A profile of circulating vascular progenitor cells in human neovascular age-related macular degeneration

Abstract

Background/objective: A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects.

Materials and methods: Blood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion, the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR.

Results: There was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media, and markers such as NG2 and αSMA in mesenchymal media. However, analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population.

Conclusions: CD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However, nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease.

Fig 1. Sorting of CD34+ cells from PBMC population.

A) Box and whisker plots comparing the numbers of PBMC per mL between refractory and non-refractory nvAMD subjects. Plots show the maximum value, 3^rd quartile, median, 1^st quartile and minimum values. Mean shown as +. B) FACS analysis of magnetic bead sorting showing percentage of CD34+ cells in unsorted PBMC population, CD34+ fraction and CD34- fraction. C) Box and whisker plots comparing the numbers of CD34+ cells isolated between refractory and non-refractory nvAMD subjects. D) Box and whisker plots comparing percentage of CD34+ cells in PBMC population between refractory and non-refractory nvAMD subjects.

Fig 2

Expansion of CD34+ cells A) Images of isolated CD34+ cells 1 day (left image) and 7 days (right image) after plating in StemSpan media. B) Box and whisker plots comparing the expansion factor of CD34+ cells between refractory and non-refractory nvAMD subjects. C) FACS analysis of CD34 expression in a CD34+ population before and after expansion in StemSpan media.

Fig 3. Differentiation of CD34+ cells.

A) Images of isolated CD34+ cells after expansion in StemSpan media (left image), culturing in EGM media (center image) and culturing in pericyte media (right image). B) Comparison of ΔC_T values of VWF, NG2 and COL1A1 (endogenous control 18S) between refractory and non-refractory subjects.

Fig 4. Variability of blood draws.

A) Bland-Altman analysis of the mean PBMC/ml for each subject vs difference in PBMC/ml between 1^st and 2^nd blood draw. B) Bland-Altman analysis of the mean no. of CD34+ cells isolated for each subject vs difference in no. of CD34+ cells isolated between 1^st and 2^nd blood draw. C) Bland-Altman analysis of the CD34% of PBMC population for each subject vs difference in CD34% between 1^st and 2^nd blood draw. D) Bland-Altman analysis of the CD34+ expansion factor for each subject vs difference in CD34 expansion factor between 1^st and 2^nd blood draw. E) Scatter plot of the difference in PBMC/ml obtained in intra-patient samples vs time elapsed between samples. F) Scatter plot of the difference in CD34-positive percentage of the PBMC population obtained in intra-patient samples vs time elapsed between samples.

Fig 5. Repeatability of CD34+ differentiation.

Comparison of CD34+ cells isolated from a single subject examining differentiation in EGM and Pericyte media from the first blood draw (A) vs the second blood draw (B).