Experimental schematic and design. A) Schematic of magnetically-steered cell delivery to the TM. As cells are injected into the anterior chamber at a low flow rate, the experimenter places the “point magnet” (15) on the limbus and carefully drags the cells towards the iridocorneal angle, targeting the trabecular meshwork (TM). Features of the figure are not drawn to scale. B) Time-line of the experiments. An ultrastructural analysis, specifically the quantification of inner wall basement membrane fenestrations, was not undertaken for eyes receiving iPSC-TMs due to their inferior performance. Additionally, cell retention in the anterior chamber was only investigated at the short-term. Note that baseline measurements were taken for WT and transgenic animals that did not necessarily receive an injection afterwards. Refer to Methods for a description of various experimental groups and further details.

IOP was reduced by targeted cell delivery to the TM. In each experimental cohort, the central white strip indicates the mean, while the darker region represents the 95% confidence interval on the mean. The colored region shows the distribution. Dots represent individual eyes, with error bars demarcating the 95% confidence intervals. For further information on experimental groups and statistical analysis refer to text. *p < 0.05 with Bonferroni correction (see Table 2). See below for a more complete statistical analysis. WT: wildtype hybrid mice (naïve control), Tg: Tg-MYOCY437H mice, Sham: Tg mice receiving saline injection, hAMSC: Tg mice receiving magnetically-steered hAMSCs, iPSC-TM: Tg mice receiving magnetically-steered iPSC-TMs. “Short”, “Mid”, and “Long” refer to time points. n is number of eyes measured in each group.

Outcome measures, shown as means and [95% confidence intervals].

Result of multiple comparison for various groups and variables, with statistically significant comparisons highlighted in orange*.

hAMSCs increase outflow facility in mice. A i-ii) IOP and flow rate vs. time for multiple pressure steps during the perfusion experiment in a representative eye. After each pressure step, the perfusion system automatically waits for a steady state inflow rate to be achieved, based on the criterion that the rate of change in the inflow rate falls below 3 nl/min/min. The steady intervals for each step are shown in green. Data has been trimmed to not include preparatory and pre-loading phases. A iii) Calculated outflow facility (red dots) vs. IOP. The solid curve shows the fitted model with the shaded region being the 95% confidence interval on the regression line. Error bars are 95% confidence intervals on individual steps. B) Outflow facilities across different experimental cohorts. Refer to Figure 2 for interpretation details. Note that outflow facilities in mice follow a log-normal distribution. C) Cross-validation of experimentally-measured and expected IOP, calculated from measured facility values. i) Regression plot of experimental vs. expected IOPs. Solid black fitted line (y = 0.94x − 0.21, R2 = 0.99) is shown along with its confidence bounds in dashed blue. Error bars show 95% confidence interval on both experimental (vertical) and expected (horizontal) IOPs. The unity line is shown as a solid red line. ii) Bland-Altman plot of IOP residuals (experimental minus expected IOPs) vs. average of experimental and expected IOPs. Individual experimental groups are indicated by colors matching those in panel B. Dashed line is shows the mean and is shown along with its 95% confidence interval (shaded). Solid line shows zero difference between the two parameters, i.e. the null hypothesis. For further information on experimental groups and statistical analysis refer to text. ∗ p < 0.05 with Bonferroni correction (see Table 2).

TM cellular density is improved by stem cell delivery. A and B) Brightfield and fluorescent micrographs of the irideocorneal angle (sagittal view) taken from a representative eye from the iPSC-TM short-term (A) and sham short-term (B) experimental groups. Green line shows the contour of the TM along the inner wall of Schlemm’s Canal used for normalizing nuclei count. DAPI-stained nuclei in the fluorescent image are shown in blue. Adjacent panels show a magnified view of the angle. C) Comparison of TM cellular density (number of nuclei per length of inner wall of Schlemm’s canal) for various experimental cohorts. Bars show mean and standard deviation. Multiple sections analyzed from each eye are coded with the same color. n = number of eyes. Linear mixed-effect model, *p < 0.05 with Bonferroni correction (see Table 2). D) Cross-comparison of TM cellularity vs. IOP for the eyes shown in panel C. The negative correlation between the variables was statistically confirmed (Pearson correlation coefficient = −0.63 and p < 10−7). Each color represents one eye, with different colors matching the experimental groups shown in Figure 2. Trend line (solid) is shown along with the 95% prediction interval (dashed).

