**4. Isolation of precursors from cultured human Corneal Endothelial cells**

#### **4.1. Culture of human Corneal Endothelial cells**

As mentioned in sections 3.1-3.4, we have isolated precursor cells from human donor corneas (Yamagami et al. 2007; Yokoo et al., 2005). However, the number of precursors that can be isolated from a cornea is insufficient for corneal endothelial regeneration, so establishment of a mass production method for precursor cells is needed before clinical application can be attempted. Accordingly, we isolated spheres from cultured HCECs and investigated whether the cells of these spheres had CE-like functions. We also tested the effect of injecting these spheres into the anterior chamber (instead of full-thickness corneal transplantation) in a rabbit model of bullous keratopathy, representing a state in which corneal endothelial defects exist.

Several groups have established HCEC culture techniques (Chen et al., 2001; Engelmann & Friedl 1989; Miyata et al., 2001; Yue et al. 1989). Various growth factors have been reported to influence the proliferation of cells cultured from human CE, including fibroblast growth factor (Chen et al., 2001; Engelmann 1988, 1989, 1995; Yue et al. 1989; Samples et al., 1991), epidermal growth factor (Chen et al., 2001; Samples et al., 1991; Schultz et al., 1992; Yue et al. 1989), nerve growth factor (Chen et al., 2001), and endothelial cell growth supplement (Blake et al., 1997; Yue et al. 1989). In addition, cell attachment and growth can be supported by seeding cells onto an artificial matrix, such as chondroitin sulfate or laminin (Engelmann et al., 1988), laminin-5 (Yamaguchi et al., 2011), extracellular matrix secreted by bovine corneal endothelial cells (Blake et al., 1997; Miyata et al., 2001), or fibronectin/type I collagen coating mix (Joyce & Zhu, 2004).

In our studies, HCECs were isolated and cultured according to the published protocols of Joyce and our laboratory with some modifications (Chen et al., 2001; Joyce & Zhu, 2004; Miyata et al., 2001). Briefly, Descemet's membrane was carefully dissected with the intact CE. After centrifugation, membrane strips were incubated in 0.02% EDTA solution at 37ºC for 1 hour to loosen intercellular junctions. Then isolated cells were plated in 6-well tissue culture plates that had been precoated with undiluted fibronectin/type I collagen coating mix, and incubation was done at 37ºC under a humidified atmosphere with 5% CO2. After primary cultures reached confluence, cells were subcultured at a 1:4 ratio, and cells from the 4th to 6th passages were used.

#### **4.2. Isolation and characterization of sphere colonies**

but cells migrated out from the spheres grown on glass coverslips coated with bovine ECM alone. After 7 days, some of the cells that had migrated from the spheres showed double immunostaining for nestin and β3-tublin (Fig. 2H), as has been reported for human scalp tag– derived cells (Toma et al., 2001). However, there was no staining of cells migrating out of the

RT-PCR was performed to examine the expression of genes governing the proteins detected by immunocytochemistry in the spheres and their progeny (Fig. 2I). GAPDH mRNA was detected in both spheres and progeny, but not in the control assay without the RT reaction. Expression of nestin,β 3-tublin, GFAP, and α-SMA mRNA was detected in the spheres and adherent progeny after 35 PCR cycles. However, mRNAs for NFM, p75NTR, and peripherin were not found under any cycling conditions. Nestin andβ 3-tublin mRNAs were also detected

These findings indicated that spheres isolated from human CE contain bipotential precur‐ sors, yielding progeny that display the morphologic characteristics of HCECs. Taken together, these results suggest that precursors from the CE remain close to the tissue of origin and undergo differentiation into CECs. Because precursors should ideally differenti‐ ate efficiently to produce their tissue of origin, precursors obtained from the CE may be more appropriate for tissue regeneration or cell transplantation than those derived from

**4. Isolation of precursors from cultured human Corneal Endothelial cells**

As mentioned in sections 3.1-3.4, we have isolated precursor cells from human donor corneas (Yamagami et al. 2007; Yokoo et al., 2005). However, the number of precursors that can be isolated from a cornea is insufficient for corneal endothelial regeneration, so establishment of a mass production method for precursor cells is needed before clinical application can be attempted. Accordingly, we isolated spheres from cultured HCECs and investigated whether the cells of these spheres had CE-like functions. We also tested the effect of injecting these spheres into the anterior chamber (instead of full-thickness corneal transplantation) in a rabbit model of bullous keratopathy, representing a state in which corneal endothelial defects exist.

