**3.3 Choroidal RALDH2+ cells: a novel cell type**

In chicks and humans, RALDH2 is synthesized by a population of extravascular choroidal stromal cells, some of which are closely associated with blood vessels (**Figure 1**) [10, 14, 44]. In chicks, RALDH2+ cells increase in number markedly over 1–7 days of recovery due, in part, to cellular proliferation (**Figure 1F** and **G**) and become concentrated on the proximal (RPE) side of the choroid [14]. Immunohistochemical analyses of chick choroids indicate that many of RALDH2+ express pro-collagen type IA (**Figure 1B** and **C**), similar to activated pericytes (a.k.a. perivascular stromal cells) within the CNS perivascular space [45]. Additionally RALDH2 is expressed in the chick

#### **Figure 1.**

*Choroidal RALDH2 positive (+) cells are heterogeneous. (A–E) RALDH2+ cells (green) are identified following labeling of 4 day recovering choroids with anti-RALDH2, together with anti-TCRγδ (γδ), pro-collagen IA (COL1A), or Ia antigen (IA) (red). Double-labeled cells are indicated by arrows. Asterisks in (C) indicate RALDH2+ cells that co-express Col1A. (F) Proliferating RALDH2+ cells were labeled with BrdU and identified with anti-BRDU (red). (G) Percentage of RALDH2+/BRDU+ cells is* ≈ *3× higher in 4 day recovering choroids, suggesting that RALDH2 activity is partially controlled by proliferation of RALDH2+ cells. Bar = 20 μM in A, 40 μM in B–F. \*\*\*Student's t test, p < 0.0001. Error bars = SEM. Nuclei in A–F are stained with DAPI. BV, blood vessel.* 

choroid by a small population of round cells that are positive for the Ia antigen [46, 47], indicating similarities with thymic macrophages/dendritic cells (**Figure 1D** and **E**), but are negative for the macrophage markers KuL01, MHC-II, and IgY [48]. A subpopulation of RALDH2+ cells also express α-smooth muscle actin (αSMA) [10, 14], but are negative for the smooth muscle/myofibroblast proteins, smoothelin, desmin and myocardin. RALDH2+ cells do not co-localize with CD-45 [14], TCRδγ (**Figure 1A**), CD5, or GRL(2) positive cells [49, 50], indicating they are not of hematopoietic origin. RALDH2+ cells also do not co-localize with neuron-specific beta III tubulin, NOS (pan), or tyrosine hydroxylase, indicating they are not of neuronal origin. Negative results were also obtained using anti-NG2 (a pericyte marker), vimentin, and PECAM-1 (an endothelial marker). Similarly, RALDH2+ cells in the human choroid were negative for the endothelial cell marker, CD31, the pericyte markers, NG2 and CD146, α-smooth muscle actin, the macrophage markers CD68 and LYVE1, IBA1 (microglia) and the pan-neuronal marker PGP9.5 [51]. Unlike results in the chick, some RALDH2+ cells in the human choroid co-localized with vimentin, suggesting a mesenchymal origin [51]. Based on the markers used in these studies, RALDH2+ cells seem to represent an independent cell-population. Studies are in progress using additional markers as well as transcriptome analyses on RALDH2+ cells isolated from chick and human choroids to further classify this new cell-population as this cell type may represent a potential target for therapies to slow or prevent myopia in children.
