Human sensory neurons derived from induced pluripotent stem cells support varicella-zoster virus infection

Abstract

After primary infection, varicella-zoster virus (VZV) establishes latency in neurons of the dorsal root and trigeminal ganglia. Many questions concerning the mechanism of VZV pathogenesis remain unanswered, due in part to the strict host tropism and inconsistent availability of human tissue obtained from autopsies and abortions. The recent development of induced pluripotent stem (iPS) cells provides great potential for the study of many diseases. We previously generated human iPS cells from skin fibroblasts by introducing four reprogramming genes with non-integrating adenovirus. In this study, we developed a novel protocol to generate sensory neurons from iPS cells. Human iPS cells were exposed to small molecule inhibitors for 10 days, which efficiently converted pluripotent cells into neural progenitor cells (NPCs). The NPCs were then exposed for two weeks to growth factors required for their conversion to sensory neurons. The iPS cell-derived sensory neurons were characterized by immunocytochemistry, flow cytometry, RT-qPCR, and electrophysiology. After differentiation, approximately 80% of the total cell population expressed the neuron-specific protein, βIII-tubulin. Importantly, 15% of the total cell population co-expressed the markers Brn3a and peripherin, indicating that these cells are sensory neurons. These sensory neurons could be infected by both VZV and herpes simplex virus (HSV), a related alphaherpesvirus. Since limited neuronal populations are capable of supporting the entire VZV and HSV life cycles, our iPS-derived sensory neuron model may prove useful for studying alphaherpesvirus latency and reactivation.

Figure 1. Conversion of human iPS cells to neural progenitor cells.

(A) Outline of the differentiation protocol. Human iPS cells were dissociated and plated on Matrigel-coated plates. For the first 10 days, they were exposed to small molecule inhibitors, followed by culturing for two weeks in growth factors. (B) Brightfield images of iPS cells after 4 and 10 days of exposure to small molecule inhibitors. After 10 days, iPS cells expressed (C) Pax6 and (D) nestin, markers of neural progenitor cells. Nuclei were visualized with DAPI. Scale for all images is 100 um.

Figure 2. Differentiation of neural progenitor cells to sensory neurons.

(A) Brightfield images of neural progenitor cells on days 2, 8 and 14 in media containing growth factors. (B) Twenty-four days after initiating differentiation of iPS cells, neurons expressed the neuronal marker βIII-tubulin. Nuclei were visualized with DAPI. (C) Approximately 80% of differentiated iPS cells are βIII-tubulin+ as determined by flow cytometry. The red tracing represents unstained cells and the blue tracing represents cells stained with an antibody to βIII-tubulin. (D) Cells also expressed peripherin and Brn3a, markers of sensory neurons. (E) The percentage of peripherin+, Brn3a+, and peripherin+/Brn3a+ cells was determined by flow cytometry. Data shown are the mean ± SEM. Co-expression of Islet-1 with (F) Brn3a and (G) peripherin. Scale for all images is 100 um.

Figure 3. Brn3a expression is increased in differentiated iPS cells.

RT-qPCR of Brn3a and GAPDH mRNA was performed. (A) Gel electrophoresis of Brn3a and GAPDH expression in undifferentiated and differentiated iPS cells. The PCR products obtained after 34 cycles was run on a 3% agarose gel. (B) The relative Brn3a gene expression in undifferentiated and differentiated iPS cells (unpaired t test, p<0.0004). Data shown are mean ± SEM of three biological replicates.

Figure 4. Neurons derived from iPS cells support action potentials.

Whole-cell patch-clamp recordings were made in current-clamp mode from the soma of neurons. Shown above are representative traces recorded from one neuron that generated action potentials in response to depolarization. The stimulus protocol is depicted in lower traces. Membrane potentials were recorded from four neurons.

Figure 5. VZV infects iPS cell-derived neural progenitor cells and sensory neurons, but not undifferentiated iPS cells.

(A–C) Undifferentiated iPS cell cultures were infected with cell-free VZV at a MOI of 0.1 for 96 hours. iPS cells grow as colonies (dotted white circles) on a monolayer of non-dividing fibroblasts. (A) DAPI staining identifies the iPS cell colonies. (B) Immunostaining for IE62 revealed that only the monolayer of fibroblasts supported VZV infection. No IE62 staining could be observed in the iPS cell colonies. (C) Merge of panels A and B. (D–F) Neural progenitor cells infected with cell-free VZV at a MOI of 0.1 for 96 hours. Staining for (D) IE62 and (E) nestin. (F) Co-expression of IE62 and nestin indicated that neural progenitors cells can be infected by VZV (merge of panels D and E ). (G–O) Differentiated iPS cell cultures containing sensory neurons were infected with cell-free VZV at a MOI of 0.1 for 96 hours. Immunostaining for Brn3a (H, K), peripherin (N), IE62 (G) and gE (J, M) revealed that Brn3a+ cells co-expressed IE62 (I) and gE (L). Peripherin+ cells also co-expressed gE (O) and IE62 (data not shown). Collectively, these data show that sensory neurons can be infected by VZV. Scale for all images is 100 um.

Figure 6. HSV infects iPS cells at all stages of differentiation.

Cells were infected with cell-free HSV at a MOI of 0.1 for 96 hours. (A) DAPI staining identifies the iPS cell colonies (highlighted by the dotted white lines). (B) Immunostaining for gD revealed that both iPS cells and the underlying fibroblasts supported HSV infection, as evidenced by abundant gD expression. (C) Merge of panels A and B. (D–F) Sensory neurons derived from iPS cells also supported HSV infection. Staining for gD (D) and Brn3a (E). (F) Merge of panels D and E. Scale for all images is 100 um.