Bachman, Lori A.

Abstract Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.g., laminin) for expansion in adherent cell culture for use in cell therapy. To address this, we investigated the potential of culturing iPSCs on fibrin hydrogels for three-dimensional applications and further examined the use of fibrinogen, the soluble precursor protein, as a coating substrate for traditional adherent cell culture. iPSCs successfully adhered to and proliferated on fibrin hydrogels. The two-dimensional culture with fibrinogen allows for immediate adaption of culture models to a nonxenogeneic model. Similarly, multiple commercially available iPSC lines adhered to and proliferated on fibrinogen coated surfaces. iPSCs cultured on fibrinogen expressed similar levels of the pluripotent stem cell markers SSea4 (98.7% ± 1.8%), Oct3/4 (97.3% ± 3.8%), TRA1-60 (92.2% ± 5.3%), and NANOG (96.0% ± 3.9%) compared with iPSCs on Geltrex. Using a trilineage differentiation assay, we found no difference in the ability of iPSCs grown on fibrinogen or Geltrex to differentiate to endoderm, mesoderm, or ectoderm. Finally, we demonstrated the ability to differentiate iPSCs to endothelial cells using only fibrinogen coated plates. On the basis of these data, we conclude that human fibrinogen provides a readily available and inexpensive alternative to laminin-based products for the growth, expansion, and differentiation of iPSCs for use in research and clinical cell therapy applications. Stem Cells Translational Medicine 2019;8:512–521

The NF-κB component RelB is essential for dendritic cell (DC) differentiation and maturation. The vitamin D receptor (VDR) is a nuclear receptor that mediates inhibition of DC maturation and transcriptional repression of relB after engagement of its ligand, 1α,25-dihydroxyvitamin D 3 , or related analogs (D 3 analogs). Ligand-dependent relB suppression was abolished by a histone deacetylase (HDAC) inhibitor. Constitutive association of VDR with the relB promoter was demonstrated in DCs by chromatin immunoprecipitation. Promoter binding by VDR was enhanced by ligand and reduced by LPS. Association of HDAC3 and HDAC1 with the relB VDR-binding site was observed, but only HDAC3 was reciprocally modulated by D 3 analog and LPS. Overexpression of HDAC3 caused relB promoter suppression, increased sensitivity to D 3 analog, and resistance to LPS. Depletion of HDAC3 attenuated relB suppression by D 3 analog. In vivo , D 3 analog resulted in reduced RelB in DCs from VDR WT mice but not VDR knockout mice. Other NF-lation of RelB and c-Rel in control animals. We conclude that vitamin D-regulated relB transcription in DCs is controlled by chromatin remodeling by means of recruitment of complexes including HDAC3.

Dendritic cells (DCs) play a central role in regulating immune activation and responses to self. DC maturation is central to the outcome of antigen presentation to T cells. Maturation of DCs is inhibited by physiological levels of 1α,25 dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] and a related analog, 1α,25(OH) 2 -16-ene-23-yne-26,27-hexafluoro-19-nor-vitamin D 3 (D 3 analog). Conditioning of bone marrow cultures with 10 −10 M D 3 analog resulted in accumulation of immature DCs with reduced IL-12 secretion and without induction of transforming growth factor β1. These DCs retained an immature phenotype after withdrawal of D 3 analog and exhibited blunted responses to maturing stimuli (CD40 ligation, macrophage products, or lipopolysaccharide). Resistance to maturation depended on the presence of the 1α,25(OH) 2 D 3 receptor (VDR). In an in vivo model of DC-mediated antigen-specific sensitization, D 3 analog-conditioned DCs failed to sensitize and, instead, promoted prolonged survival of subsequent skin grafts expressing the same antigen. To investigate the physiologic significance of 1α,25(OH) 2 D 3 /VDR-mediated modulation of DC maturity we analyzed DC populations from mice lacking VDR. Compared with wild-type animals, VDR-deficient mice had hypertrophy of subcutaneous lymph nodes and an increase in mature DCs in lymph nodes but not spleen. We conclude that 1α,25(OH) 2 D 3 /VDR mediates physiologically relevant inhibition of DC maturity that is resistant to maturational stimuli and modulates antigen-specific immune responses in vivo .