- Email: [email protected]
American Academy of Dermatology 2003 Awards for Young Investigators in Dermatology
FROM
THE
ACADEMY
American Academy of Dermatology 2003 Awards for Young Investigators in Dermatology Supported by an educational grant from Janssen Pharmaceutica Research Foundation Topographic differentiation of human fibroblasts Howard Chang, MD, PhD, Stanford University, Stanford, California Nominated by Alfred T. Lane, MD, Stanford University One of the essential features of cutaneous biology is the regional variation of skin anatomy and physiology. Clinically, the distributions of lesions offer dermatologists many clues to the differential diagnoses of skin diseases, and the specific location of the lesions often dictates the strategy and course of treatment. My research seeks to understand the molecular basis of site-specific differences in skin using genome-wide patterns of gene expression. Sequencing of the human genome and microarray technology have allowed the expression level of tens of thousands of genes to be quantitated simultaneously.1 The comprehensive view of gene expression patterns can often reveal unanticipated but fundamental insights into biology. Although topographic differences in epidermal structures (eg, hairs) on different anatomic sites are easily appreciated, embryologic experiments have demonstrated that it is the underlying mesenchymal tissue that dictates the epithelial fates that develop.2 The factors or cells that endow positional identity in mesenchyme are incompletely understood. Fibroblasts are mesenchymal cells with many vital functions during development and in adult organisms, including extracellular matrix synthesis and wound healing. Although fibroblasts are among the most accessible normal mammalian cell types, they remain poorly defined in molecular terms and are often identified on the basis of the lack of other lineage markers. Thus I hypothesized that fibroblasts as they are traditionally defined may contain J Am Acad Dermatol 2004;50:e1. Published online ●●● 0197-6787/$30.00 © 2004 by the American Academy of Dermatology, Inc. doi:10.1016/S0190-9622(03)02723-3
several separable cell types based on their gene expression profiles. I have discovered that purified fibroblasts from different anatomic sites of the skin exhibit systematic and characteristic differences in gene expression such that they should be considered different cell types3 (see http://genome-www.stanford.edu/fibroblast/). I examined the expression of more than 36,000 mRNAs using cDNA microarrays in each of 50 fibroblast cultures. Although all fibroblasts are morphologically similar, the gene expression patterns of cultured fibroblasts from different sites are strikingly distinct. Approximately 8% of all transcribed genes in fibroblasts are regulated in a site-specific fashion. The variation and magnitude of gene expression differences among fibroblasts are comparable to the variation observed among different types of white blood cells. When their gene expression patterns were grouped by similarity, fibroblasts from the same topographic sites of the skin were consistently grouped together, and the distinctiveness of topographic gene expression was not obscured among different donors, by passage in tissue culture, or by environmental changes such as serum starvation. These results demonstrate that in fact there are many different cell types that go under the traditional heading of “fibroblasts.” The main rule of differentiation appears to be dictated by the anatomic site of origin, a phenomenon we term “topographic differentiation.” The topographically regulated genes suggest distinct choreographed programs in extracellular matrix synthesis, lipid metabolism, and signaling pathways controlling cell migration and cell fate specification. Moreover, the expression domains of genes underlying several genetic diseases affecting skin or musculoskeletal connective tissue correlated closely with the phenotypic defects, suggesting that the gene expression map of fibroblasts may help to identify new disease genes. In addition, we observed that adult fibroblasts maintain the expression of HOX genes encode a family of transcription factors that control positional identity during development.4 Gain or loss of function in HOX genes causes homeotic mutations, E1
E2
where one anatomic structure is dramatically transformed into another fully formed structure. Fibroblasts from each site were found to have a unique pattern of HOX genes such that they could be distinguished from each other on the basis of HOX genes alone.3 These results suggest a model whereby positional identity in fibroblasts is established during development and encoded in HOX genes; the Hox transcription factors then activate distinct target genes to give rise to topographic differentiation in fibroblasts. REFERENCES 1. Brown PO, Botstein D. Exploring the new world of the genome with DNA microarrays. Nat Genet 1999;21:33-7. 2. Dhouailly D. Specification of feather and scale patterns. In: Malincinski G, Bryant S, editors. Pattern formation. New York: Macmillan, 1984. p. 581-601. 3. Chang HY, Chi JT, Dudoix S, Bondre C, van de Rijn M, Botstein D, et al. Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci USA 2002;99:12877-82. 4. Krumlauf R. Hox genes in vertebrate development. Cell 1994;78: 191-201.
