J. Michael Sorrell

Fibroblast heterogeneity: more than skin deep

Dermal fibroblasts are a dynamic and diverse population of cells whose functions in skin in many respects remain unknown. Normal adult human skin contains at least three distinct subpopulations of fibroblasts, which occupy unique niches in the dermis. Fibroblasts from each of these niches exhibit distinctive differences when cultured separately. Specific differences in fibroblast physiology are evident in papillary dermal fibroblasts, which reside in the superficial dermis, and reticular fibroblasts, which reside in the deep dermis. Both of these subpopulations of fibroblasts differ from the fibroblasts that are associated with hair follicles. Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of skin. They also play an important role in cutaneous wound repair and an ever-increasing role in bioengineering of skin. Bioengineered skin currently performs important roles in providing (1) a basic understanding of skin biology, (2) a vehicle for testing topically applied products and (3) a resource for skin replacement.

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.

Chapter 4 Fibroblasts—A Diverse Population at the Center of It All

The capacity of fibroblasts to produce and organize the extracellular matrix and to communicate with other cells makes them a central component of tissue biology. Even so, fibroblasts remain a somewhat enigmatic population. Our inability to fully comprehend these cells is in large part due to the paucity of unique cellular markers and to their pervasive diversity. Much of our understanding of fibroblast diversity has evolved from studies where subpopulations of these cells have been produced without resorting to cell surface markers. In this regard, cloning and mechanical separation of tissues prior to establishing cultures has provided multiple subpopulations. Nonetheless, in isolated situations, the expression or lack of expression of Thy-1/CD90 has been used to separate fibroblast subsets. The role of fibroblasts in intercellular communication is emerging through the implementation of organotypic studies in which three-dimensional fibroblast culture are combined with other populations of cells. Such studies have revealed critical paracrine loops that are essential for organ development and for wound repair. These studies also provide a backdrop for the emerging field of tissue engineering. The participation of fibroblasts in the regulation of tissue homeostasis and their contribution to the aging process are emerging issues that require better understanding. In short, fibroblasts represent a multifaceted, complex group of cells.

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.

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.

Age-related differences in human skin proteoglycans

Previous work has shown that versican, decorin and a catabolic fragment of decorin, termed decorunt, are the most abundant proteoglycans in human skin. Further analysis of versican indicates that four major core protein species are present in human skin at all ages examined from fetal to adult. Two of these are identified as the V0 and V1 isoforms, with the latter predominating. The other two species are catabolic fragments of V0 and V1, which have the amino acid sequence DPEAAE as their carboxyl terminus. Although the core proteins of human skin versican show no major age-related differences, the glycosaminoglycans (GAGs) of adult skin versican are smaller in size and show differences in their sulfation pattern relative to those in fetal skin versican. In contrast to human skin versican, human skin decorin shows minimal age-related differences in its sulfation pattern, although, like versican, the GAGs of adult skin decorin are smaller than those of fetal skin decorin. Analysis of the catabolic fragments of decorin from adult skin reveals the presence of other fragments in addition to decorunt, although the core proteins of these additional decorin catabolic fragments have not been identified. Thus, versican and decorin of human skin show age-related differences, versican primarily in the size and the sulfation pattern of its GAGs and decorin in the size of its GAGs. The catabolic fragments of versican are detected at all ages examined, but appear to be in lower abundance in adult skin compared with fetal skin. In contrast, the catabolic fragments of decorin are present in adult skin, but are virtually absent from fetal skin. Taken together, these data suggest that there are age-related differences in the catabolism of proteoglycans in human skin. These age-related differences in proteoglycan patterns and catabolism may play a role in the age-related changes in the physical properties and injury response of human skin.

Production of a monoclonal antibody, DF‐5, that identifies cells at the epithelial–mesenchymal interface in normal human skin. APN/CD13 is an epithelial–mesenchymal marker in skin

Abstract: Epithelial–mesenchymal interactions play a critical role in skin development and differentiation, and similar interactions may also regulate the day‐to‐day proliferation and differentiation events of the epidermis that occur in normal adult skin. This study was directed at identifying molecules that are selectively located at the dermal–epidermal junction in normal adult skin as they may be involved in regulating these homeostatic events. To this end, monoclonal antibodies were raised against the crude cell membrane fraction of cultured human dermal fibroblasts. Screening of antibodies that recognized cell surface antigen on cultured human dermal fibroblasts was followed by determining which of these antibodies selectively localized cells at sites of epithelial–mesenchymal interactions. Antibody DF‐5 fit these criteria and was further characterized. This antibody was found to recognize the cell surface ectopeptidase aminopeptidase N (APN), a molecule homologous to the cluster differentiation antigen CD13. Antibody DF‐5 and anti‐CD13 antibodies both identified cells at sites of epithelial–mesenchymal interactions in fetal, neonatal, and adult human skin, and the APN/CD13 enzyme activity was also identified at these sites. A second ectopeptidase, dipeptidyl peptidase IV (DPPIV) or CD26, presented a significantly different immunohistochemical and histochemical pattern in skin samples, confirming the specificity of the APN/CD13 studies. The function of APN/CD13 in skin has yet to be determined. Its invariant localization at sites of epithelial–mesenchymal interactions argues for a role particular to this region. It may play a role in regulating the activity of neuropeptides or other signaling peptides that are released in this region of skin or it may have an as yet undefined role in mediating communication between dermal and epidermal cells.

The creation of an in vitro adipose tissue that contains a vascular-adipocyte complex.

An increased demand for soft-tissue substitutes has impelled the development of an in vitro adipose tissue. Ideally, such a tissue should contain a vascular network that can deliver blood throughout the construct following its engraftment. This study describes the in vitro fabrication of a pre-vascularized adipose tissue entirely using a self-assembly approach. Adult human adipose stromal cells (ASCs) provided the foundation for this construct. These cells were cultured at high density in the presence of elevated levels of ascorbate prior to adipocytic induction. Vascular support cells consisting of dermal fibroblasts, mixtures of adipose stromal cells and bone marrow mesenchymal stem cells (MSCs) were introduced to sustain an extensive vascular network formed by human umbilical vein endothelial cells (HUVECs). MSCs were introduced to serve as perivascular cells. The resulting construct contained a vascular-adipose tissue continuum that was held together by basement membrane molecules. This construct contains multiple cell types that are typically found in adipose tissue: adipocytes, pre-adipocytes, stem cells, fibroblasts, vascular cells, and perivascular support cells. As such, these constructs can be employed both for in vitro studies to assay cellular interactions between vasculature and other components of adipose tissue. Further, they can also be engrafted into athymic hosts to study vascular and adipocyte stability.