A SINGLE-CELL RNA-SEQ ANALYSIS UNRAVELS THE HETEROGENEITY OF PRIMARY CULTURED HUMAN CORNEAL ENDOTHELIAL CELLS
The corneas is a transparent and avascular tissue located in front of the eye. Its inner surface is lined by a monolayer of corneal endothelial cells (CECs), which maintain the cornea transparent. CECs remain arrested at a non-proliferative state and damage to these cells can compromise their function leading to corneal opacity. The primary culture of donor-derived CECs is a promising cell therapy. It confers the potential to treat multiple patients from a single donor, alleviating the global donor shortage. Nevertheless, this approach has limitations preventing its adoption, particularly culture protocols allow limited expansion of CECs and there is a lack of clear parameters to identify therapy-grade CECs. To address this limitation, a better understanding of the molecular changes arising from the primary culture of CECs is required. Using single-cell RNA sequencing on primary cultured CECs, we identify their variable transcriptomic fingerprint at the single cell level, provide a pseudo temporal reconstruction of the changes arising from primary culture, and suggest markers to assess the quality of primary CEC cultures. This research depicts a deep transcriptomic understanding of the cellular heterogeneity arising from the primary expansion of CECs and sets the basis for further improvement of culture protocols and therapies. Overall design: Research-grade donor human corneas and ethical statement This study was performed in compliance with the tenets of the Declaration of Helsinki. All research-grade human donor corneas used for primary culture were obtained from the Lions Eye Institute for Transplant & Research (Tampa, USA), with informed consent from the next of kin. The research involving human-derived corneas was performed in accordance to Maastricht University and Dutch national regulations. All corneas had an endothelial cell density of at least 2800 cells/mm2, deemed unsuitable for transplantation, and were preserved in Optisol-GS at 4°C for up to 14 days prior to their use (Table 1). No corneas were procured from prisoners. Isolation and culture of primary human corneal endothelial cells Six paired corneas, from two male and one female donor aged 27 to 34 years, were used for isolation and primary culture of endothelial cells. Donors had no history of ocular disease, chronic systemic disease, or pathological infection such as HIV, Hepatitis B and C, HTLV-I/II, syphilis, or SARS-CoV-2. Prior to isolation, the endothelial–Descemet's corneal layer was manually stripped as follows: the corneas were vacuum fixed in a punch base (e.janach) endothelial cell side up and trephined with a 10 mm Ø corneal punch at a fixed depth of 100 µm (e.janach). To delimit the endothelial trephined line, corneas were stained with a trypan blue solution (0.4%) for 30 s, and washed with balanced salt solution sterile irrigating solution (BSS; Alcon). The corneal endothelium was then gently lifted using a DMEK cleavage hook (e.janach) and fully stripped using angled McPherson tying forceps.  Human corneal endothelial cells were isolated and cultured as previously reported.5 Briefly, the stripped endothelium–Descemet's layer was incubated with 2 mg/ml collagenase A (Roche) solution in human endothelial serum free media (SFM) (Thermo Fisher Scientific) for 2–5 h at 37°C followed by a 5 min incubation in TrypLE express (Thermo Fisher Scientific) to generate small clumps of corneal endothelial cells. Cells from each cornea were seeded equally across 2 wells of a 24-well plate coated with fibronectin collagen (FNC) coating mix (Athena Enzyme Systems) in M5 stabilization media (human endothelial SFM (Thermo Fisher Scientific) supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin–streptomycin, and 0.25 µg/mL amphotericin B) supplemented with 10 µM Y-27632 (STEMCELL Technologies). Subsequently, corneal endothelial cells were cultured in M4 proliferation medium (1:1 Ham's F12 (Thermo Fisher Scientific) and M199 (Thermo Fisher Scientific) supplemented with 5% FBS, 20 µg/mL ascorbic acid (Sigma), 1 × ITS (Thermo Fisher Scientific), 10 ng/mL human recombinant bFGF (Sigma), and 10 µM Y-27632 (STEMCELL Technologies)); media was refreshed every other day. Upon reaching 90% confluency, after approximately 8–10 days of culture, cells were cultured in M5 stabilization media for 7 days. After this, corneal endothelial cells were treated with TripLE express and passaged into wells pre-coated with FNC-coating mix at a seeding density of 10,500 cells/cm2 in M5 stabilization medium. All cell culture was performed in incubators with humidified atmosphere of 37°C and 5% CO2. Preparation of single cell suspension and methanol fixation Cells from five different culture time points were methanol fixed for sequencing. Namely, cells at days 2 and 5 of culture after isolation in M4 proliferation media at passage 0, and cells at confluency after 7 days of culture in M5 stabilization media, at passages 0, 1, and 2.  To generate a single cell suspension, primary cultured corneal endothelial cells were treated with TripLE express for approximately 30 min at 37°C. Then cells were centrifuged for 5 min at 800 × g and resuspended in 1 mL ice-cold Dulbecco's phosphate-buffered saline (DPBS). Next, the cells were centrifuged for 5 min at 800 × g and resuspended in ice-cold DPBS at a ratio of 200 µL DPBS/1 × 106 cells, followed by the dropwise addition of ice-cold methanol at a ratio of 800 µL DPBS/ 1 × 106 cells. The fixed cell suspensions were stored at -80°C until sequencing. Single-cell RNA sequencing scRNAseq of primary cultured CECs was performed at Single Cell Discoveries (Utrecht, the Netherlands) following standard 10× Genomics 3' V3.1 chemistry protocol. Cells were rehydrated and loaded on the 10× Chromium controller as follows. Approximately 10,000 cells were loaded per each sample specified in Table 2. The resulting sequencing libraries were prepared following a standard 10× Genomics protocol and sequenced with an Illumina NovaSeq 6000 platform; read length: 150 bp, paired-end.