Original research•

Embryoid body-based differentiation of human-induced pluripotent stem cells into cells with a corneal stromal keratocyte phenotype

Abstract

Objective The transparency of the cornea is determined by the extracellular matrix, which is secreted by corneal stromal keratocytes (CSKs). Human-induced pluripotent stem cell (hiPSC)-derived keratocytes (hiPSC-CSKs) can be used in cell-based therapy for treating corneal blindness. Our goal was to develop an effective small molecule-based technique for differentiating hiPSCs into keratocytes.

Methods and analysis hiPSCs were cultured in chemically defined medium, and embryoid bodies (EBs) were generated; these EBs were induced into CSKs using keratocyte-differentiated medium. The expression of keratocyte-specific markers was assessed using quantitative RT-PCR, immunostaining and Western blotting.

Results We found that the expression of genes encoding keratocyte markers, including aldehyde dehydrogenase 1 family member A1 (ALDH1A1), lumican and keratocan, was upregulated. Immunostaining showed positive staining for ALDH1A1 and keratocan in the hiPSC-CSK samples. Similarly, western blot analysis indicated that ALDH1A1 and keratocan expression levels were significantly greater in the hiPSC-CSKs than in the control cells. In addition, hiPSC-CSKs were not transformed into fibroblasts or myofibroblasts.

Conclusion We established an innovative and effective method to generate CSKs via the EB-based differentiation of hiPSCs, which might be employed for cell-based therapy of corneal stromal opacities.

What is already known on this topic

  • Corneal stromal keratocytes (CSKs) are essential for maintaining the cornea’s structural integrity and transparency by secreting stromal matrix. The loss or dysfunction of these cells can lead to corneal diseases. Human-induced pluripotent stem cell (hiPSC)-derived keratocytes offer a promising cell-based therapy for treating such corneal stromal opacities.

What this study adds

  • We have developed an embryoid body-based approach differentiating hiPSCs into cells with a CSK phenotype.

How this study might affect research, practice or policy

  • The embryoid body-based derivation of cells exhibiting a CSK phenotype holds potential for treating corneal stromal opacities.

Introduction

The cornea is the outermost portion of the eye, and eyesight depends on the transparency of the cornea. The cornea comprises three layers, the epithelial layer, stromal layer and endothelial layer, among which the stromal layer is the main part of the cornea.1 Corneal stromal keratocytes (CSKs) are the primary cell type of the stroma that produces and organises extracellular matrix (ECM) proteins, including collagens (types I, V, VI and XII) and proteoglycans (keratocan, keratan sulfate, decorin, mimecan and lumican).2 Morphologically, CSKs are thin, spindle-shaped and dendritic, containing distinct nuclei and cytoplasm comprising the majority of the cell volume.3

Under normal conditions, CSKs are derived from neural crest cells and mitotically quiescent cells.4 However, quiescent keratocytes proliferate and differentiate into fibroblasts and myofibroblasts in corneal diseases, such as inflammatory and traumatic disorders. As a result, transformed keratocytes exhibit changes in cell morphology and deposit fibronectin 1 (FN1), alpha-smooth muscle actin (α-SMA), tenascin-C, collagen III and collagen VIII to repair the ECM.5 The deposition of these proteins leads to disruption of the structure of the fibril, which causes the loss of corneal transparency and vision.

