1 - Towards regenerative medicine and human therapeutics using human induced Pluripotent Stem Cells (iPSC) cells – Our goal is to develop efficient ways to derive functional cell types form induced pluripotent stem cells to generate tissues that demonstrate a broad range of biological functions that can be used to replace or regenerate human cells and tissue to restore normal function. We have shown that iPSC can differentiate towards fibroblast lineage fates (Figure 1) and that demonstrate functions similar to adult-derived fibroblasts within 3D tissues that mimic the properties of human skin. We are currently studying how epigenetic regulation acts to control iPSC of differentiation and have developed 3D tissues harboring these cells that can predict how these iPSC-derived cells may behave in in vivo after future transplantation.
Figure 1 - ESC and iPSCs differentiation to to fibroblast fate. ESC and iPSC were differentiated and monitored for cell morphology at various stages of differentiation. Representative images show the morphology of ESC and 2 iPSC lines after days 1, 4, 7, 10, 14, 21 and 28 of differentiation. Early morphologic changes were analogous, with differentiation beginning at the periphery of colonies (day 1). At later stages cells acquired features of elongated, stellate cells (day 10) similar to fibroblasts that became the predominant cell type.
2 - Tissue Engineering Meets iPSC-Derived Cells: From “Disease in a Dish” to “Disease in a Tissue” – We have developed 3D skin-like tissues that mimic the form and function of human skin. By growing these 3D, human skin equivalents (HSE) at an air-liquid interface on a connective tissue harboring fibroblasts, we engineer fully differentiated, skin-like tissues displaying normal tissue architecture (Figure 2). We use iPSC that have been reprogrammed directly from patient tissues to study disease phenotypes upon their differentiation from iPSC back to fibroblasts. We incorporate these iPSC-derived fibroblasts into 3D skin-like tissues models to study their possible role in disease pathogenesis and to assess their biological potential. By incorporating iPSC- or ESC-derived fibroblasts into skin, we can better understand how these cells integrate in these complex tissue microenvironments in order to better predict how they may behave after in vivo transplantation.
Figure 2 - Schematic of changes in DNA methylation following differentiation. In the pluripotent state, lineage-specific promoters are generally in a methylated, repressed state, while genes needed to maintain the pluripotent state, such as OCT4, are demethylated and expressed (left). Following differentiation, lineage-specific genes are demethylated and expressed, while, pluripotency genes and genes associated with alternative lineages are methylated and repressed (right. Methylation is marked by dark circles, and unmethylated is marked by open circles. Arrows indicate active transcription, and blocks indicate repression.
3 - Studying Epigenetic Signatures Linked to Chronic, Diabetic Ulcers: Can iPSC reprogramming “rejuvenate” cell phenotypes to improve wound repair? – We have shown that the step-wise differentiation from pluripotency to mature fibroblasts is accompanied by an improved biological potency that suggests that normal skin fibroblasts can augment their function through reprogramming. We are now studying if acquisition of an improved biological potential can be used to treat disease conditions. We are studying if reprogramming of fibroblasts from chronic diabetic ulcers and their subsequent differentiation to fibroblasts can improve their ability to heal these wounds. We are also studying the epigenetic mechanisms that control this biological potential to improve the therapy of these chronic disease states. For example, chronic wounds do not undergo proper healing that may be linked to methylation changes that cause the aberrant expression of genes needed for wound repair. Since DNA methylation marks are reset following reprogramming, it may be possible to modify epigenetic control of genes linked to a “repair-deficient” phenotype in wound-derived fibroblasts that can be reverted following reprogramming to “normalize” their cellular phenotype upon subsequent differentiation to repair-competent fibroblasts (Fig. 3).
Figure 3 - Schematic model for reversing changes in DNA methylation due to chronic wound environment. Somatic fibroblasts have a characteristic methylation signature that consists gene promoters that are methylated and repressed (e.g. OCT-4), as well as other promoters that are unmethylated and expressed (e.g. PDGFRβ). Following reprogramming, these methylation marks are reversed, and upon differentiation they are re-established to resemble the original somatic fibroblasts. In a diseased state there are changes in methylation that allow expression of repressed genes and repress genes that should be expressed, resulting in aberrant phenotypes. Upon reprogramming, methylation is “reset” to allow for differentiation to fibroblasts with a normalized epigenetic signature.