Production of human T cells from bone marrow progenitor cells using a skin-derived thymic construct Rachael A. Clark, MD, PhD, Harvard Skin Disease Research and Brigham and Women’s Department of Dermatology, Boston, Massachusetts Nominated by Thomas S. Kupper, MD, Harvard Skin Disease Research and Brigham and Women’s Department of Dermatology The development of T cells requires a period of specialized education in the thymus, an organ that largely undergoes involution during late adolescence. This requirement for thymic education has been a barrier to the laboratory production of T cells that can be given to immunocompromised patients
J AM ACAD DERMATOL MARCH 2004
to treat infection or malignancies. In vitro systems for T-cell development have thus far required the use of either human or animal thymus tissue, thereby preventing their use in adult human patients. Utilizing the natural similarities between skin and the thymus, we have developed a system in which cells from the skin are reprogrammed to act as a surrogate thymus, supporting the in vitro development of mature T cells from bone marrow precursor cells. Keratinocytes and fibroblasts from adult human skin were expanded separately in culture and then seeded onto a 3-dimensional tantalumcoated carbon framework. AC133⫹ (CD133⫹) human bone marrow precursor cells were then added, and the resultant construct was cultured for 3 to 4 weeks. Cells produced by the construct were analyzed by flow cytometry and T-cell recombination excision circles (TREC) analysis. Using this system, we have produced T cells that have surface markers consistent with mature, functional T cells. The cells were CD3hi, either CD8 or CD4 single-positive, and a significant proportion were CD45RA⫹, suggesting a naı¨ve phenotype. Input hematopoietic precursor cells were negative for CD3 and for TREC expression, where output cells were CD3⫹ and positive for TREC expression, indicating that these cells are newly developed T cells. Lymphocytes produced in this construct proliferated in response to phytohemagglutinin and produced tumor necrosis factor– g␣ and expressed CD69 in response to treatment with concanavalin A, suggesting that these cells are mature and functional. The ultimate goal of this project is to develop a method by which newly generated T cells, either naı¨ve or with engineered T-cell receptors, can be produced for the treatment of diseases of individual patients using autologous bone marrow and skin biopsies.
THE
ACADEMY
American Academy of Dermatology 2003 Awards for Young Investigators in Dermatology Supported by an educational grant from Janssen Pharmaceutica Research Foundation Topographic differentiation of human fibroblasts Howard Chang, MD, PhD, Stanford University, Stanford, California Nominated by Alfred T. Lane, MD, Stanford University One of the essential features of cutaneous biology is the regional variation of skin anatomy and physiology. Clinically, the distributions of lesions offer dermatologists many clues to the differential diagnoses of skin diseases, and the specific location of the lesions often dictates the strategy and course of treatment. My research seeks to understand the molecular basis of site-specific differences in skin using genome-wide patterns of gene expression. Sequencing of the human genome and microarray technology have allowed the expression level of tens of thousands of genes to be quantitated simultaneously.1 The comprehensive view of gene expression patterns can often reveal unanticipated but fundamental insights into biology. Although topographic differences in epidermal structures (eg, hairs) on different anatomic sites are easily appreciated, embryologic experiments have demonstrated that it is the underlying mesenchymal tissue that dictates the epithelial fates that develop.2 The factors or cells that endow positional identity in mesenchyme are incompletely understood. Fibroblasts are mesenchymal cells with many vital functions during development and in adult organisms, including extracellular matrix synthesis and wound healing. Although fibroblasts are among the most accessible normal mammalian cell types, they remain poorly defined in molecular terms and are often identified on the basis of the lack of other lineage markers. Thus I hypothesized that fibroblasts as they are traditionally defined may contain J Am Acad Dermatol 2004;50:e1. Published online ●●● 0197-6787/$30.00 © 2004 by the American Academy of Dermatology, Inc. doi:10.1016/S0190-9622(03)02723-3
