Heterogeneity and neurovascular integration of intraportally transplanted islets revealed by 3-D mouse liver histology

Supplemental Materials

Supplemental Figure Legends

Supplemental Fig. S1 (related to Fig. 1). Clot-like islet graft and the peri-graft ischemic injury. (a) Panoramic image of intraportally transplanted islets. The scattered islet grafts are detected in the transparent mouse liver (map). Graft 1 and 2 are enlarged in the insets (i, ii) and b-i to identify the graft microstructure and peri-graft microenvironment. Yellow: b-cells (insulin). Green: a-cells (glucagon). Red: perfusion labeling of blood vessels. (b-e) Graft 1 and the severe peri-graft ischemic injury. Transmitted light signals in b identify the peri-graft lesion (arrow heads) and steatosis. Overlay of fluorescence and transmitted light signals at three focal depths (c-e) confirms the clot-like graft lodging inside the portal venule (PV). Blue: nuclear staining. (f-i) Graft 2 and the mild peri-graft ischemic injury. Unlike the severe injury caused by Graft 1, Graft 2 at the end of a portal venule (PV) causes a mild tissue injury (arrow head).

Supplemental Fig. S2 (related to Fig. 2). Plaque-like islet graft and the graft revascularization. (a, b) Map of liver and zoom-in examination of a plaque-like graft in the peri-portal space. Yellow: b-cells (insulin). Green: a-cells (glucagon). Red: perfusion labeling of blood vessels. Blue: nuclear staining. The microvessels in b (zoom-in, arrows) are further enlarged in e for examination. (c-e) 3-D projection and high-definitional 2-D image of plaque-like graft. The arrows in e indicate the graft blood microvessels, which contact the islet a- and b-cells, confirming the graft revascularization.

Supplemental Fig. S3 (related to Fig. 5). Mouse hepatic sympathetic neural network and its connection to intraportally transplanted islets. (a-d) Connection of peri-portal sympathetic nerves with islet grafts. a: map of liver showing the intraportally transplanted islets (yellow arrows). b-d: projection of islet grafts and their close association with peri-portal sympathetic nerves. c, d reveal that the a-cells of clot-like graft (yellow) are scattered and lodge inside a portal vessel. In comparison, the a-cells of plaque-like graft deposit on the vessel wall. The islet grafts at the two locations are integrated via the hepatic neurovascular networks. Yellow: a-cells (glucagon). Green: TH⁺ sympathetic nerves. Red: perfusion labeling of blood vessels. White/blue: nuclear staining. (e, f) Sympathetic innervation of clot-like graft. Overlay of fluorescence and transmitted light signals confirm the clot-like graft and the graft-lesion association (oval) downstream of a portal vein (PV). The sympathetic axons and varicosities (green) contact the graft a-cells (yellow) as well as the portal vessels. Blue: nuclear staining. Depth-1 and -2 indicate that the images were taken from two optical depths.

Supplemental Fig. S4 (related to Fig. 5). VAChT⁺ parasympathetic innervation of mouse gallbladder but not liver. (a-c) Map of liver and gallbladder. a: stereomicroscopic image. b: panoramic confocal image (nuclear staining). a and b examine the same area and enlarged in c. Gallbladder is used as a positive control of vesicular acetylcholine transporter (VAChT) staining to identify the parasympathetic innervation (gallbladder vs. liver; enlarged in d-f, arrows). (d-f) High-definitional 3-D neurohistology to compare liver (TUJ1⁺, VAChT^-) and gallbladder (TUJ1⁺, VAChT⁺) parasympathetic innervation. The 3-D projections at the gallbladder-liver boundary (dotted yellow line in e, f) show that the VAChT⁺ parasympathetic nerves (red) associate with the muscle layer, lamina propria, and epithelium of gallbladder, but they are not in the liver domain (f). In the liver, the pan-neuronal marker TUJ1 staining (green) identifies the peri-portal innervation, but the nerve bundle does not include the VAChT⁺ parasympathetic nerves (e, f). In comparison, in the gallbladder, the overlap of TUJ1 (green) and VAChT (red) signals confirms the parasympathetic innervation and the reactivity of the immunohistochemical assay. However, we cannot rule out that the specific liver environment may interfere with the VAChT staining, leading to the negative result.