The only way to restore corneal clarity and function is through corneal transplantation.6 However, this surgery has certain drawbacks, including immunological rejection, graft failure and restricted availability of high-quality donor tissue. Therefore, corneal tissue engineering and stem cell-based regenerative therapies are alternative corneal transplantation methods for restoring stromal transparency. Stem cell-based treatment using autologous cell sources has the advantage of avoiding immunological reactions.7 Human-induced pluripotent stem cells (hiPSCs) can be generated from patients, and different cell types can be obtained for autologous transplantation, decreasing the risk of rejection and increasing the supply of donor tissue.8–10 Foster et al successfully generated corneal organoids that closely resemble the native cornea, by differentiating hiPSCs through a sequential differentiation protocol. These organoids developed key features of the corneal epithelium, stroma and endothelium, offering a new model to explore development, disease mechanisms and potential therapeutic strategies for conditions like corneal dystrophies or injuries.11 Another study applied single-cell transcriptomics to reveal the cellular heterogeneity in the human cornea, with a focus on identifying novel markers for specific regions, including the stroma, which is important in understanding corneal diseases and repair mechanisms.12 Specially, three-dimensional (3D) aggregates generated in suspension from induced pluripotent stem cells (iPSCs) are called embryoid bodies (EBs), and EB differentiation is a typical method for producing particular cell lineages.13 To regulate the fate of stem cells, EBs are thought to be a possible biomimetic body since they resemble the early phases of embryogenesis.14

Differentiation protocols have been introduced to obtain corneal stromal cells from hiPSCs; however, these protocols have several problems, such as low differentiation efficiency, long differentiation time and medium quality with an unknown chemical composition. In this study, we provide an effective method to differentiate hiPSCs into CSKs with chemically defined media. We first generated EBs from hiPSCs and subsequently induced them to differentiate into CSKs in keratocyte-differentiated medium (KDM). These cells expressed specific keratocyte markers, including aldehyde dehydrogenase 1 family member A1 (ALDH1A1), lumican and keratocan. Our research offers a novel method for producing CSKs, which might aid in cell-based treatments for corneal disorders.

Materials and methods

Cell culture

The hiPSCs used for differentiation were a gift from Professor Jin Ying (Chinese Academy of Sciences, China). The hiPSCs were grown on Matrigel-coated (Corning, Corning, New York, USA) plates and kept in a 37°C incubator with 5% CO2 using basal medium supplemented with Dulbecco's modified Eagle's medium: nutrient mixture F-12 (DMEM/F12, Thermo Fisher, USA), 1×N-2 supplement (Thermo Fisher), 1× B-27 supplement (Thermo Fisher), 0.1 mM minimum essential medium supplemented with non-essential amino acids (Gibco, USA), 0.1 mM 2-mercaptoethanol (Gibco, USA), and 10 ng/mL bFGF (TargetMol Boston, USA), as previously described. The medium was changed every day.

CSK differentiation

For CSK differentiation, the hiPSCs were trypsinised into a single-cell suspension with accutase (Thermo Fisher), and 2000 hiPSCs in 20 µL were cultured through the hanging drop method to generate EBs. Through EB formation suspension culture and subsequent attachment, CSKs were differentiated from iPSCs with Advanced DMEM (Thermo Fisher) supplemented with 10 ng/mL FGF2 (TargetMol Boston, USA) and 0.1 mM ascorbic acid-2-phosphate (Sigma-Aldrich, Germany). After 21 days of culture, the cells surrounding the EBs were digested and then transferred to a fresh dish for additional culture, after which the medium was changed every 2 days.

Quantitative RT-PCR

Total RNA was isolated from differentiated cells at various time points following differentiation (days 0–21) using RNAiso Plus reagent (TaKaRa, Japan) and chloroform according to the manufacturer’s protocol. The purity of the isolated RNA was tested by spectrophotometry (Thermo, USA), aiming for A260/A280 ratios between 1.8 and 2.0. Total RNA (1 µg) was reverse transcribed to cDNA using a PrimeScript RT Master Mix kit (Takara, Japan). qPCR was carried out using SYBR Green qPCR Master Mix (Vazyme, China) according to the manufacturer’s instructions. The primers used in our research are summarised in the supplementary materials (online supplemental table 1).

