Bovine posterior limbus: an evaluation of an alternative source for corneal endothelial and trabecular meshwork stem/progenitor cells

Abstract

A growing body of evidence has revealed that stem-like cells in the posterior limbus of the eye between the corneal endothelium (CE) and trabecular meshwork (TM) may be able to rejuvenate these tissues in disease. However, these cells have not been clearly defined and we have named them PET cells (progenitor cells of the endothelium and trabeculum). A good and inexpensive animal model for PET cells is lacking, so we investigated bovine eyes as an effective large tissue source. We showed the presence of stem/progenitor cells in the bovine CE, transition zone, and TM in situ. Floating spheres cultured from the CE and TM showed similar stem cell marker expression patterns. Both the CE and TM spheres were bipotent and highly proliferative, but with limited secondary sphere-forming capability. They were highly prone to differentiate back into the cell type of their tissue of origin. It is speculated that the PET cells become more tissue-specific as they migrate away from their niche. Here, we showed that PET cells are present in the posterior limbus of bovine eyes and that they can be successfully cultured and expanded. PET cells represent an attractive target for developing new treatments to regenerate both the CE and TM, thereby reducing the requirement for donor tissue for corneal transplant and invasive treatments for glaucomatous patients.

FIG. 1.

Comparative anatomy of the human and bovine posterior limbus. (A) Anatomy of the human chamber angle. The posterior limbus starts near Schwalbe's line. It contains the TZ where the anterior TM inserts into the peripheral CE. (B) A macro photograph of a human dissection for confocal microscopy. Arrowheads indicate TM. Inset shows higher magnification. (C) Scanning electron micrographs show a smooth TZ, from 80 to 130 μm in width, between the human CE and TM. The change from CE to TM cells is rapid. (D, E) Confocal pictures illustrate an irregular outline of peripheral CE cells demarcating the anterior margin of the human TZ, an area rich in cells. The posterior border is bound by insertions of TM beams. (F) Anatomy of the bovine chamber angle. There are PLs linking the peripheral CE and iris. (G) A macro photograph of a bovine dissection. (H) Scanning electron micrograph of the bovine TZ. There is no smooth zone and the transition from CE to TM is abrupt. (I) Confocal picture of the bovine TZ. Peripheral CE cells form the anterior border, followed by remnants of pectinate ligaments and a rapid change into TM. (J, K) Meridional examination of the bovine TZ by scanning electron microscopy (J) and vimentin staining (red) (K). Arrowheads depict the outline of the anterior TM, which inserts beneath Descemet's membrane. This insertion varied from 125 to 350 μm measured from the termination of Descemet's membrane (arrow). CC, ciliary cleft; CE, corneal endothelium; PL, pectinate ligament; TM, trabecular meshwork; TZ, transition zone.

FIG. 2.

Immunolocalization of stem cell markers in the bovine posterior limbus. (A–C) Stem cell markers ABCG2, nestin, Oct4, Pax6, Sox2, STRO-1, and telomerase were expressed in the bovine (A) CE, (B) transition zone, and (C) TM. The immunopositive cells in the transition zone were found in deep limbal layers just beneath the end of the peripheral CE. They appeared to be continuous with the anterior TM and comprised of 1%–5% of the total TM population. Asterisk marks the termination of Descemet's membrane. Arrowheads indicate immunopositive cells. Isotype controls showed no staining. (D) AnkG was exclusively expressed in the transition zone, but not in the CE or the TM. Nuclei were counterstained with DAPI. Scale bar=50 μm. AnkG, Ankyrin G; DAPI, 4′,6-diamidino-2-phenylindole; Oct4 octamer-binding transcription factor 4; Pax6, paired box gene 6; Sox2, sex-determining region Y-related box gene.

FIG. 3.

Characterization of the CE spheres. (A) Comparison of central and peripheral CE cells on primary sphere formation. CE cells from the peripheral cornea (>12 mm in diameter) generated significantly higher number of spheres than those from the central (<12 mm in diameter). Error bars indicate standard error of mean. *P<0.05, Student's t-test. Scale bar=100 μm. (B) Immunocytochemical studies on the CE spheres showed positive expression of a panel of stem cell markers, including ABCG2, nestin, Oct4, Pax6, Sox2, STRO-1, and telomerase, and the insert cell marker AnkG. However, primary CE cells only stained positively with Pax6, but not with the other stem cell markers nor with AnkG. Isotype controls showed no staining. Nuclei were counterstained with DAPI. Scale bar=50 μm. (C) Quantitative RT-PCR results showed that the abcg2, ankg, nes, oct4, pax6, sox2, and tert gene transcripts were significantly higher in the CE spheres than primary CE cells. Error bars indicate standard deviation from at least three individual biological samples. *P<0.05, **P<0.01, Student's t-test. (D) Proliferative capacity of the CE spheres. Greater than 90% of the cells within the CE spheres were positively stained with Ki67, while 20.1%±2.6% of the cells in the spheres incorporated BrdU (n=27). Scale bar=20 μm. BrdU, bromodeoxyuridine; RT-PCR, reverse transcription–polymerase chain reaction.

