Endothelial progenitor cells (EPCs) and endothelial cells (ECs) have been applied in the clinic to treat pulmonary arterial hypertension (PAH), a disease characterized by disordered pulmonary vasculature. However, the lack of sufficient transplantable cells before the deterioration of disease condition is a current limitation to apply cell therapy in patients. It is necessary to differentiate pluripotent stem cells (PSCs) into EPCs and identify their characteristics.
Comparing previously reported methods of human PSCs-derived ECs, we optimized a highly efficient differentiation protocol to obtain cells that match the phenotype of isolated EPCs from healthy donors. The protocol is compatible with chemically defined medium (CDM), it could produce a large number of clinically applicable cells with low cost. Moreover, we also found PSCs-derived EPCs express CD133, have some characteristics of mesenchymal stem cells and are capable of homing to repair blood vessels in zebrafish xenograft assays. In addition, we further revealed that IPAH PSCs-derived EPCs have higher expression of proliferation-related genes and lower expression of immune-related genes than normal EPCs and PSCs-derived EPCs through microarray analysis.
In conclusion, we optimized a highly efficient differentiation protocol to obtain PSCs-derived EPCs with the phenotypic and molecular characteristics of EPCs from healthy donors which distinguished them from EPCs from PAH.

Plasmacytoid dendritic cells [pDCs] represent a rare innate immune subset uniquely endowed with the capacity to produce substantial amounts of type-I interferons. This function of pDCs is critical for effective antiviral defenses and has been implicated in autoimmunity. While IFN-I and select cytokines have been recognized as pDC secreted products, a comprehensive agnostic profiling of the pDC secretome in response to a physiologic stimulus has not been reported. We applied LC-MS/MS to catalogue the repertoire of proteins secreted by pDCs in the unperturbed condition and in response to challenge with influenza H1N1. We report the identification of a baseline pDC secretome, and the repertoire of virus-induced proteins including most type-I interferons, various cytokines, chemokines and granzyme B. Additionally, using single-cell RNA-seq [scRNA-seq], we perform multidimensional analyses of pDC transcriptional diversity immediately ex vivo and following stimulation. Our data evidence preexisting pDC heterogeneity, with subsequent highly specialized roles within the pDC population upon stimulation ranging from dedicated cytokine super-producers to cells with APC-like traits. Dynamic expression of transcription factors and surface markers characterize subclusters within activated pDCs. Integrating the proteomic and transcriptomic datasets confirms the pDC-subcluster origin of the proteins identified in the secretome. Our findings represent the most comprehensive molecular characterization of primary human pDCs at baseline, and in response to influenza virus, reported to date.
Copyright © 2022 Ghanem, Shih, Khalili, Werth, Chakrabarty, Brown, Simpfendorfer and Gregersen.

Endothelial progenitor cells (EPCs) and endothelial cells (ECs) have been applied in the clinic to treat pulmonary arterial hypertension (PAH), a disease characterized by disordered pulmonary vasculature. However, the lack of sufficient transplantable cells before the deterioration of disease condition is a current limitation to apply cell therapy in patients. It is necessary to differentiate pluripotent stem cells (PSCs) into EPCs and identify their characteristics. Comparing previously reported methods of human PSCs-derived ECs, we optimized a highly efficient differentiation protocol to obtain cells that match the phenotype of isolated EPCs from healthy donors. The protocol is compatible with chemically defined medium (CDM), it could produce a large number of clinically applicable cells with low cost. Moreover, we also found PSCs-derived EPCs express CD133, have some characteristics of mesenchymal stem cells and are capable of homing to repair blood vessels in zebrafish xenograft assays. In addition, we further revealed that IPAH PSCs-derived EPCs have higher expression of proliferation-related genes and lower expression of immune-related genes than normal EPCs and PSCs-derived EPCs through microarray analysis. In conclusion, we optimized a highly efficient differentiation protocol to obtain PSCs-derived EPCs with the phenotypic and molecular characteristics of EPCs from healthy donors which distinguished them from EPCs from PAH.

h4>Aims: /h4> Endothelial cells (ECs) have been applied in clinic to treat pulmonary arterial hypertension (PAH), a disease characterized by disordered pulmonary vasculature. However, lack of enough transplantable cells before the deterioration of patient condition is current limitation to apply cell therapy in cardiovascular diseases. So, we thought it necessary to continue to differentiate embryo stem cells (ESCs)/induced-pluripotent stem cells (iPSCs) into endothelial cells (ECs) and identify their characteristics. Methods and results Comparing previous reported methods of human pluripotent cell differentiation toward vascular cells/ Hemogenic Endothelial cells/ endothelial colony-forming cells, we established a highly efficient differentiation protocol to get cells that match phenotype of isolated ECs from health donors. This protocol, including two stages, early mesoderm endothelial progenitor stage and EC marker expression stage. In the first stage, Rock inhibitor Y27632 and DMSO plays an important role in inducing-APLNR + mesoderm and promotes EC differentiation potential; later on SB431542 and BMP4 drives cells toward EC lineage. Meanwhile, an improved protocol with chemically-defined medium (CDM) has similar differentiation efficiency,again demonstrating that a large number of clinically needed cells could be obtained with simple factors. ESC/iPSC-ECs, normal EPCs and IPAH-EPCs have the characteristics of early EPCs marked by CD133 and mesenchymal stem cells. Microarray analysis further revealed IPAH-derived EPCs features of rapid proliferation and weak immune regulation. At last, a model Zebrafish xenograft was utilized to assess the functionality of differentiated ESC/iPSC-ECs. ConclusionsWe established a highly efficient differentiation protocol to get ESC/iPSC-ECs with characteristics of phenotype and molecular matched with early-EPCs from health donors, and revealed the molecular pathogenesis in PAH.

To reduce the use of non-specific immunosuppressive drugs detrimental to transplant patient health, therapies in development aim to achieve antigen-specific tolerance by promoting antigen-specific regulatory T cells (Tregs). However, identification of the natural antigens recognized by Tregs and the contribution of their dominance in transplantation has been challenging. We identify epitopes derived from distinct major histocompatibility complex (MHC) class II molecules, sharing a 7-amino acid consensus sequence positioned in a central mobile section in complex with MHC class I, recognized by cross-reactive CD8+ Tregs, enriched in the graft. Antigen-specific CD8+ Tregs can be induced in vivo with a 16-amino acid-long peptide to trigger transplant tolerance. Peptides derived from human HLA class II molecules, harboring the rat consensus sequence, also activate and expand human CD8+ Tregs, suggesting its potential in human transplantation. Altogether, this work should facilitate the development of therapies with peptide epitopes for transplantation and improve our understanding of CD8+ Treg recognition.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.