Immunofluorescence

The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Germany), treated with 0.3% Triton X-100 (Sigma-Aldrich, Germany) for 7 min, and then blocked with 3% bovine serum albumin (BSA, Sangon Biotech, China) for 1 hour at room temperature. The cells were subsequently incubated with primary antibodies overnight at 4 °C and then incubated with Alexa Fluor 555-conjugated donkey anti-mouse (1:800; Thermo Fisher) or Alexa Fluor 488-conjugated donkey anti-mouse (1:800; Thermo Fisher) antibodies for 1 hour at room temperature. The cell nuclei were stained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich, Germany) and observed by an Olympus Microscope. For figures 1A, B, 2C-G and 3A,B, cells were visualised using an Olympus DP26 camera (Olympus, Japan) with objectives ranging from 4× to 40×. For figures 1C, 3B, 4C, cells were imaged using an Olympus IX70 fluorescence microscope (Olympus, Japan) with Fluor objectives ranging from 10× to 40×.

Figure 1
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Figure 1

Characterisation of human-induced pluripotent stem cells (hiPSCs). (A) Images of hiPSCs under a light microscope. Scale bar: 200 µm. (B) Alkaline phosphatase (AKP) staining of hiPSCs. hiPSCs were positively stained with AKP. Scale bar: 200 µm. (C) Immunofluorescence staining of the hiPSCs showing the expression of pluripotency markers, including NANOG, OCT4, SOX2 and SSEA4. Scale bar: 100 µm. DAPI, 4,6-diamidino-2-phenylindole.

Figure 2
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Figure 2

Differentiation of corneal stromal keratocytes (CSKs) from human-induced pluripotent stem cells (hiPSCs). (A) Schematic diagram of the differentiation procedure. (B–D) Images demonstrating embryoid body (EB) formation. (E–G) Bright-field photographs of cells at various stages of CSK differentiation. Scale bar: 100 µm.

Figure 3
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Figure 3

The expression of alpha-smooth muscle actin (α-SMA) and fibronectin 1 (FN1) in human-induced pluripotent stem cell (hiPSC)-derived corneal stromal keratocytes (CSKs). (A, B) Bright-field photographs of CSKs stimulated with fetal bovine serum (FBS) or transforming growth factor β1 (TGF-β1). Scale bar: 100 µm. (C) Representative image of α-SMA staining in hiPSC-derived CSKs and in CSKs treated with TGF-β1, wherein the latter served as the control. Scale bar: 50 µm. (D) Western blots showing the expression of FN1 and α-SMA in hiPSC-CSKs, FBS-activated keratocytes and TGF-β1-activated keratocytes. GAPDH served as a normalizing control. (E) Quantification of the western blotting results in (D). n=3 biologically independent samples per group. For panel E, statistical analysis was performed using one-way analysis of variance and Dunnett multiple comparisons test; data are presented as the means±SD. ***P < 0.001, ****p< 0.0001 versus FBS.

Figure 4
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Figure 4

Characterisation of the differentiated corneal stromal keratocyte (CSK). (A) qRT-PCR analysis of human-induced pluripotent stem cell (hiPSC)-related genes (OCT4) and CSK-specific genes (aldehyde dehydrogenase 1 family member A1 (ALDH1A1), Lumican and Keratocan). (B) Immunofluorescence staining of hiPSC-CSKs showing positive staining for ALDH1A1, Lumican and Keratocan, which are all CSK-specific markers. Scale bar: 100 µm. (C) Western blots demonstrating greater expression levels of ALDH1A1 and Keratocan in comparison to hiPSCs (N1). β-Actin served as a normalizing control. (D–E) Quantification of the western blotting results in (C). n=3 biologically independent samples per group. For panel A, statistical analysis was performed using one-way analysis of variance and Dunnett multiple comparisons test; for panel E, statistical analysis was performed using unpaired, two-tailed Student’s t-test; data are presented as the means±SD. For panels A, D and E, **p< 0.01, ***p< 0.001 versus iPSC.