Several groups have established HCEC culture techniques (Chen et al., 2001; Engelmann & Friedl 1989; Miyata et al., 2001; Yue et al. 1989). Various growth factors have been reported to influence the proliferation of cells cultured from human CE, including fibroblast growth factor (Chen et al., 2001; Engelmann 1988, 1989, 1995; Yue et al. 1989; Samples et al., 1991), epidermal growth factor (Chen et al., 2001; Samples et al., 1991; Schultz et al., 1992; Yue et al. 1989), nerve growth factor (Chen et al., 2001), and endothelial cell growth supplement (Blake et al., 1997; Yue et al. 1989). In addition, cell attachment and growth can be supported by seeding cells onto an artificial matrix, such as chondroitin sulfate or laminin (Engelmann et al., 1988), laminin-5 (Yamaguchi et al., 2011), extracellular matrix

spheres for α-SMA, p75NTR, NFM, peripherin, GFAP, or O4.

in HCECs from primary culture.

436 Regenerative Medicine and Tissue Engineering

the multipotential stem cells.

**4.1. Culture of human Corneal Endothelial cells**

Cells from the 4th or 5th passages were used in this study. HCECs were incubated in 0.2% EDTA at 37ºC for 5 minutes and then were dissociated into single cells by pipetting with a flame-polished Pasteur pipette. The viability of the isolated HCECs was >90% as shown by trypan blue staining. The sphere-forming assay was used for primary culture (Rey‐ nolds & Weiss, 1992). Cells were plated at a density of 10 viable cells/μL (40,000 cells per well or 1,420 cells/cm2 ) in the uncoated wells of 60-mm culture dishes. The basal medi‐ um was Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with B27, epidermal growth factor (EGF, 20 ng/mL), and basic fibroblast growth factor (bFGF, 20 ng/ mL). A methylcellulose gel matrix (1.5%; Wako) was added to the medium to prevent reaggregation of the cells (Gritti et al., 1999; Kawase et al., 2004). To distinguish growing spheres from dying cell clusters, only spheres with a diameter of more than 50 μm were counted. For passaging, primary spheres were harvested on day 7 and treated with 0.5% EDTA for dissociation into single cells, which were plated in 24-well culture plates at a density of 10 cells/μL. Then culture was continued for another 7 days in basal medium containing the methylcellulose gel matrix.

Spheres formed after 7 days of culture (Fig. 3A), while nonproliferating cells were eliminated. Many of the cells in each sphere were BrdU-positive (Fig. 3B), indicating that such cells were proliferating. These findings suggested that the spheres had developed from single HCECs and that the sphere-forming cells displayed proliferative activity. The number of sphere colonies obtained after 7 days of culture was 44 ± 10 per 10,000 cells (mean ± SD). Replating of primary spheres to generate secondary sphere colonies was less efficient, indicating that the cells only had limited self-renewal capacity.

On immunostaining, the spheres were positive for nestin (Fig. 3C), which is a marker of immature cells (Lendahl et al., 1990), and for α-SMA (Fig. 3D), a mesenchymal myofibro‐ blast marker. We previously demonstrated that primary spheres derived from human donor CE express β-III tubulin and GFAP, a mature glial cell marker, as well as nestin and α-SMA (Fig. 2), but β-III tubulin and GFAP were negative in the spheres derived from cultured HCECs.

from cultured HCECs.

of sphere colonies obtained after 7 days of culture was 44 10 per 10,000 cells (mean SD). Replating of primary spheres to generate secondary sphere colonies was less efficient,

On immunostaining, the spheres were positive for nestin (Fig. 3C), which is a marker of immature cells (Lendahl et al., 1990), and for -SMA (Fig. 3D), a mesenchymal myofibroblast marker. We previously demonstrated that primary spheres derived from

indicating that the cells only had limited self-renewal capacity.

