Transition toward Human Cytomegalovirus Susceptibility in Early Human Embryonic Stem Cell-Derived Neural Precursors

Abstract

Congenital human cytomegalovirus (HCMV) infection is associated with neurodevelopmental disabilities. To dissect the earliest events of infection in the developing human brain, we studied HCMV infection during controlled differentiation of human embryonic stem cells (hESC) into neural precursors. We traced a transition from viral restriction in hESC, mediated by a block in viral binding, toward HCMV susceptibility in early hESC-derived neural precursors. We further revealed the role of plateletderived growth factor receptor alpha (PDGFR␣) as a determinant of the developmentally acquired HCMV susceptibility. Human cytomegalovirus (HCMV) is a leading cause of congenital infection (1), associated with neurodevelopmental disabilities (2, 3). The ability of the virus to infect the developing fetal brain is a key factor in its neuropathogenesis (3)(4)(5)(6)(7)(8). While considerable experimental data were obtained from newborn and embryonic mouse models (6, 9-15), the strict species specificity precludes animal models of HCMV. Recent studies in human neuronal progenitor cells (NPC) derived from fetal and neonatal brains have revealed productive HCMV infection of NPC, with resultant functional alterations (16)(17)(18)(19)(20). Notably, NPC do not represent early neural embryonic development but rather a more advanced stage along the neuronal differentiation route. Hence, the earliest events defining the susceptibility of the developing human nervous system to HCMV have remained largely unknown.Human embryonic stem cells (hESC) provide an opportunity to study early human neural development (21-23). We have developed highly reproducible protocols for controlled induction of differentiation of hESC toward a neural lineage, giving rise to enriched populations of proliferating, developmentally multipotent early NPC (24,25).Here, we have employed experimental HCMV infection in a dynamic model of controlled differentiation of hESC into neural precursors, to gain insight into the molecular events mediating HCMV infection during early human neurodevelopment.We first sought to track the earliest stage of neural differentiation at which the cells become susceptible to HCMV. To this end, differentiation of hESC was induced in floating cell clusters toward the formation of neural spheres (Fig. 1A) as detailed previously (24, 25). The emerging neural spheres were infected at sequential time points along the neural differentiation process with the broadly tropic HCMV TB40/E strain expressing green fluorescent protein (GFP) (26,27) (Fig. 1B). In accordance with previous studies (25), immunostaining analysis for the pluripotent stem cell marker TRA 1-81 and the neural differentiation marker PSA-NCAM showed that the majority of the cells underwent early neural differentiation within 11 days (Fig. 1C). Importantly, we have identified a transition toward HCMV susceptibility occurring between 4 and 11 days post-differentiation induction (Fig. 1B). A similar susceptibility pattern was observed for low-passage-number, cell-associated, clinic...

Human Cytomegalovirus Compromises Development of Cerebral Organoids

“…Fortunately, large populations of almost pure cell types can be generated using either embryonic stem (ES) cell or induced pluripotent stem cell (iPSC) technologies. Interestingly, NPCs derived from 10-week gestation tissue were substantially more permissive to infection (30) than either ES-or iPS-derived NPCs (31)(32)(33)(34)(35)(36)(37)(38); also our unpublished results). This suggests that the more primitive stage of development of the ES-and iPS-derived cells influences their interactions with the virus.…”

“…All of the infection studies performed to date on undifferentiated ES or iPS cells (including ours described herein) find both cell types to be essentially nonpermissive for infection, as measured by de novo expression of immediate early (IE) antigens (Ags) (31,33,35,37). However, the susceptibility to infection of these cells is much less clear.…”

Pluripotent Stem Cell-Based Models: A Peephole into Virus Infections during Early Pregnancy

“…This notion of a gradual release of viral restriction blocks along neuronal differentiation was corroborated by the transition to an increased susceptibility to HCMV during neural differentiation of ESCs into neural spheres [103]. Susceptibility to HCMV appeared to be correlated to expression of the platelet-derived growth factor receptor alpha (PDGFRα) [103]. Brown et al showed correspondingly a lack of permissiveness of iPSCs to HCMV, but also identified iPSCs to be susceptible to HCMV infection through positive staining for the input tegument proteins [104].…”

“…Due to their similarity to cells in the developing neural tube, they are suggestive of the presence of a cellular harbor at a very primitive neuronal developmental stage for virus persistence along further neuronal differentiation stages [101]. This notion of a gradual release of viral restriction blocks along neuronal differentiation was corroborated by the transition to an increased susceptibility to HCMV during neural differentiation of ESCs into neural spheres [103]. Susceptibility to HCMV appeared to be correlated to expression of the platelet-derived growth factor receptor alpha (PDGFRα) [103].…”

Herpesviruses and Their Host Cells: A Successful Liaison

“…Progenitors of the myeloid lineage are hosts for latent HCMV, and differentiation to macrophages or dendritic cells results in reactivation of the virus. Recently, Berger et al investigated the transition from restriction of HCMV infection in human embryonic stem cells (hESC) toward susceptibility in hESC-derived neural precursors [36]. Using protocols for controlled induction of differentiation of hESC into neural precursors, they discovered PDGFR-/ as a determinant of HCMV susceptibility.…”