Western blot analysis

Total protein was extracted using RIPA lysis buffer (Beyotime, China) containing protease and phosphatase inhibitor cocktails (TargetMol, USA). The protein concentration was measured with a BCA assay (Pierce, USA). The proteins were electrophoresed using a PAGE Gel Quick Preparation Kit (8%–15%, Yeasen, China), and 40 μg of protein were transferred to a polyvinylidene difluoride membranes (Millipore, Germany). A total protein stain with Ponceau S was performed to confirm equal loading and transfer efficiency. After then, the membranes were blocked with 5% BSA in TBS-Tween 20 (0.1%) and incubated with primary antibodies (listed in online supplemental table 2 of the supplementary materials) overnight at 4°C. Next, the corresponding Horseradish Peroxidase-conjugated secondary antibodies were incubated with the blots. The blots were visualised using a Tanon system with enhanced chemiluminescence reagent (Thermo Fisher). Uncropped western blot images are available in online supplemental materials.

Statistical analysis

All experiments were performed in triplicate at least three times, and the data are presented as the mean±SD. All the statistical analyses were performed using Prism V.9 (GraphPad) software. Unpaired, two-tailed Student’s t-test or one-way analysis of variance with Dunnett’s multiple comparisons test was performed as indicated to determine significant differences. P < 0.05 was considered to indicate statistical significance.

Results

Characterisation of the hiPSCs

The generated iPSC lines had a typical human embryonic stem cell (ESC) morphology and consisted of closely packed cells encircled by a clear boundary (figure 1A). Additionally, iPSC colonies were positive for the genetic integrity marker alkaline phosphatase (figure 1B). Using immunofluorescence staining, the expression of pluripotency markers, such as NANOG, OCT4, SOX2 and SSEA4, was confirmed in these iPSCs (figure 1C).

Differentiation of hiPSCs into CSKs

The EB is an aggregate of three-dimensional PSCs, including both ESCs and iPSCs. EB-based differentiation is frequently used to produce specific cell lineages from PSCs.15 We modified previous methods to obtain CSKs from hiPSCs.16–18 In this study, we first produced EBs from hiPSCs with fully chemically defined medium and then used KDM supplemented with 10 ng/mL FGF2 and 0.1 mM ascorbic acid-2-phosphate to induce the differentiation of hiPSCs into CSKs in an ordered manner, as shown in figure 2A. In brief, EB formation occurred via a hanging drop (figure 2B), after which the cells were further cultured in suspension for another 2–3 days (figure 2C). Then, the EBs were cultured in KDM on Matrigel-coated plates (figure 2D), and the media was changed every 2 days. The cells were continually cultured for an additional 21 days. Cells began climbing out of the EB after a 4-day attachment period (figure 2E). As shown in figure 2F, a bright-field image of CSK differentiation was taken on day 14. hiPSC-derived CSKs exhibited a thin, dendritic morphology and had long processes in contact with adjacent cells, similar to the shape of human stromal keratocytes on day 21 (figure 2).

CSK marker expression in differentiated CSKs

CSKs express specific markers, including keratocan, lumican and ALDH1A1, which were used for CSK characterisation in this study. To evaluate the characteristics of the hiPSC-CSKs, qRT-PCR, immunofluorescence staining and western blotting were performed. Using qRT-PCR, we revealed constant upregulation of ALDH1A1, lumican and keratocan expression. Conversely, a substantial reduction in the expression of the pluripotency marker OCT4 was observed (figure 3A). Moreover, the hiPSC-CSKs were positively stained for ALDH1A1 and keratocan after 21 days of induction (figure 3B). Similarly, the hiPSC-CSKs had markedly greater ALDH1A1 and keratocan expression levels than did the hiPSC control (figure 3C,D). These results demonstrate a CSK phenotype of the obtained CSKs derived from hiPSCs.