Fig. 3. Immunocytochemistry (A-F) and RT-PCR analysis (G) of sphere colonies derived from cultured HCECs were their progeny. Cultured HCECs were disaggregated into single cells and plated at a density of 10 viable cells/L in basal medium containing a methylcellulose gel matrix to prevent reaggregation. (A) A representative day 7 sphere. (B) Cells in a sphere colony labeled by BrdU on day 7. A total of 44 10 primary spheres were generated per 10,000 cells (mean SD). Scale bar=50 m. (C-F) A day 7 sphere shows staining for nestin (C) and -SMA (D). Less than 5% of the sphere progeny cells were stained by the mesenchymal cell marker -SMA (E, arrow). There is no staining by control IgG (F). Scale bar=100 µm. (G) RT-PCR of spheres and progeny. cDNA was obtained from spheres and from their progeny cultured in 1% FBS or 15% FBS. GAPDH was detected in all **Figure 3.** Immunocytochemistry (A-F) and RT-PCR analysis (G) of sphere colonies derived from cultured HCECs were their progeny. Cultured HCECs were disaggregated into single cells and plated at a density of 10 viable cells/μL in bas‐ al medium containing a methylcellulose gel matrix to prevent reaggregation. (A) A representative day 7 sphere. (B) Cells in a sphere colony labeled by BrdU on day 7. A total of 44± 10 primary spheres were generated per 10,000 cells (mean ± SD). Scale bar=50 μm. (C-F) A day 7 sphere shows staining for nestin (C) and α-SMA (D). Less than 5% of the sphere progeny cells were stained by the mesenchymal cell marker α-SMA (E, arrow). There is no staining by control IgG (F). Scale bar=100 µm. (G) RT-PCR of spheres and progeny. cDNA was obtained from spheres and from their prog‐ eny cultured in 1% FBS or 15% FBS. GAPDH was detected in all samples, except those reacted without reverse tran‐ scriptase. Nestin mRNA expression was detected in cultured spheres, but not in their progeny cultured in either 1% or 15% FBS. Both the spheres and progeny were positive forα -SMA mRNA. Figures are modified from Mimura et al. (2005b) with permission.

#### **4.3. Differentiation of sphere colonies**

Individual primary spheres (day 7) were transferred to 13-mm glass coverslips coated with 50 μg/mL PLL and 10 μg/mL fibronectin in separate wells (Mimura et al., 2005a). To promote differentiation, 1% or 15% FBS was added to the basal medium, after which culture was continued for another 7 days.

Then the spheres were transferred to PLL/fibronectin-coated glass coverslips in 24-well plates and were cultured in a differentiation medium containing 1% or 15% fetal bovine serum (FBS). After 7 days, many cells were found to have migrated out of the spheres. Fewer than 5% of these cells were α-SMA-positive (Fig. 3E), whether cultured with 1% or 15% FBS. All of these cells were negative for control IgG (Fig. 3F) and for the differentiated epithelial cell marker cytokeratin 3, as well as for nestin, β-III tubulin, and GFAP (not shown). These findings indicated that a single sphere colony could give rise to a small population of mesenchymal cells under clonogenic conditions. Expression of nestin and α-SMA by the spheres, as well as α-SMA expression by their progeny, was confirmed using RT-PCR (Fig. 3G). Positivity for β-III tubulin mRNA was only detected in cultures with 1% FBS.

Spheres derived from donor CE expressed an immature cell marker (nestin), an immature neuronal marker (β-III tubulin), and a mature glial cell marker (GFAP), while their progeny expressedβ -III tubulin and nestin, but not GFAP. In contrast, the spheres and progeny obtained from cultured HCECs did not express neuronal markers and showed decreased expression of immature cell markers. These findings suggested that the precursors were close in nature to the original tissue and underwent differentiation during culture. Thus, precursors obtained from cultured HCECs may be a more appropriate cell source than cells from donor CE, because precursors that efficiently differentiate into the tissue of origin are ideal for tissue regeneration or cell transplantation.

#### **4.4. Assessing the pump function of cells derived from spheres**

of sphere colonies obtained after 7 days of culture was 44 10 per 10,000 cells (mean SD). Replating of primary spheres to generate secondary sphere colonies was less efficient,

On immunostaining, the spheres were positive for nestin (Fig. 3C), which is a marker of immature cells (Lendahl et al., 1990), and for -SMA (Fig. 3D), a mesenchymal myofibroblast marker. We previously demonstrated that primary spheres derived from human donor CE express -III tubulin and GFAP, a mature glial cell marker, as well as nestin and -SMA (Fig. 2), but -III tubulin and GFAP were negative in the spheres derived

**G**

Fig. 3. Immunocytochemistry (A-F) and RT-PCR analysis (G) of sphere colonies derived from cultured HCECs were their progeny. Cultured HCECs were disaggregated into single cells and plated at a density of 10 viable cells/L in basal medium containing a methylcellulose gel matrix to prevent reaggregation. (A) A representative day 7 sphere. (B) Cells in a sphere colony labeled by BrdU on day 7. A total of 44 10 primary spheres were generated per 10,000 cells (mean SD). Scale bar=50 m. (C-F) A day 7 sphere shows staining for nestin (C) and -SMA (D). Less than 5% of the sphere progeny cells were stained by the mesenchymal cell marker -SMA (E, arrow). There is no staining by control IgG (F). Scale bar=100 µm. (G) RT-PCR of spheres and progeny. cDNA was obtained from spheres and from their progeny cultured in 1% FBS or 15% FBS. GAPDH was detected in all

