Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells

Abstract

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

“…Cells were reprogrammed with Sendai viral vectors expressing the transcription factors Sox2, c-Myc, Klf4, and Oct4. Following QC measures described earlier, 4 iPSCs were maintained in 6-well plates with the supplemented mTeSR TM 1 medium (Stem Cell Technologies) changed daily, allowing for manual cleaning of differentiated cells. The protocol was approved by the University of Pittsburgh Institutional Review Board and written informed consent was obtained from all participants.…”

“…A small fraction of neurons were GAD1 positive. 4 No dopaminergic neurons were observed following immunocytochemistry. A subpopulation of neurons stained for CALBINDIN (Fig.…”

“…Human embryonic stem (hES) cells and human iPSCs 1 have revolutionized such research, as they can be differentiated into neurons in vitro from specific individuals [2][3][4] , potentially enabling personalized medicine by overriding the problems of allogenic recognition. Compelling evidence now indicates that iPSC-based models can be used to model selected aspects of neurological and neurodegenerative disorders.…”

Human Cytomegalovirus Infection of Human Embryonic Stem Cell-Derived Primitive Neural Stem Cells Is Restricted at Several Steps but Leads to the Persistence of Viral DNA

“…Permissivity increases with extended passaging of the cells (9). Recently, rosette-type neural stem cells (R-NSCs) and neural progenitor cells (NPCs) generated in vitro from induced pluripotent stem cells (iPSCs) were also used as models to study HCMV rep-lication in neural cells, and similar results were obtained (10). One difficulty in interpreting the results of these studies is that the cell populations were poorly characterized, contained cells at different neural developmental stages, and could not be stably maintained in culture.…”

Peroxisome proliferator-activated receptor γ (PPARγ) activation: A key determinant of neuropathogeny during congenital infection by cytomegalovirus

“…12 Two other studies used neural stem cells generated from human induced pluripotent stem (iPS) cells and reported that HCMV infection impaired neuronal differentiation. 13,14 In order to perform molecular investigations on the outcomes of HCMV infection on neural progenitor cells, we used a new model, highly neuronogenic neural stem cells from embryonic stem cells (NSCs) (Fig. 1).…”