Figure 1.
NHNP TLAs are genetically stable for at least 180 days and form tissue like assemblies.
(A) mFISH analysis of passage 1 NHNP cells (left panel) and (B) NHNP TLAs after 180 days in culture (right panel). Representative metaphase chromosomes labeled with Spectra Vysion Tm probes are shown. (C–F) Environmental scanning electron micrographs with 150× (C, D), 650× (E) and 2000× (F) magnifications illustrate the relative size, density, and complexity of TLAs. Scale bars are shown in each image.
Figure 2.
Expression of neuronal markers in NHNP TLAs and human trigeminal ganglia (TG).
(A) The neuronal markers Neuron-Specific Nuclear Protein (NeuN), Microtubule Associated Protein A&B (MAP2 A/B), β-Tubulin-III and Glial Fibrillary Acidic Protein (GFAP) were detected in NHNP TLAs by confocal microscopy (green). Nuclei were visualized using Alexa Fluor 488 (red). Representative images are shown at 400× magnification. (B) Expression of Nestin and β-Tubulin-III in human TGs at indicated magnification. (C) Expression levels of CXCR4, CD133, Nestin, CD105, CD90, CD49f and β-Tubulin-III in three primary TGs (white bars) obtained from two donors or NHNP TLAs (black bars). Data is shown as mean mRNA copies relative to GAPDH with standard deviations (error bars).
Table 1.
Marker protein expression as measured by flow cytometry demonstrates consistent expression of early progenitor and neuronal cell markers during the course of the 180 day experiment.
Figure 3.
Generation of fluorescently labeled VZV P-Oka facilitates analysis of ORF63 and ORF70 expression.
(A) Schematic representation of the VZV genome with a focus on the regions containing ORF63 and ORF70. Insertion of eGFP (green arrow) and mRFP (red arrow) at the C-terminus of ORF63 and ORF70 are indicated respectively. (B) Restriction fragment length polymorphism (RFLP) analysis of parental pP-Oka (lane 1 and 3) and p63G/70R (lane 2 and 4) BAC DNA digested with indicated enzymes. Green and red asterisks indicate fragments containing ORF63 and ORF70 respectively that increase in size upon insertion of eGFP and mRFP. (C) Florescence microscopy images of VZV plaques detecting ORF63-eGFP and ORF70-mRFP expression after BAC DNA transfections (upper panel) and upon passaging of the virus (middle and lower panel).
Figure 4.
v63G/70R replicates comparable to parental virus in vitro.
(A) Replication kinetics of parental P-Oka (rOKA) and v63G/70R in MeWo cells. Data is shown as mean viral titers with standard deviations (error bars). (B) Replication of v63G/70R in NHNP TLAs. NHNP TLAs were infected with v63G/70R in triplicate, samples removed at the indicated time points and analyzed by qPCR. Data is shown as mean viral genome copies with standard deviations (error bars). (C) Daily glucose utilization of MeWo cells and NHNP TLAs prior and post infection with v63G/70R at day 39. Passages of MeWo cells are indicated with an arrow. Data is shown as mean glucose utilization levels with standard deviations (error bars).
Figure 5.
VZV gene transcription in infected NHNP TLAs.
Two independent NHNP TLAs were established, infected with v63G/70R and sampled at indicated times post infection. A) qPCR analysis of VZV DNA genome copies (dORF63) and qRT-PCR of ORF63/70 transcript copies (tORF63) normalized against corresponding GAPDH copies. (B) qRT-PCR analysis of VZV ORF40 (tORF40) and ORF9 (tORF9) transcript copies normalized against GAPDH mRNA copies. Data of two independent experiments analyzed in triplicate is shown as mean relative copies and was analyzed using ΔΔCt analyses.
Table 2.
Release of infectious virus into VZV infected NHNP TLA tissue culture fluid.
Figure 6.
Infected NHNP TLA cells express progenitor and mature neuronal markers.
NHNP TLAs were infected with v63G/70R, fixed 21 days post-infection and stained for the progenitor marker Nestin (upper panels, A–C) and the mature marker β-Tubulin-III (lower panels, D–F). Representative confocal images are shown at 40× magnification. The outline of the TLAs (white line) and scale bars are indicated.
Figure 7.
The VZV genome is genetically stable in infected NHNP TLAs.
(A) MeWo cells and NHNP TLAs were infected with v63G/70R and propagated for the indicated time frames. Infected MeWo cells were passaged every 3–4 days, while infected NHNP TLAs were maintained by the replenishment of medium as required. At indicated time points, samples from the infected MeWo cells and NHNP TLAs were removed, total DNA extracted and DNA copies of ORF63-eGFP and ORF70-mRFP determined by qPCR. Data are shown as mean ratio of ORF63-eGFP to ORF70-mRFP determined in triplicate from two independent experiments in MeWo cells and 4 independent NHNP TLAs. (B) ORF63-eGFP and ORF70-mRFP expression (upper row) in a representative NHNP TLA at 27 days post infection with v63G/70R. An overlay of eGFP and mRFP fluorescence as well as a bright field image (lower row) of the NHNP TLA is shown at 40× magnification.
Table 3.
Primers used for qPCR and the construction of virus mutants.