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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 9, 2004 as doi:10.1096/fj.04-1725fje. |
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Department of Cell Biology and Neuroscience, University of California, Riverside, California, USA
1 Correspondence: Department of Cell Biology and Neuroscience, Spieth Hall, University of California Riverside, 900 University Ave., Riverside, CA 92521, USA. E-mail: mmgreen{at}ucrac1.ucr.edu
SPECIFIC AIMS
The inability to directly experiment with humans creates the need to develop culture systems that mimic human tissues and organs in order to understand biological processes. The availability of primary human cells now enables the engineering of such tissues and organs. The aim of this study was to develop an organ culture to allow investigation of physiological/pathological processes in a human tissue under conditions that are well controlled and yet mimic in vivo.
PRINCIPAL FINDINGS
The organ culture presented here was prepared by culturing together the three primary cell types of skin (fibroblasts, microvascular endothelial cells, and keratinocytes) in a basic extracellular matrix composed of interstitial collagen. This organ culture matures into a tissue that 1) contains a well-developed epidermis and a network of microvessels, 2) produces human cytokines, growth factors, and extracellular matrix (ECM) molecules characteristic of human skin, 3) contains new cell types, responds normally to stimulation by cytokines, and 5) persists over a long period in culture.
1. Structural characteristics
A comparison of the mature skin cultures with normal human skin (Fig. 1
A–F) shows they have a similar morphology in both the epidermis and dermis. In the epidermis of cultured skin, keratin 1 and basal lamina components such as collagen IV are differentially expressed in the basal vs. apical layers, as they are in vivo. Keratin 16 and keratin 6 (not shown), which are expressed in injured tissue but not in normal skin, are expressed in young cultures, but expression disappears as the cultures mature. The cells staining for keratin 16 are found only in the superficial (older) layers of the epidermis of a mature culture; in the epidermis of wounds it is expressed in all layers.
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The connective tissue of this "skin" organ contains an interconnected network of mature microvessels resembling that of normal human skin (Fig. 2
A–C). The microvessels are well defined by a single layer of endothelial cells that label for placental endothelial cell adhesion molecule (PECAM, Fig. 2D
), a specific marker of endothelial cell junctions; they are surrounded by a complete basal lamina (Fig. 2E
) and a layer of collagen VII (Fig. 2G
) and are associated with peri-endothelial cells (Fig. 2H
), all characteristics of well-developed, functional microvessels. ECM molecules normally present in skin are deposited as in vivo; for example, fibronectin is deposited within the collagen matrix, primarily in association with the basal lamina of microvessels, and fibroblasts are the major producers. Similarly, collagen III is produced by fibroblasts and deposited throughout the tissue. Hyaluronic acid and tenascin, which in adults are present in areas of tissue remodeling, are only found locally, primarily in areas surrounding microvessels. Staining with wheat germ agglutinin (WGA) and concanavalin A (Con A), two lectins that label ECM glycoproteins, showed that the proper ECM molecules are deposited in the cultured tissue and that glycoproteins are properly glycosylated, reflecting functionality.
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2. Functional characteristics
Earlier work has shown that if microvascular endothelial cells are cultured alone in collagen gels, they self-organize into microvessel-like structures. However, the cells begin to undergo apoptosis 3 days after plating if not treated with tumor promoters. In our cocultures, we achieve maturation in 10 days and culture stability for several weeks without any artificial treatment. As is the case in vivo, mature cultures produce cytokines and growth factors known, at low levels, to be common components of normal tissues and survival factors for endothelial cells. VEGFs are deposited in the immediate vicinity of the vessels; bFGFs are expressed primarily by fibroblasts and are broadly distributed.
Matrix metalloproteinase-2 (MMP-2), found in most tissues and thought to be important in ECM remodeling, is produced and activated as these cultures mature. Our organ cultures do not produce MMPs absent in natural skin (as happens in phorbol ester-treated cells). For example, MMP-9, an enzyme important in angiogenesis and produced upon stimulation by a variety of cell-activating agents, is not normally expressed in our cultures, but expression can be stimulated by treatment of the cultures with an angiogenic factor.
3. Development of new cell types
In addition to fibroblasts, microvascular endothelial cells, and keratinocytes we introduced, two new cell types appear as the cultures mature. Cells expressing 
SMA are initially absent, but with time fibroblasts close to microvessels appear to differentiate into cells expressing this protein. When endothelial cells are plated at high density, larger blood vessels develop; after 8 days, we observed the presence of leukocytes that have the morphological characteristics of monocyte/macrophages. These cells immunolabel with antibodies to macrophage scavenger receptor, a specific marker for macrophages, and with antibodies to CD68, a cell surface marker for mononuclear leukocytes.
CONCLUSIONS AND SIGNIFICANCE
We have developed the first of a new generation of organ cultures made possible by the availability of primary human cells. This generation of cultures provides a new dimension to address a variety of importantscientific and medical subjects (see Fig. 3
). 1) "Direct" experimentation on human tissues becomes available. Investigation of basic biological questions of human cell-cell and cell-microenvironment interactions and how they affect general organ development, differentiation, and maintenance of the differentiated state can be addressed. Transdifferentiation of one cell type to another can be studied as well as the existence of stem cells in specific populations of adult primary cells. 2) Genetic manipulation, restricted primarily to rodents, can be directly applied in an all-human system. Because the cellular components of this "skin" can potentially be manipulated using molecular/biological techniques, it is possible to increase or decrease expression of specific human genes by stimulation of endogenous genes or introduction of exogenous ones. 3) Genetically modified "living bandages" can be developed that can later be removed from the patient. 4) More complex tissue and organ cultures can be developed with adult human primary cells for studies of disease, testing of drugs, and potential application as replacement organs.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1725fje;
2 Present address: Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. 
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