File S1 - Regenerative Therapeutic Potential of Adipose Stromal Cells in Early Stage Diabetic Retinopathy

Combined file of supporting figures and tables. Figure S1: Decreased pancreatic β-cell mass in diabetic athymic nude rat. (A) Pancreata from STZ treated diabetic rats and non-diabetic controls were sectioned and stained for insulin (red) and counterstained with hematoxylin (blue). Original magnification, ×100. β cell mass in diabetic rats was quantitated by MetaMorph software. Note a significant decrease in β cell mass in diabetic rats compared to non-diabetic controls (*p<0.05). Data shown is a representative of n = 5 per group. Figure S2: Body weights unchanged in the diabetic athymic nude rat with intravitreal injection of ASC. Two months post diabetes induction intravitreal ASC injections were performed. Body weight was measured daily for the next three weeks. Insulin was given periodically. Intravitreal injection of ASC had no effect on elevated blood glucose after 3 weeks post transplantation but demonstrated a slight natural expected increase in body weights in these rats. The data shown is from a group size of n = 6–8 animals. Figure S3: Retinal trypsin digests reveal acellular capillaries and pericyte ghosts. Two months post diabetes induction trypsin digests were performed as described in methods. Acellular capillaries (red arrows) were identified as capillary-sized vessel tubes having no nuclei anywhere along their length. Pericyte ghosts (black arrow) were estimated from the prevalence of protruding ―bumps in the capillary basement membranes from which pericytes had disappeared. At least 1,000 capillary cells (endothelial cells and pericytes) in 5 field areas in the mid-retina (400× magnifications) in a masked manner were examined for quantification. Data is a representative photomicrograph from n = 6–8 per group. Figure S4: Long term improvement in the retinal function in the diabetic athymic nude rat with intravitreal injection of ASC. Two months post diabetes induction ERG was recorded in anesthetized rats at day 0 (green line) and performed intravitreal injections of either saline (left) or ASC (right). At day 7 and day 21 post ASC injections, ERG was measured. A representative ERG waves from dim flash to bright flash over time is computed (A). Typical b-wave amplitudes plotted against time clearly demonstrated a decreased in amplitudes with saline at day-7 (red line; left) while animals that received ASC (right), clearly had an increase. This increase in amplitudes measured on day-21 (blue line) remained high in ASC group suggesting a long lasting effect of ASC treatment in diabetic retinal function. The data shown is from a group size of n = 6–8 animals. Figure S5: Increased retinal vascular permeability in diabetic athymic nude rat. Fluorescein angiography (FA) was performed to assess the leaky vessels (Micron III retinal imaging system, Phoenix Research Labs) based on standard procedures. Sodium fluorescein (0.05 ml of 25%) injected through tail vein was captured at the same time frame between diabetic and non-diabetic rats clearly revealed a significant leakage of fluorescein. In addition, fundus examination of live anesthetized diabetic and non-diabetic rats using bright field imaging revealed hemorrhages in diabetic rats that were near completely absent in non-diabetic rats. Data shown is a representative of n = 3–6 per group. Table S1: Realtime RT-qPCR primer pairs. Rat gene specific primers were designed using Primer3, a widely used program for designing PCR primers available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html.
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