Marilyn A. Baber

Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: Effects of dexamethasone and IL-1α

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‐1α (IL‐1α), 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‐β2, 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‐1α increased the expression of G‐CSF, M‐CSF, LIF, IL‐6, and IL‐11 and induced the expression of GM‐CSF. IL‐1α 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‐1α, identify specific cytokines whose levels of expression change as MPCs differentiate or modulate their phenotype during osteogenic or stromagenic lineage entrance/progression.

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.

Influence of Adult Mesenchymal Stem Cells on In Vitro Vascular Formation

The effective delivery of bioactive molecules to wound sites hasten repair. Cellular therapies provide a means for the targeted delivery of a complex, multiple arrays of bioactive factors to wound sites. Thus, the identification of ideal therapeutic populations is an essential aspect of this approach. In vitro assays can provide an important first step toward this goal by selecting populations that are likely suitable for more expensive and time-consuming in vivo assays. In this study, bone marrow-derived mesenchymal stem cells (BM-MSCs) were integrated into a three-dimensional coculture system that supports the development and stabilization of vascular tube-like structures. The presence of a limited number of BM-MSCs resulted in their coalignment with vascular structures, and it further resulted in increased tubule numbers and complexity. Thus, these studies suggest that BM-MSCs functionally interacted with and were attracted to in vitro formed vascular structures. Further, these cells also provided sufficient bioactive factors and matrix molecules to support the formation of tubular arrays and the stabilization of these arrays. This in vitro system provides a means for assessing the function of BM-MSCs in aspects of the angiogenic component of wound repair.

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 Self-Assembled Fibroblast-Endothelial Cell Co-Culture System That Supports in vitro Vasculogenesis by both Human Umbilical Vein Endothelial Cells and Human Dermal Microvascular Endothelial Cells

The construction of vascularized connective tissues is an important goal in tissue engineering in that the presence of a patent bio-engineered vasculature should facilitate vascularization of an implant. Fibroblasts play an essential role in the angiogenic process through their production of extracellular matrix molecules and through their release of essential growth factors. Therefore, the aim of this study is to develop a thin 3-dimensional model in which fibroblasts support endothelial cells in the formation of tube-like structures. Macro- and microvascular endothelial cells were seeded onto confluent lawns of human fibroblasts and were cultured in the presence of high levels of ascorbate 2-phosphate to create a tissue-like structure in which endothelial cell organized into tube-like structures. The process was visualized in the culture dish through labeling of cells with a long-lasting fluorescent vital dye. Intact sheet-like structures were created in which endothelial cell tube-like structures were encased by fibroblasts and were surrounded by a basement membrane. These structures appeared to contain a lumen and remained stable for up to 5 weeks in culture. This culture system provides an in vitro method to study fibroblast-endothelial cell interactions and to study the effects of pro- and anti-angiogenic factors on endothelial cell differentiation. This system also provides an experimental basis for developing vascularized tissue-engineered connective tissue.

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.

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.

Human dermal fibroblast subpopulations; differential interactions with vascular endothelial cells in coculture : Nonsoluble factors in the extracellular matrix influence interactions

The superficial dermis of adult human skin contains a complex arcading microvasculature that provides nutrient support to the overlying epidermis. We propose that the unique subpopulations of dermal fibroblasts located in the superficial dermis contribute to the organization and maintenance of this elaborate microvasculature. This possibility was tested in a coculture system in which distinct subpopulations of adult human dermal fibroblasts were grown to form high‐density lawns that were then seeded with human umbilical vein vascular endothelial cells (EC). The fibroblast subpopulation cultured specifically from the papillary dermis supported a robust array of highly branched tube‐like structures. In contrast, fibroblasts cultured from the reticular dermis provided an anemic level of support for the formation of tube‐like structures. These varied interactions with vascular EC were not due to the differential production of the potent pro‐angiogenic factors vascular endothelial growth factor‐A or fibroblast growth factor‐2. Instead, the extracellular matrix and/or molecules bound to this matrix appeared to contain instructions that modulated these differential fibroblast–vascular EC interactions. One matrix‐binding growth factor, hepatocyte growth factor/scatter factor, was identified that was both differentially expressed by papillary and reticular dermal fibroblasts and which was shown to be physiologically relevant in the coculture model. These studies highlight the importance of fibroblasts in supporting and maintaining vascular integrity. Furthermore, these studies have important implications for wound repair and may help to explain how fibroblasts contribute to the etiology of nonhealing wounds.

Clonal characterization of fibroblasts in the superficial layer of the adult human dermis

The dermis of adult human skin contains a physiologically heterogeneous population of fibroblasts that interact to produce its unique architecture and that participate in inflammatory and wound repair functions in vivo. This heterogeneity has been well documented for fibroblasts located in the superficial papillary dermis and the deep reticular dermis. However, the existence of diverse fibroblast subpopulations within a given region of the dermis has not been explored. In this study, fibroblast cultures have been established from the superficial dermis following enzymatic dissociation of the tissue. These fibroblasts have been cloned by limiting dilution and initially selected on the basis of morphology and proliferation kinetics. Fibroblasts in some of the clones selected for study express α-smooth muscle actin, a myofibroblast characteristic. Significant differences for fibroblast clones obtained from the same piece of skin have been observed with regard to their rate of collagen lattice contraction, their ability to organize a fibronectin matrix, their release of specific growth factors/cytokines into culture medium, and their response to interleukin-1α. These differences in both morphological and physiological characteristics indicate that the superficial papillary dermis contains a heterogeneous population of fibroblasts. This heterogeneity might indicate that diverse subpopulations of fibroblasts are required to interact in both homeostatic and pathological situations in skin.

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.