Ultrastructural analysis of ECM underlying the inner wall of Schlemm’s canal (basement membrane material, or BMM). A) Greater amounts of BMM are evident immediately adjacent to the inner wall of Schlemm’s canal (arrowheads) in a saline-injected eye (top row) vs. in a hAMSC-treated eye (bottom row) at the mid-term time point. The images at right are a zoomed view of the orange boxed areas in the left panels. B) The normalized length of BMM directly in contact with the IW (length of BMM material divided by length of inner wall of Schlemm’s canal) for the experimental groups represented in panel A. Dots represent the average value between annotators for each measured section. Multiple sections analyzed from each eye are coded with the same color. n = number of eyes. Linear mixed-effect model. ∗ p < 0.05.

Retention of exogenous cells in the anterior segment 3 weeks after injection. Distribution of both hAMSC and iPSC-TM cells (red) are shown in A) en face images of the anterior segments and B) sagittal sections. In panel B), insets show a magnified view of the sites with the most intense fluorescent signal (green boxes). Autofluorescence can be seen in the posterior chambers as well as exterior to the corneoscleral shell. A cell mass, possibly a growing tumor, can be seen over the iris in the iPSC-TM injected eye.

Histopathological assessment of tumors in eyes receiving transplanted cells. iPSC-TM- and hAMSC-transplanted eyes were stained with hematoxylin and eosin (H&E). iPSC-TM sections show distinct tumor characteristics in the anterior chamber, including the presence of rosettes (black arrowheads), densely packed cells with high nuclear-cytoplasmic ratios (red arrowhead), and more loosely coherent cells (green arrowhead). Note that eyes were collected immediately after showing visible signs of tumor growth (usually within a month post-transplantation) and not at a pre-defined time point. hAMSC eyes at long-term time point showed no sign of tumor growth. In all panels, the green boxes provide a magnified view of the areas where tumor growth or the accumulation of exogenous cells occurred.

Quantification procedure for the amount of basement membrane materials (BMM) underlying to the inner wall of Schlemm’s canal. The red and blue line segments mark the basement membrane materials adjacent to the inner wall of Schlemm’s canal for each annotator. The summed length of these segments was then normalized by the overall length of the inner wall (yellow) for quantifications. The yellow line is slightly shifted from the blue and red segments for easier visualization.

Complementary micrographs to Figure 4 used for TM cellularity quantifications. Overview micrographs are shown in the left and right columns (see labels above images), with zoomed regions in the central column as indicated by the red arrows. Regions of interest (ROI), encompassing the parts of TM used for nuclei counting (demarcated in red) are outlined by green dashed boxes and the DAPI-stained nuclei (blue) are shown in a zoomed-in fluorescent micrograph of the ROI (green solid box). In overview images in which the ROI is tilted, the corresponding fluorescent micrograph has been rotated counterclockwise so that it is horizontal, thus enabling a more compact presentation.

Autofluorescence from various ocular tissues at the same fluorescence settings as used in Figure 6. The autofluorescence profile is dependent on the quality of dissection. In A, a signal is evident in insufficiently removed orbital tissue (yellow arrow) and at the limbus (green arrow). In B, autofluorescence is localized within the remnants of the retina (yellow arrow) and ciliary body (green arrow).

Prussian blue staining to locate SPIONs within the anterior segment after cell transplantation. The left column shows overview images of the anterior segment, while green boxes in the right column show a zoomed view of the region with strongest Prussian blue staining, corresponding to the green dashed boxes in the left column. Top row: No Prussian blue staining could be found in the saline injection control. Middle row: Prussian blue stain is challenging to distinguish from melanin, but accumulation of blue label (red arrowheads) can be seen to coincide with the locations of exogenous cells visualized in Figure 6. In particular, injected hAMSCs primarily accumulated close to the TM, corresponding to the location of Prussian blue stain. A small region of Prussian blue staining can be observed in the TM (green arrowhead). Bottom row: Similarly, in eyes receiving iPSC-TMs, most of the Prussian blue staining was found within the TM, corresponding to the location of injected cells (Figure 6). Unfortunately, the fluorescent signal in Figure 6 was significantly attenuated after Prussian blue staining and could not be overlaid on these images to assist with interpretation. Iris degradation, notable in the middle row, is an undesirable artifact of the cryosectioning or staining process (Figure 6).