Summary• Under cadmium (Cd) stress, Solanum nigrum accumulated threefold more Cd in its leaves and was tolerant to Cd, whereas its low Cd-accumulating relative, Solanum torvum, suffered reduced growth and marked oxidative damage. However, the physiological mechanisms that are responsible for differential Cd accumulation and tolerance between the two Solanum species are largely unknown.• Here, the involvement of antioxidative capacity and the accumulation of organic and amino acids in response to Cd stress in the two Solanum species were assessed.• Solanum nigrum contains higher antioxidative capacity than does S. torvum under Cd toxicity. Metabolomics analysis indicated that Cd treatment also markedly increased the production of several organic and amino acids in S. nigrum. Pretreatment with proline and histidine increased Cd accumulation; moreover, pretreatment with citric acid increased Cd accumulation in leaves but decreased Cd accumulation in roots, which indicates that its biosynthesis could be linked to Cd long-distance transport and accumulation in leaves.• Our data provide novel metabolite evidence regarding the enhancement of citric acid and amino acid biosynthesis in Cd-treated S. nigrum, support the role of these metabolites in improving Cd tolerance and accumulation, and may help to provide a better understanding of stress adaptation in other Solanum species.

To expand multi-potent progenitors from human trabecular meshwork (TM), we have created a new optimized method on two-dimensional (2D) followed by three–dimensional (3D) Matrigel in modified embryonic stem cell medium supplemented with 5% fetal bovine serum (MESCM + 5% FBS). The expanded TM cells were small cuboidal cells expressing TM markers such as AQP1, MGP, CHI3L1, and AnkG, embryonic stem cell (ESC) markers such as Oct4, Sox2, Nanog, and ABCG2, and neural crest (NC) markers such as p75NTR, FOXD3, Sox9, Sox10, and MSX1. Although expanded cells lost expression of these markers after passage, the cells regained the markers when Passage 2 cells were seeded on 3D Matrigel through activation of canonical BMP signaling. Such restored progenitors could differentiate into corneal endothelial cells, adipocytes, and chondrocytes but not keratocytes or osteocytes. Therefore, we have concluded that human TM harbors multipotent progenitors that can be effectively isolated and expanded using 2D Matrigel in MESCM + 5% FBS. This unique in vitro model system can be used to understand how TM is altered in glaucoma and whether such TM progenitor cells might one day be used for treating glaucoma or corneal endothelial dysfunction.

Proliferative vitreoretinopathy (PVR) is mediated by proliferation and epithelial mesenchymal transition (EMT) of retinal pigment epithelium (RPE). Because heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) purified from human amniotic membrane exerts anti-inflammatory and anti-scarring actions, we hypothesized that HC-HA/PTX3 could inhibit these PVR-related processes in vitro. In this study, we first optimized an ARPE-19 cell culture model to mimic PVR by defining cell density, growth factors, and cultivation time. Using this low cell density culture model and HA as a control, we tested effects of HC-HA/PTX3 on the cell viability (cytotoxicity), proliferation (EGF + FGF-2) and EMT (TGF-β1). Furthermore, we determined effects of HC-HA/PTX3 on cell migration (EGF + FGF-2 + TGF-β1) and collagen gel contraction (TGF-β1). We found both HA and HC-HA/PTX3 were not toxic to unstimulated RPE cells. Only HC-HA/PTX3 dose-dependently inhibited proliferation and EMT of stimulated RPE cells by down-regulating Wnt (β-catenin, LEF1) and TGF-β (Smad2/3, collagen type I, α-SMA) signaling, respectively. Additionally, HA and HC-HA/PTX3 inhibited migration but only HC-HA/PTX3 inhibited collagen gel contraction. These results suggest HC-HA/PTX3 is a non-toxic, potent inhibitor of proliferation and EMT of RPE in vitro, and HC-HA/PTX3’s ability to inhibit PVR formation warrants evaluation in an animal model.

In this article, human limbal niche cells (LNC) or bone marrow derived mesenchymal stem cells (BMMSC) were used to prevent limbal stem cell deficiency (LSCD) in an alkali burn rabbit model and their results were compared. The epithelial cell defect area, corneal neovascularization, and the print cell cytometry were quantified to grade the severity of LSCD. Three months after the alkali burn, a partial LSCD was observed in the control group (no treatment) indicated by chronic corneal epithelial defects, positive corneal fluorescein staining, neovascularization and goblet cell migration. In contrast, the severity of LSCD in both the LNC and BMMSC transplantation groups was dramatically reduced as shown by smaller epithelial cell defects, decreased fluorescein sodium staining, decreased neovascularization and decreased goblet cell density. Interestingly, the LNC group was shown to more effectively prevent LSCD than the BMMSC group. Further analysis indicated subconjunctivally transplanted LNCs were more powerful than BMMSCs to prevent LSCD, at least partially, due to increased activation of SCF-c-Kit signal. We conclude that LNCs are a more powerful resource than BMMSCs to prevent LSCD in an alkali burn rabbit model, at least partially due to increased activation of SCF signaling.

On ocular surface, corneal epithelial stem cells (SC) reside in limbus between cornea and conjunctiva. Pax6, an evolutionally conserved transcription factor essential for eye development, is expressed in post-natal corneal and limbal epithelia progenitors (LEPC) but not in underlying stroma. Because Pax6 is transiently expressed in developing corneal stroma and a subset of limbal and corneal stromal progenitors, we examined the role of Pax6 in limbal niche cells (LNC) in maintaining the phenotype of neural crest (NC) progenitors to support LEPC. Our results showed that nuclear Pax6 staining was found in freshly isolated LNC but not corneal stromal cells. Serial passaged LNC resulted in gradual loss of nuclear Pax6 (46 kDa) staining and neural crest progenitor status defined by the expression of embryonic SCs and NC markers, neurosphere formation, and differentiation into neurons, oligodendrocytes and astrocytes. Gain of function of 46 kDa Pax6 in late-passaged LNC resulted in nuclear Pax6 staining and promotion of the aforementioned NC progenitor status. In an in vitro reunion assay, early passaged LNC and late passaged LNC with overexpression of Pax6 inhibited the expression of corneal epithelial differentiation marker and promoted holoclone by LEPC. Therefore, expression of nuclear 46 kDa Pax6 in LNC plays an important developmental role in maintaining NC progenitor status to support self-renewal of corneal epithelial SCs in the limbal niche.

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Human corneal endothelial cells are notorious for their restricted proliferative ability in vivo and in vitro. Hence, injury or dysfunction of these cells may easily result in blindness. Currently, the only treatment is to transplant a donor cornea that contains a healthy corneal endothelium. However there is a severe global shortage of donor corneas and there remains an unmet clinical need to engineer human corneal grafts with healthy corneal endothelium. In this review, we present current advances in the culture, expansion, and molecular understandings of corneal endothelial cells in vitro in order to help establish methods of engineering human corneal endothelial grafts.