Isolation of Diverse Simian Arteriviruses Causing Hemorrhagic Disease.

Genetically diverse simian arteriviruses (simarteriviruses) naturally infect geographically and phylogenetically diverse monkeys, and cross-species transmission and emergence are of considerable concern. Characterization of most simarteriviruses beyond sequence analysis has not been possible because the viruses fail to propagate in the laboratory. We attempted to isolate 4 simarteriviruses, Kibale red colobus virus 1, Pebjah virus, simian hemorrhagic fever virus, and Southwest baboon virus 1, by inoculating an immortalized grivet cell line (known to replicate simian hemorrhagic fever virus), primary macaque cells, macrophages derived from macaque induced pluripotent stem cells, and mice engrafted with macaque CD34+-enriched hematopoietic stem cells. The combined effort resulted in successful virus isolation; however, no single approach was successful for all 4 simarteriviruses. We describe several approaches that might be used to isolate additional simarteriviruses for phenotypic characterization. Our results will expedite laboratory studies of simarteriviruses to elucidate virus-host interactions, assess zoonotic risk, and develop medical countermeasures.

SOX18-enforced expression diverts hemogenic endothelium-derived progenitors from T towards NK lymphoid pathways.

Hemogenic endothelium (HE) is the main source of blood cells in the embryo. To improve blood manufacturing from human pluripotent stem cells (hPSCs), it is essential to define the molecular determinants that enhance HE specification and promote development of the desired blood lineage from HE. Here, using SOX18-inducible hPSCs, we revealed that SOX18 forced expression at the mesodermal stage, in contrast to its homolog SOX17, has minimal effects on arterial specification of HE, expression of HOXA genes and lymphoid differentiation. However, forced expression of SOX18 in HE during endothelial-to-hematopoietic transition (EHT) greatly increases NK versus T cell lineage commitment of hematopoietic progenitors (HPs) arising from HE predominantly expanding CD34+CD43+CD235a/CD41a-CD45- multipotent HPs and altering the expression of genes related to T cell and Toll-like receptor signaling. These studies improve our understanding of lymphoid cell specification during EHT and provide a new tool for enhancing NK cell production from hPSCs for immunotherapies.

Generation of SIV-resistant T cells and macrophages from nonhuman primate induced pluripotent stem cells with edited CCR5 locus.

Adoptive therapies with genetically modified somatic T cells rendered HIV resistance have shown promise for AIDS therapy. A renewable source of HIV-resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian cynomolgus macaques (MCM), using CRISPR-Cas9 technology. We found that CCR5 editing does not affect hematopoietic and T cell differentiation potentials of fib-iPSCs. However, T-iPSCs with edited CCR5 lost their capacity to differentiate into CD4+CD8+ T cells while maintaining myeloid differentiation potential. T cells and macrophages produced from CCR5-edited MCM iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T cell tropic) and SIVmac316 (macrophage-tropic). Overall, these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in a nonhuman primate model.