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The advent of induced pluripotent stem cells (iPSCs) has begun to revolutionize cell therapy by providing a convenient source of rare cell types not normally available from patients in sufficient numbers for therapeutic purposes. In particular, the development of protocols for the differentiation of populations of leukocytes as diverse as naïve T cells, macrophages, and natural killer cells provides opportunities for their scale-up and quality control prior to administration. One population of leukocytes whose therapeutic potential has yet to be explored is the subset of conventional dendritic cells (DCs) defined by their surface expression of CD141. While these cells stimulate cytotoxic T cells in response to inflammation through the cross-presentation of viral and tumor-associated antigens in an MHC class I-restricted manner, under steady-state conditions CD141+ DCs resident in interstitial tissues are focused on the maintenance of homeostasis through the induction of tolerance to local antigens. Here, we describe protocols for the directed differentiation of human iPSCs into a mixed population of CD11c+ DCs through the spontaneous formation of embryoid bodies and exposure to a cocktail of growth factors, the scheduled withdrawal of which serves to guide the process of differentiation. Furthermore, we describe the enrichment of DCs expressing CD141 through depletion of CD1c+ cells, thereby obtaining a population of "untouched" DCs unaffected by cross-linking of surface CD141. The resulting cells display characteristic phagocytic and endocytic capacity and acquire an immunostimulatory phenotype following exposure to inflammatory cytokines and toll-like receptor agonists. Nevertheless, under steady-state conditions, these cells share some of the tolerogenic properties of tissue-resident CD141+ DCs, which may be further reinforced by exposure to a range of pharmacological agents including interleukin-10, rapamycin, dexamethasone, and 1α,25-dihydoxyvitamin D3. Our protocols therefore provide access to a novel source of DCs analogous to the CD141+ subset under steady-state conditions in vivo and may, therefore, find utility in the treatment of a range of disease states requiring the establishment of immunological tolerance. [ABSTRACT FROM AUTHOR]

In lung tissue, dendritic cells (DC) are found in close association with the epithelial cell layer, and there is evidence of DC regulation by the epithelium; that epithelial dysfunction leads to overzealous immune cell activation. However, dissecting basic mechanisms of DC interactions with epithelial cells in human tissue is difficult. Here, we describe a method to generate a three-dimensional organotypic model of the human airway mucosa in which we have implanted human DC. The model recapitulates key anatomical and functional features of lung mucosal tissue, including a stratified epithelial cell layer, deposition of extracellular matrix proteins, and the production of tight junction and adherence junction proteins. Labeling of fixed tissue model sections and imaging of live tissue models also revealed that DC distribute in close association with the epithelial layer. As functional properties of DC may be affected by the local tissue microenvironment, this system provides a tool to study human DC function associated with lung mucosal tissue. As an example, we report that the lung tissue model regulates the capacity of DC to produce the chemokines CCL17, CCL18, and CCL22, leading to enhanced CCL18 expression and reduced CCL17 and CCL22 expression. This novel tissue model thus provides a tool well suited for a wide range of studies, including those on the regulation of DC functional properties within the local tissue microenvironment during homeostasis and inflammatory reactions. [ABSTRACT FROM AUTHOR]

The advent of induced pluripotent stem cells (iPSCs) has begun to revolutionize cell therapy by providing a convenient source of rare cell types not normally available from patients in sufficient numbers for therapeutic purposes. In particular, the development of protocols for the differentiation of populations of leukocytes as diverse as naïve T cells, macrophages, and natural killer cells provides opportunities for their scale-up and quality control prior to administration. One population of leukocytes whose therapeutic potential has yet to be explored is the subset of conventional dendritic cells (DCs) defined by their surface expression of CD141. While these cells stimulate cytotoxic T cells in response to inflammation through the cross-presentation of viral and tumor-associated antigens in an MHC class I-restricted manner, under steady-state conditions CD141 + DCs resident in interstitial tissues are focused on the maintenance of homeostasis through the induction of tolerance to local antigens. Here, we describe protocols for the directed differentiation of human iPSCs into a mixed population of CD11c + DCs through the spontaneous formation of embryoid bodies and exposure to a cocktail of growth factors, the scheduled withdrawal of which serves to guide the process of differentiation. Furthermore, we describe the enrichment of DCs expressing CD141 through depletion of CD1c + cells, thereby obtaining a population of "untouched" DCs unaffected by cross-linking of surface CD141. The resulting cells display characteristic phagocytic and endocytic capacity and acquire an immunostimulatory phenotype following exposure to inflammatory cytokines and toll-like receptor agonists. Nevertheless, under steady-state conditions, these cells share some of the tolerogenic properties of tissue-resident CD141 + DCs, which may be further reinforced by exposure to a range of pharmacological agents including interleukin-10, rapamycin, dexamethasone, and 1α,25-dihydoxyvitamin D 3 . Our protocols therefore provide access to a novel source of DCs analogous to the CD141 + subset under steady-state conditions in vivo and may, therefore, find utility in the treatment of a range of disease states requiring the establishment of immunological tolerance.

In lung tissue, dendritic cells (DC) are found in close association with the epithelial cell layer, and there is evidence of DC regulation by the epithelium; that epithelial dysfunction leads to overzealous immune cell activation. However, dissecting basic mechanisms of DC interactions with epithelial cells in human tissue is difficult. Here, we describe a method to generate a three-dimensional organotypic model of the human airway mucosa in which we have implanted human DC. The model recapitulates key anatomical and functional features of lung mucosal tissue, including a stratified epithelial cell layer, deposition of extracellular matrix proteins, and the production of tight junction and adherence junction proteins. Labeling of fixed tissue model sections and imaging of live tissue models also revealed that DC distribute in close association with the epithelial layer. As functional properties of DC may be affected by the local tissue microenvironment, this system provides a tool to study human DC function associated with lung mucosal tissue. As an example, we report that the lung tissue model regulates the capacity of DC to produce the chemokines CCL17, CCL18, and CCL22, leading to enhanced CCL18 expression and reduced CCL17 and CCL22 expression. This novel tissue model thus provides a tool well suited for a wide range of studies, including those on the regulation of DC functional properties within the local tissue microenvironment during homeostasis and inflammatory reactions.