Supplemental Video Legends

Supplemental Video S1 (Related to Fig. 4)

Stereo projection of peri-portal innervation of plaque-like graft. Yellow/blue: pooled insulin and glucagon signals of plaque-like graft around a portal vein. Green: TUJ1 staining of hepatic neural network. Note that the islet endocrine cells are also TUJ1⁺ as illustrated in Fig. 4h-m.

Supplemental Video S2 (Related to Fig. 4)

In-depth recording of clot-like graft innervation. Blue: pooled insulin and glucagon signals of clot-like graft. Green: TUJ1 staining of hepatic nerves. Red: perfusion labeling of blood vessels. The transmitted light signals (gray) reveal the peri-graft tissue injury (black spots) and steatosis (lipid droplets). The close association and signal overlap of islet cells (blue), TUJ1 (green, nerves), and blood vessels (red) create the rainbow colors of the graft.

Supplemental Video S3 (Related to Fig. 5)

Sympathetic innervation of clot-like graft. TH⁺ sympathetic nerves (green) reach the clot-like graft (a-cells, yellow; lodging inside a portal venule, 00:03-00:07) via the peri-portal space. The integrative 2-D/3-D stereo projection (00:11-00:24) shows that the sympathetic axons and varicosities (green dots around portal vein) infiltrate the graft and contact the a-cells. Yellow: glucagon staining of a-cells. Red: perfusion labeling of blood vessels. Blue: nuclear staining. High-definitional tile scanning (1×2) was used to acquire the image stack.

Supplemental Video S4 (Related to Fig. 5)

Sympathetic innervation of plaque-like graft. Similar to the clot-like graft presented in Video S3, TH⁺ sympathetic nerves (green) reach the plaque-like graft (a-cells, yellow) via the peri-portal space. The lumen of the portal vein is clearly seen at 00:04-00:06 with a-cells on the vessel walls. The spatial association among the portal vein, islet graft (a-cells), and sympathetic nerves are illustrated in the integrative 2-D/3-D stereo projection (00:11-00:24). Red: perfusion labeling of blood vessels. Blue: nuclear staining. High-definitional tile scanning (1×2) was used to acquire the image stack.

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Supporting Fig. 1 (related to Fig. 1 and 5). Detection and confirmation of clot-like islet graft. (a) Map of liver showing the location of clot-like grafts. Yellow: insulin staining of graft b-cells. PV: portal venule. (b, c) Zoom-in confirmation of clot-like graft with standard H&E histology. After 3-D fluorescence imaging (Fig. 1e-h), the same liver specimen was processed with microtome sectioning and H&E staining to confirm the islet embolus (aggregation of nuclear signals, c) lodging in the portal venule as presented in Fig. 1e (Depth-1). (d-f) Second example of clot-like islet graft confirmed by H&E histology. Yellow: glucagon staining of graft a-cells. Green: TH⁺ sympathetic nerves. White: nuclear staining. The detail of the graft a-cell sympathetic innervation is presented in Supplemental Fig. S3 (related to Fig. 5). The results in a-c and d-f confirm the clot-like islet graft and demonstrate the compatibility of the modern 3-D and the classic 2-D histology.

Note that the classic H&E histology is the gold standard in clinical tissue analysis. However, using the microtome-based H&E or immunohistochemical assay alone cannot efficiently detect and analyze the peri-graft microenvironment and neurovascular networks.

Supporting Table 1 (Related to Materials and Methods).  Summary of primary antibodies used in illustrations.

Supporting Table 2 (Related to Materials and Methods).  Summary of color codes presented in illustrations.