Fig 2: Microsphere-based scaffolds support the coculture of NSCs, astrocytes, and HUVECs. (A) Cell seeding and ICC analysis timeline. Confocal images are displayed as maximum projections of iPSC-derived NSCs, astrocytes, and HUVECs within the scaffold. (B–E) Day 7 ICC for neurons (ßIII-tubulin) and astrocytes (GFAP). (F) Day 28 ICC for neurons (ßIII-tubulin) and astrocytes (GFAP). (G–J) Day 7 ICC for HUVECs (CD31) and astrocytes (GFAP). (K) Day 28 ICC for HUVECs (CD31) and cytoskeletal formation (F-actin). (L–O) Day 7 ICC for cytoskeletal formation (F-actin) and mature neurons (NF-M). (P) Day 28 ICC for neural progenitors (hNestin) and mature neurons (NF-M). DAPI nuclear counterstain is also shown. Scale bars = 300 µm.

Fig 3: Loss of hematopoietic cells in the developing retina delays retinal vascular development and increases inflammation. Representative images (A,B) showing retinal flat mounts of WT and Vav1 KO retinas stained with collagen IV which labels the vasculature, and the corresponding outputs from VESGEN (C,D) showing the different vessel generations color coded (E) from 1–9. (F–I) Graphs showing VESGEN quantifications comparing total vessel number density, large vessel, medium-sized vessel and small vessel densities between the two groups. (J,K) Representative images showing GFAP expression (green) in the retinas of WT and Vav1 KO mice and the corresponding quantification in (L). White arrows indicate retinal astrocytes.

Fig 4: Selective hACE2 expression in OE and neurons is sufficient to confer hypoxemia and death following SARS-CoV-2 infection.(A-C) Immunohistochemistry of WT and Foxg1Cre/+;LSL-hACE2+/0 mouse OE (A), OB, and cerebral cortex (B) and lung (C) 6 days after infection with 105 PFU of SARS-CoV-2 using antibodies that recognize viral nucleocapsid, sustentacular cell E-cadherin, olfactory sensory neuron ßIII-tubulin, neuronal NeuN, glial GFAP, AT1 PDPN, or AT2 DC-LAMP. Asterisk indicates OB with nucleocapsid staining. Arrows indicate nucleocapsid staining colocalized with ßIII-tubulin+ OSNs (OE in A) or NeuN+ neurons (OB and cerebral cortex in B). White dotted lines trace the border between the OE and OB. Representative of n = 5 per genotype. (D) HE staining of WT and Foxg1Cre/+; hACE2fl/y lung tissue 6 days after exposure to 105 PFU of SARS-CoV-2 virus. Arrows, sites of focal consolidation. Asterisks, intravascular thrombi. Representative of n = 5 animals per genotype. (E, F) Immunohistochemistry of WT and Foxg1Cre/+; hACE2fl/y lung tissue 6 days after exposure to 105 PFU of SARS-CoV-2 virus using antibodies against ICAM-1 and PDPN, or vWF and PECAM. Arrows in I identify vWF-positive microvasculature of the lung in Foxg1Cre/+; hACE2fl/y animals. Representative of n = 5 animals per genotype. (G, H) Weight loss and survival of LSL-hACE2+/0 and Foxg1Cre/+; LSL-hACE2+/0 mice after infection with 105 or 104 PFU of SARS-CoV-2. N = 4 (LSL-hACE2+/0), 8 (Foxg1Cre/+; LSL-hACE2+/0 105 PFU), 5 (Foxg1Cre/+; LSL-hACE2+/0 104 PFU) and two independent experiments. Asterisks indicate time points at which significant differences in weight (C) or survival (D) were observed between infected Foxg1Cre/+; LSL-hACE2+/0 and LSL-hACE2+/0 mice animals. (I) Pulse oximetry of WT (LSL-hACE2+/0 or Foxg1Cre/+) and Foxg1Cre/+; LSL-hACE2+/0 mice infected with 104 viral titer at the time of harvest (Day 6–8). ***p < 0.001; ****p < 0.0001 determined by unpaired, two-tailed t test or log-rank Mantel Cox test. Scale bars in all images 50 µm. Numerical data in corresponding S1 Metadata tab. hACE2, human ACE2; ICAM-1, intracellular adhesion marker 1; OB, olfactory bulb; OE, olfactory epithelium; OSN, olfactory sensory neuron; PDPN, Podoplanin; PECAM, platelet endothelial cell adhesion molecule; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; vWF, von Willebrand’s Factor; WT, wild-type.

Fig 5: High-plex 4i tissue profiling of human postmortem spinal cord. Fresh frozen tissue sections from ALS patient spinal cords were subjected to 14-plex profiling over six 4i cycles. (A) K-means clustering of 4i data was used to visualize tissue domains defined by all 14 channels across the entire tissue section. Each of 7 clusters is assigned a unique color and inset locations are noted (B–E). (B') Higher magnification image of anterior horn inset region (B). Cell type marker MBP (cyan) is expressed by oligodendrocytes, MAP2 (magenta) is expressed in neurons and neuronal processes, and ELAVL3 (yellow) is expressed by neurons, including motor neurons. (B?) Same as (B'), but yellow is used to visualize tissue autofluorescence. (B'?) Pathological markers P62 (cyan), TDP-43 (magenta), and hyperphosphorylated TDP43 (yellow) are visualized in the same FOV as in (B',B?). (C) Higher magnification image of lateral white matter inset region (C). Cell type markers NFH expressed in neurons (cyan), GFAP expressed in astrocytes (magenta), and CD68 expressed in activated microglia (yellow) are visualized. (D) Higher magnification image of ventral white matter inset region (D). Cell type markers NFH expressed in neurons (cyan), IBA1 expressed in microglia (yellow), and nuclear envelope marker LMNB1 (magenta), are visualized. (E) Higher magnification image of dorsal horn inset region (E). Cell type markers CD34 expressed in endothelial cells (cyan), ALDH1L1 expressed in astrocytes (yellow), and nuclear envelope marker LMNB1 (magenta), are visualized.