown to contain mesenchymal cells, which fabricate the connective tissue network of the marrow called the stroma. A subset of these marrow-derived mesenchymal cells can be isolated, expanded in culture, and then induced to differentiate into bone-producing osteoblasts and ultimately osteocytes when placed in the proper environment. At present, there are no methods for definitively identifying these cells in human marrow tissue or following their differentiation into osteogenic phenotypes. Therefore, we culture-expanded, marrow-derived mesenchymal cells from human donors and used these cells to immunize cells from human donors and used these cells to immunize mice whose spleens were used to generate hybridoma cell lines, which secrete antibodies to antigens on the cell surface of these culture-expanded mesenchymal cells. Hybridoma culture supernatants were successively screened against highly enriched samples of culture-expanded, marrow-derived mesenchymal cells in cryosections and live cell cultures to identify unique cell surface antigens. Positive clones were then screened against cell suspensions of whole and fractionated marrow to identify hybridomas whose supernatants were nonreactive with marrow hemopoietic cells. Three hybridoma cell lines, SH2, SH3, and SH4, were identified; these hybridomas secrete antibodies that recognize antigens on the cell surface of marrow-derived mesenchymal cells, but fail to react with marrow-derived hemopoietic cells. Additional tissue screening reveals unique tissue distributions for each of the recognized antigens, which suggests different antigen recognition for each antibody. However, all three antibodies fail to react with the cell surface of osteoblasts or osteocytes, suggesting that the antigens recognized by these antibodies are developmentally regulated and specific for primitive or early-stage cells of the osteogenic lineage.

Generation of a monoclonal antibody against avian small dermatan sulfate proteoglycan: immunolocalization and tissue distribution of PG-II (decorin) in embryonic tissues.

Chick embryonic skeletal muscle synthesizes three major types of proteoglycans: large chondroitin sulfate proteoglycans, small dermatan sulfate proteoglycans and small heparan sulfate proteoglycans. A monoclonal antibody has been raised which recognizes the small dermatan sulfate proteoglycan. Immunoblot analysis of a partially purified preparation of skeletal muscle proteoglycans indicates that the antibody reacts with a molecule which migrates with an estimated Mr of 100,000. Prior treatment of the proteoglycans with chondroitinase results in immunostaining of a species of estimated Mr 45,000. These values for the intact proteoglycan and its core protein suggest that the antibody is directed against a proteoglycan of the PG-II or decorin class. Immunohistochemistry indicates a widespread distribution of the proteoglycan, which is localized in connective tissue septa of skeletal and cardiac muscle, dermis, tendon, bone, perichondrium and cornea. Immunoblot analysis of the proteoglycan core proteins from these tissues demonstrates that the antibody recognizes the same 45,000-dalton band in each tissue. The widespread tissue distribution is also consistent with the antibody being directed against an epitope of PG-II. Neither the glycosaminoglycan chains nor N-linked oligosaccharides are required for reactivity and the antibody cross-reacts with other avian material, but not mammalian. This antibody, which has been designated CB-1, reveals developmental stage-specific changes in the deposition of PG-II in embryonic limb bud and skeletal muscle.

Bone and cartilage formation in diffusion chambers by subcultured cells derived from the periosteum.

Periosteal cells were enzymatically isolated from the tibiae of young chicks, introduced into cell culture, allowed to reach confluence, and subcultured. The freshly isolated or subcultured cells were loaded into diffusion chambers and implanted into the peritoneal cavity of athymic mice to test their osteo-chondrogenic potential in a contained in vivo location. Freshly isolated periosteal cells formed both bone and cartilage tissue in such test chambers, but with a relatively low incidence. In contrast, cultured periosteal cells consistently gave rise to bone and cartilage even after 10 population doublings. With further passages of cells, the osteo-chondrogenic potential diminished substantially, until complete loss of expressivity at 16 population doublings or longer. Cultured muscle fibroblasts, when loaded into diffusion chambers under identical conditions to those of cultured periosteal cells, formed neither bone nor cartilage. These observations suggest that periosteal cells of young chicks contain subsets of progenitor cells or mesenchymal stem cells which possess the potential to differentiate into osteoblasts or chondrocytes, and this potential is retained after enzymatic isolation and for several population doublings in culture.