Directed Differentiation of Human Induced Pluripotent Stem Cells into Dendritic Cells Displaying Tolerogenic Properties and Resembling the CD141+ Subset.

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.

Rapamycin conditioning of dendritic cells differentiated from human ES cells promotes a tolerogenic phenotype.

While human embryonic stem cells (hESCs) may one day facilitate the treatment of degenerative diseases requiring cell replacement therapy, the success of regenerative medicine is predicated on overcoming the rejection of replacement tissues. Given the role played by dendritic cells (DCs) in the establishment of immunological tolerance, we have proposed that DC, rendered tolerogenic during their differentiation from hESC, might predispose recipients to accept replacement tissues. As a first step towards this goal, we demonstrate that DC differentiated from H1 hESCs (H1-DCs) are particularly responsive to the immunosuppressive agent rapamycin compared to monocyte-derived DC (moDC). While rapamycin had only modest impact on the phenotype and function of moDC, H1-DC failed to upregulate CD40 upon maturation and displayed reduced immunostimulatory capacity. Furthermore, coculture of naïve allogeneic T cells with rapamycin-treated H1-DC promoted an increased appearance of CD25(hi) Foxp3+ regulatory T cells, compared to moDC. Our findings suggest that conditioning of hESC-derived DC with rapamycin favours a tolerogenic phenotype.

Pharmacological manipulation of dendritic cells in the pursuit of transplantation tolerance.

PURPOSE OF REVIEW: Although long-term immune suppression remains the intervention of choice for the treatment of allograft rejection, transplantation tolerance would achieve graft survival with fewer inherent risks. Although the use of dendritic cells for the induction of tolerance might confer antigen specificity, factors determining the balance between tolerogenicity and immunogenicity remain uncertain, as does the stability of the functional phenotype. Here, we review recent studies suggesting that pharmacological agents may profoundly influence this delicate balance and outline the insights they provide into parameters that contribute to the tolerogenic state. RECENT FINDINGS: Recent findings have revealed that the inhibition of dendritic cell maturation by pharmacological intervention is not a prerequisite for the acquisition of tolerogenicity, but that susceptibility to a tolerogenic phenotype may vary between dendritic cell subsets and depend on the nature of maturation stimuli to which the cells are exposed. Furthermore, such studies have highlighted the degree to which the maintenance of tolerogenicity is influenced by local environmental factors, such as the cytokine milieu. SUMMARY: Although the rational design of tolerogenic dendritic cells for modulating the outcome of organ transplantation remains ambitious, the use of pharmacological agents to influence their functional phenotype continues to illuminate the basic biology of this critical cell type.