Maintaining the CSK phenotype of hiPSC-derived CSKs

To further characterise hiPSC-CSKs, we assessed fibroblast and myofibroblast markers in these cells using immunofluorescence staining and western blotting. The control CSKs were cultured with fetal bovine serum (FBS) and transforming growth factor β1 (TGF-β1), which represent CSK-activated fibroblasts and myofibroblasts, respectively. Morphological analysis revealed the loss of dendritic morphology in both the FBS-activated and TGF-β1-activated conditioned media (figure 4A,B). In contrast, no substantial expression of αSMA was detected in hiPSC-CSKs (figure 4C). As expected, compared with those in FBS-activated keratocytes and TGF-β1-activated keratocytes, FN1 and α-SMA were barely expressed in hiPSC-CSKs according to western blot analysis (figure 4D,E). Thus, we obtained relatively pure CSKs from differentiated hiPSCs.

Discussion

Herein, we established a novel method for differentiating hiPSCs into CSKs by employing an EB-based approach in vitro, which might potentiate cell-based treatments for corneal stromal opacities. The protocol included a total differentiation period of 21 days, during which fully chemically defined media were used. Using RT-PCR, immunostaining and western blotting, the mRNA and protein expression of keratocyte markers, including ALDH1A1, lumican and keratocan, was detected in differentiated CSKs. Furthermore, we did not detect fibroblast or myofibroblast markers in our hiPSC-CSKs, indicating purer CSKs.

Recently, several types of stem cells, including ESCs, hiPSCs, adipose-derived stem cells (ADSCs), and mesenchymal stem cells, have been differentiated into CSKs. Using substratum-independent pellet culture in ascorbate-containing medium at the neural crest stage, ESCs were differentiated into CSKs.19 As with ESCs, hiPSCs can be produced from several somatic cell types and then used for CSK differentiation. Numerous cell types, such as corneal epithelial cells, retinal pigment epithelium, corneal endothelium and other cell types, are generated from hiPSCs.20–22 Moreover, the use of hiPSCs has fewer ethical issues. A recent study used a method similar to that used in our study, wherein ADSCs were induced to differentiate into CSKs expressing keratocyte-specific markers through pellet culture under keratocyte differentiation conditions.16 However, performing pellet culture is more complex than our EB-based protocol, which makes the latter more reproducible. Dos Santos reported that MSCs can differentiate into keratocyte lineages by upregulating TNFα-stimulated gene-6.23 Therefore, we established an EB-based approach for CSK differentiation.

CSKs are quiescent under normal conditions and are transformed into fibroblasts on corneal injury or FBS induction. Transformed CSKs exhibit altered dendritic morphology, decreased expression of CSK markers, upregulated expression of stress proteins and repair-type ECM deposition.3 24 Fibroblasts can turn into myofibroblasts when substantial damage is sustained or exposed to TGF-β.25 We detected fibroblast and myofibroblast markers in hiPSC-CSKs using immunofluorescence staining and western blotting. Compared with FBS-activated and TGF-β1-activated keratocytes, hiPSC-CSKs barely expressed FN1 and α-SMA. Therefore, we obtained relatively pure CSKs from hiPSCs differentiation. Intrastromal injection of cultured CSKs was reported to be an effective and safe method for prospective cell therapy for treating corneal opacity disorders.26 Research has indicated the ability of human limbal biopsy-derived stromal cells embedded in fibrin gel to treat corneal scarring in a mouse corneal debridement model.27 Further in vivo investigations using differentiated CSKs are warranted to determine the role of these cells in the treatment of corneal stromal disease.

The present study has several limitations. First, the present experiments were mainly conducted in vitro, and functional assessments of hiPSC-CSKs in vivo are needed. Future studies will focus on assessing their behaviour in a 3D matrix in vitro and following in vivo transplantation, evaluating CSKs for their ability to organise collagen fibrils properly and support corneal transparency post transplantation. Second, our study compared the CSK cell line with our hiPSC-CSK cell line, but further comparisons involving primary human CSKs are warranted in the future.

In summary, our work established an EB-based method for directly differentiating hiPSCs into CSK, which is an attractive source for use in cell-based treatment of corneal stromal opacities.

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