FIG. 4.

Differentiation properties of the CE spheres. (A) Non-directed differentiation of the CE spheres. A cell monolayer with cobblestone appearance was formed after 7 days. The CE sphere progenies expressed CE markers AQP1, N-cadherin, ZO-1, Na⁺/K⁺ ATPase, and connexin 43. Scale bar=50 μm. (B) The mRNA expression of abcg2, nes, and oct4 were significantly higher in the CE spheres than the differentiated sphere progenies. The mRNA levels of CE markers were similar between primary CE cells and differentiated sphere progenies. Error bars indicate standard deviation from at least three biological samples. *P<0.05, Student's t-test. (C) Transendothelial electrical resistance. The differentiated progenies formed better tight junctions than the primary CE cells. (n=10 for each group). *P<0.05, one-way ANOVA, Bonferroni's post hoc test. (D) Neural differentiation of CE spheres. In neural medium, more than 50% of the sphere progenies were positive for ZO-1 (cytoplasmic), GFAP, and TUJ1. With controls cultured in DMEM/10% FBS, the cells formed a monolayer of polygonal cells expressing ZO-1 (cell membrane). About 4.5%±0.9% and 2.9%±1.2% of the cells expressed GFAP and TUJ1 respectively. Telomerase was expressed when the cells were kept in stem cell medium. Scale bar=50 μm. (E) Mesenchymal differentiation of CE spheres. The CE spheres did not differentiate toward the mesenchymal lineages. However, mouse mesenchymal stem cells readily differentiated into adipocytes, osteocytes, and chondrocytes with positive oil red O, alizarin red S, and alcian blue staining respectively. Scale bar=100 μm. ANOVA, analysis of variance; AQP1, aquaporin-1; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; ZO-1, zonula occludens-1.

FIG. 5.

Characterization of the TM spheres. (A) The TM spheres showed positive expression of a panel of stem cell marker proteins, including ABCG2, nestin, Oct4, Pax6, Sox2, STRO-1, and telomerase, and the insert cell marker AnkG. However, primary TM cells only stained positively with Pax6, but not with the other stem cell markers or with AnkG. Isotype controls showed no staining. Nuclei were counterstained with DAPI. Scale bar=50 μm. (B) The abcg2, ankg, nes, oct4, pax6, sox2, and tert transcripts were significantly greater in TM spheres than the primary TM cells. Error bars indicate standard deviation from at least three individual biological samples. *P<0.05, Student's t-test. (C) Proliferative capacity of the TM spheres. Greater than 90% of the cells within the TM spheres were positively stained with Ki67. 25.4%±3.1% of the cells in the spheres incorporated BrdU (n=25). Scale bar=20 μm.

FIG. 6.

Differentiation properties of the TM spheres. (A) Non-directed differentiation. A cell monolayer showing spindle-shaped morphology was formed. The differentiated sphere progenies expressed TM markers AQP1 and CHI3L1, and readily ingested DiI-labeled Ac-LDL. Scale bar=100 μm. (B) The mRNA expression of stem cell markers was significantly higher in TM spheres than differentiated progenies. TM spheres expressed lower levels of TM genes than differentiated progenies. No significant difference of TM marker expression was found between primary TM cells and sphere progenies. Error bars indicate standard deviation from at least three biological samples. *P<0.05, **P<0.01, Student's t-test. (C) Melanin phagocytosis by TM sphere progenies. They showed a strong capacity to phagocytose melanin in a concentration-dependent manner. Nuclei were counterstained with hematoxylin. Scale bar=50 μm. (D) The mean percentage area of phagocytosed melanin of the progenies was 79% of that of the primary TM cells. Error bars indicate standard error of mean. Scale bar=100 μm. (E) Upon neural induction, the cells derived from the TM spheres showed weak staining of CHI3L1. Majority of them expressed GFAP and TUJ1. However, cells from the TM spheres that were cultured in DMEM/10% FBS medium grew as a monolayer and expressed CHI3L1. About 6.9%±2.1% and 4.8%±1.4% of them expressed GFAP and TUJ1 respectively. Telomerase was expressed when the cells were kept in stem cell medium. Scale bar=50 μm. (F) Mesenchymal differentiation. The TM spheres did not differentiate toward the mesenchymal lineages. However, mouse mesenchymal stem cells readily differentiated into adipocytes, osteocytes, and chondrocytes. Scale bar=100 μm. Ac-LDL, acetylated low density lipoprotein; CHI3L1, chitinase-3-like protein-1.

FIG. 7.

Schematic diagram of the distribution of stem/progenitor cells in the bovine anterior chamber angle. It is speculated that the PET cells resides in a niche at the TM insertion within the transition zone. As they migrate away from the niche towards the CE or TM, they become more tissue-specific. PET cells, progenitor cells of the endothelium and trabeculum.