We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency – as seen with the loss of markers OCT3/4 and TRA-1-81 – and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.
Plasmacytoid dendritic cells (pDC) are essential for immune competence. Here we show that pDC precursor differentiated from human CD34+ hematopoietic stem and progenitor cells (HSPC) has low surface expression of pDC markers, and has limited induction of type I interferon (IFN) and IL-6 upon TLR7 and TLR9 agonists treatment; by contrast, cGAS or RIG-I agonists-mediated activation is not altered. Importantly, after priming with type I and II IFN, these precursor pDCs attain a phenotype and functional activity similar to that of peripheral blood-derived pDCs. Data from CRISPR/Cas9-mediated genome editing of HSPCs further show that HSPC-pDCs with genetic modifications can be obtained, and that expression of the IFN-α receptor is essential for the optimal function, but dispensable for the differentiation, of HSPC-pDC percursor. Our results thus demonstrate the biological effects of IFNs for regulating pDC function, and provide the means of generating of gene-modified human pDCs.
TLR3 and TLR9 recognize the pathogen-associated microbial patterns dsRNA and unmethylated DNA, respectively. The recent discovery that these receptors also recognize endogenous ligands from necrotic material has drawn increased attention to their involvement in autoimmunity. Th cell cytokines IL-17A and IL-21 have been assigned with pivotal roles in the regulation of such autoimmune diseases. IL-17A is the hallmark cytokine of the recently discovered proinflammatory Th cell subset TH17. By contrast, the expression of IL-21 does not seem to be limited to a single distinct Th cell subset. We investigated the expression of IL-17A and IL-21 in human CD4+ T cells in response to stimulation with the TLR3 ligand polyinosinic:polycytidylic acid (poly(I:C)) and the TLR9 ligand CpG. We discovered that poly(I:C) induced synthesis of both IL-17A and IL-21. Moreover, we found that poly(I:C) was able to drive the differentiation of naive Th cells into an IL-21 but not into an IL-17A-producing phenotype and did this without affecting the levels of transcription factors T-bet, GATA-3, or retinoic acid receptor-related orphan receptor C. Finally, we found that the IL-21-producing cells that were differentiated in response to poly(I:C) expressed the chemokine receptor CXCR3, which is important in the recruitment of T cells into inflamed joints in rheumatoid arthritis. This is the first report to show that the TLR3 ligand poly(I:C) can directly induce the synthesis of IL-17A and IL-21 and drive differentiation of human naive CD4+ T cells.
Our genes are post-transcriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNA Pol II)-transcribed tough decoy (TuD) miRNA inhibitors are particularly attractive. Here, we demonstrate augmented miRNA suppression capacity of TuD RNA hairpins linked to the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). This effect is position-dependent and evident only when the WPRE is positioned upstream of the TuD. In accordance, inclusion of the WPRE does not change nuclear export, translation, total levels of TuD-containing RNA transcripts, or cytoplasmic P-body localization, suggesting that previously reported WPRE functions are negligible for improved TuD function. Notably, deletion analysis of TuD-fused WPRE unveils truncated WPRE variants resulting in optimized miRNA suppression. Together, our findings add to the guidelines for production of WPRE-supported anti-miRNA TuDs.
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