Gene Regulation of Stem Cell Maintenance and Differentiation
My lab leverages human skin as the primary research platform. Skin is the biggest organ of our body. The outmost skin tissue, known as epidermis, continuously regenerates owning to the actions of adult stem cells to both proliferate and differentiate. These epidermal stem cells can be isolated from surgically discarded patient skin, and be cultured ex vivo under both undifferentiated and differentiation conditions.
For functional genetic analysis, multiple genes can be altered simultaneously in organotypically regenerated epidermal tissue within as short as one week, or be grafted onto immune-deficient mice for in vivo studies. This platform thus allows rapid functional analysis in intact three-dimensional human tissue.
Primary human epidermal cells can be expanded in large quantities to allow mechanistic investigation using genomic and proteomic approaches, providing opportunities to uncovering novel gene regulatory mechanisms.
Multi-Functional Genetics in Human Tissue: A New Experimental Paradigm
Multi-Functional Genetics in Human Tissue (MFG-H) refers to the ability to alter the function of multiple genes simultaneously within 3-dimensionally intact human tissue. This new platform has been developing in the lab to enable studies on epithelial stem cell biology, cancer and therapeutics. Because of its versatility, MFG-H represents a new experimental platform designed to advance mechanistic biological studies to a degree similar to that which occurred with the prior advent of genetic models in yeast, Drosophila, and C. elegans. Because of its relevance to human disease, MFG-H also offers the potential for dramatically faster and more accurate translation of experimental findings to the treatment of human disease.
MFG-H arose to address several key problems facing current biomedical research.
The questionable relevance of many experimental model systems to human biology and disease: A recurrent theme in biomedicine over the past 3 decades has been the failure of findings in conventional model systems to translate into clinically useful progress in the treatment of human disease. Central to this failure are the major limitations intrinsic in current experimental systems. For example, most human cancer studies rely on studying transformed cell lines or purely murine models. However, most cells lines are uniquely adapted to long-term cell culture, have undergone genomic catastrophe and are aneuploid, rendering each cancer cell line unique in terms of genomic structure, gene regulation, signaling and biological behavior. Often, different cell lines of the same tissue origin provide directly contradictory results. This has made extrapolation of data with cell lines to general biologic functional relationships and clinical meaningfulness doubtful. Purely murine studies using transgenic and knockout mice, while extremely powerful, also suffer from substantial different limitations, including the fact that murine and human tissue differ in a host of ways, including oncoprotein signaling targets, carcinogen function and susceptibility to malignant transformation.
The need to perform higher complexity genetic experiments that can alter function of multiple genes and gene networks simultaneously within intact tissue. Development of the capability to assay expression of numerous genes simultaneously using microarray transcript profiling underscored the need for higher complexity experiments that could alter the function of multiple gene regulatory networks simultaneously. Such experimental ability would allow more rapid definition of epistatic relationships among genes in a pathway as well as hitherto unapproachable identification of interaction between multiple distinct regulatory pathways. It also offered to ability to reconstruct the signaling network alterations sufficient to drive human cells into cancer or to maintain them in the undifferentiated stem cell state. In mice, altering function of 4 to 6 alleles represents a practical upper limit but generating such multi-allele mice can take many years and often fails due to a variety of technical limitations. The development of multiplex serial gene transfer (MSGT) in the lab using high efficiency retroviral gene transfer into primary human cells laid the foundation for complex genetic experiments in which the function of previously unattainable numbers of alleles could be simultaneously altered within intact tissue. The combination of new viral and nonviral gene transfer capabilities with human tissue regeneration approaches provided the technological foundation for MFG-H.
MFG-H: New models
MFG-H has been developed using 2 major experimental platforms, human skin tissue regenerated in vivo on immune deficient mice and organotypic tissue generated wholly in vitro using human epithelial and mesenchymal cells within an intact dermal stromal architecture. In vivo genetically modified human tissue regenerated in the context of full-thickness human skin on immune deficient mice offers a potent new model for the study of human stem cell biology, cancer and molecular therapeutics. The in vivo human tissue model displays unique strengths that include: Capability to simultaneously alter function of 10 or more alleles genetically within architecturally faithful human tissue, providing “Multi-Functional Genetics” capabilities as well as the ability to alter the function of multiple additional proteins by antibody targeting
Experimentation within the context of multiple non-transformed human cell lineages, including epithelial and mesenchymal cells, as well as 3-dimensionally intact human extracellular matrix. Study of endogenous human genes and proteins, which represent the actual molecular targets for potential human clinical application
Capacity for long-term experiments in that human tissue can be followed out for over 1 year, a timepoint that permits rigorous assessment of stem cell function and tissue self-renewal.
Faithful recapitulation of features of intact human skin
Ability to recapitulate skin-intrinsic human genetic skin disease; grafting tissue from the >100 monogenic human skin diseases can directly generate a human tissue model of the disease in question, opening the way for analysis of new therapeutics.
Ability to generate human tissue mosaics to distinguish cell autonomous from non-cell autonomous mechanisms in stem cell biology and cancer
Expression of conditional and constitutively active or dominant negative alleles as well as shRNAs via high efficiency retroviruses.
Organotypic tissue in culture is a complementary MFG-H platform, which uses intact human mesenchymal dermis, mesenchymal fibroblasts and overlying stratified epithelial cells. This forms skin tissue veridical to skin on humans at levels of gene expression, morphology and tissue architecture. Although lacking the durability and link to systemic circulation and non-amamnestic inflammatory cells that characterize the in vivo model, the organotypic model incorporates many of its advantages and also has unique strengths that include: 1) Experimental speed; organotypic experiments commonly take 7 to 10 days, a time period that is now also reachable with the new in vivo rapid grafting model developed in the lab.; 2) Freedom from animal use; 3) Accessibility to pharmacologic agents that often cannot be used in animal models due to cost, systemic toxicity and drug absorption, distribution, metabolism and dosing; 4) Accessibility to antibody agents for inhibitor and activations studies that can use far smaller amounts than would be needed for animal studies; 5) Ability to rapidly alter function of a larger number of target genes via RNAi, CRISPR, antibodies, drugs, and gene delivery; 4) Tractability to the use of knock down or knock out reagents.
MFG-H offers a scope for experimental genetic complexity and human clinical relevance not previously available in prior models. Just as early research groups that pioneered use of powerful genetic models in yeast, Drosophila, C. elegans and zebrafish prepared cohorts of young scientists to start new laboratories using these paradigm-changing genetic models, so MFG-H is providing a novel and powerful experimental paradigm for use by current graduate students and postdoctoral fellows in their future independent research careers.