several separable cell types based on their gene expression profiles. I have discovered that purified fibroblasts from different anatomic sites of the skin exhibit systematic and characteristic differences in gene expression such that they should be considered different cell types3 (see http://genome-www.stanford.edu/fibroblast/). I examined the expression of more than 36,000 mRNAs using cDNA microarrays in each of 50 fibroblast cultures. Although all fibroblasts are morphologically similar, the gene expression patterns of cultured fibroblasts from different sites are strikingly distinct. Approximately 8% of all transcribed genes in fibroblasts are regulated in a site-specific fashion. The variation and magnitude of gene expression differences among fibroblasts are comparable to the variation observed among different types of white blood cells. When their gene expression patterns were grouped by similarity, fibroblasts from the same topographic sites of the skin were consistently grouped together, and the distinctiveness of topographic gene expression was not obscured among different donors, by passage in tissue culture, or by environmental changes such as serum starvation. These results demonstrate that in fact there are many different cell types that go under the traditional heading of “fibroblasts.” The main rule of differentiation appears to be dictated by the anatomic site of origin, a phenomenon we term “topographic differentiation.” The topographically regulated genes suggest distinct choreographed programs in extracellular matrix synthesis, lipid metabolism, and signaling pathways controlling cell migration and cell fate specification. Moreover, the expression domains of genes underlying several genetic diseases affecting skin or musculoskeletal connective tissue correlated closely with the phenotypic defects, suggesting that the gene expression map of fibroblasts may help to identify new disease genes. In addition, we observed that adult fibroblasts maintain the expression of HOX genes encode a family of transcription factors that control positional identity during development.4 Gain or loss of function in HOX genes causes homeotic mutations, E1
E2
where one anatomic structure is dramatically transformed into another fully formed structure. Fibroblasts from each site were found to have a unique pattern of HOX genes such that they could be distinguished from each other on the basis of HOX genes alone.3 These results suggest a model whereby positional identity in fibroblasts is established during development and encoded in HOX genes; the Hox transcription factors then activate distinct target genes to give rise to topographic differentiation in fibroblasts. REFERENCES 1. Brown PO, Botstein D. Exploring the new world of the genome with DNA microarrays. Nat Genet 1999;21:33-7. 2. Dhouailly D. Specification of feather and scale patterns. In: Malincinski G, Bryant S, editors. Pattern formation. New York: Macmillan, 1984. p. 581-601. 3. Chang HY, Chi JT, Dudoix S, Bondre C, van de Rijn M, Botstein D, et al. Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci USA 2002;99:12877-82. 4. Krumlauf R. Hox genes in vertebrate development. Cell 1994;78: 191-201.
Production of human T cells from bone marrow progenitor cells using a skin-derived thymic construct Rachael A. Clark, MD, PhD, Harvard Skin Disease Research and Brigham and Women’s Department of Dermatology, Boston, Massachusetts Nominated by Thomas S. Kupper, MD, Harvard Skin Disease Research and Brigham and Women’s Department of Dermatology The development of T cells requires a period of specialized education in the thymus, an organ that largely undergoes involution during late adolescence. This requirement for thymic education has been a barrier to the laboratory production of T cells that can be given to immunocompromised patients
J AM ACAD DERMATOL MARCH 2004
to treat infection or malignancies. In vitro systems for T-cell development have thus far required the use of either human or animal thymus tissue, thereby preventing their use in adult human patients. Utilizing the natural similarities between skin and the thymus, we have developed a system in which cells from the skin are reprogrammed to act as a surrogate thymus, supporting the in vitro development of mature T cells from bone marrow precursor cells. Keratinocytes and fibroblasts from adult human skin were expanded separately in culture and then seeded onto a 3-dimensional tantalumcoated carbon framework. AC133⫹ (CD133⫹) human bone marrow precursor cells were then added, and the resultant construct was cultured for 3 to 4 weeks. Cells produced by the construct were analyzed by flow cytometry and T-cell recombination excision circles (TREC) analysis. Using this system, we have produced T cells that have surface markers consistent with mature, functional T cells. The cells were CD3hi, either CD8 or CD4 single-positive, and a significant proportion were CD45RA⫹, suggesting a naı¨ve phenotype. Input hematopoietic precursor cells were negative for CD3 and for TREC expression, where output cells were CD3⫹ and positive for TREC expression, indicating that these cells are newly developed T cells. Lymphocytes produced in this construct proliferated in response to phytohemagglutinin and produced tumor necrosis factor– g␣ and expressed CD69 in response to treatment with concanavalin A, suggesting that these cells are mature and functional. The ultimate goal of this project is to develop a method by which newly generated T cells, either naı¨ve or with engineered T-cell receptors, can be produced for the treatment of diseases of individual patients using autologous bone marrow and skin biopsies.