**Figure 3.** Immunocytochemistry (A-F) and RT-PCR analysis (G) of sphere colonies derived from cultured HCECs were their progeny. Cultured HCECs were disaggregated into single cells and plated at a density of 10 viable cells/μL in bas‐ al medium containing a methylcellulose gel matrix to prevent reaggregation. (A) A representative day 7 sphere. (B) Cells in a sphere colony labeled by BrdU on day 7. A total of 44± 10 primary spheres were generated per 10,000 cells (mean ± SD). Scale bar=50 μm. (C-F) A day 7 sphere shows staining for nestin (C) and α-SMA (D). Less than 5% of the sphere progeny cells were stained by the mesenchymal cell marker α-SMA (E, arrow). There is no staining by control IgG (F). Scale bar=100 µm. (G) RT-PCR of spheres and progeny. cDNA was obtained from spheres and from their prog‐ eny cultured in 1% FBS or 15% FBS. GAPDH was detected in all samples, except those reacted without reverse tran‐ scriptase. Nestin mRNA expression was detected in cultured spheres, but not in their progeny cultured in either 1% or 15% FBS. Both the spheres and progeny were positive forα -SMA mRNA. Figures are modified from Mimura et al.

Individual primary spheres (day 7) were transferred to 13-mm glass coverslips coated with 50 μg/mL PLL and 10 μg/mL fibronectin in separate wells (Mimura et al., 2005a). To promote differentiation, 1% or 15% FBS was added to the basal medium, after which culture was

Then the spheres were transferred to PLL/fibronectin-coated glass coverslips in 24-well plates and were cultured in a differentiation medium containing 1% or 15% fetal bovine serum (FBS). After 7 days, many cells were found to have migrated out of the spheres. Fewer than 5% of these cells were α-SMA-positive (Fig. 3E), whether cultured with 1% or 15% FBS. All of these cells were negative for control IgG (Fig. 3F) and for the differentiated epithelial cell marker cytokeratin 3, as well as for nestin, β-III tubulin, and GFAP (not shown). These findings indicated that a single sphere colony could give rise to a small population of mesenchymal cells under clonogenic conditions. Expression of nestin and α-SMA by the spheres, as well as α-SMA expression by their progeny, was confirmed using RT-PCR (Fig. 3G). Positivity for β-

indicating that the cells only had limited self-renewal capacity.

from cultured HCECs.

438 Regenerative Medicine and Tissue Engineering

(2005b) with permission.

**4.3. Differentiation of sphere colonies**

III tubulin mRNA was only detected in cultures with 1% FBS.

continued for another 7 days.

The pump function of four collagen sheets seeded with cells derived from HCEC spheres was measured in an Ussing chamber, as reported previously with some modifications (Wigham, 1981, 2000; Hodson & Wigham 1983). The collagen sheets were obtained from the Nippi Biomatrix Research Institute (Tokyo, Japan). Cells from HCEC spheres were suspended at 5.0 × 106 cells in 1.5 mL of culture medium and transferred to circular collagen sheets (10 mm in diameter). Each sheet was placed in one well of a 24-well plate, and the plate was centrifuged at 1,000 rpm (176 g) for 10 minutes to enhance cell attachment. Then the sheets were incubated in culture medium for 2 days, after which nonadherent cells and debris were removed (Fig. 4A). Human donor corneas with the epithelium removed mechanically (n=4), plain collagen sheets (n=4), or HCEC-coated collagen sheets (n=4) were mounted in the Ussing chamber.

Changes of the potential difference (Fig. 4B) and short circuit current (Fig. 4C) were compared between human donor corneas without epithelium and HCEC-coated collagen sheets con‐ structed with cells from spheres. The average potential difference and short circuit current of the HCEC-coated sheets ranged from 81% to 100% at 1, 5, and 10 minutes, corresponding to the results for normal human donor corneas denuded of epithelium. These findings suggested that the cultured HCEC spheres could generate CE-like cells with adequate transport activity.

#### **4.5. Migration and proliferation of spheres on rabbit descemet's membrane**

Animals were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Primary HCEC spheres (cultured for 7 days) were labeled with a fluorescent cell tracker (CM-DiI). After the endothelium was gently scraped off four freshly excised rabbit corneas with a sterile cotton swab, HCEC spheres were applied to the posterior surface of each cornea. Then the corneas were placed in 24-well plates and main‐ tained in culture medium for 7 days. HCECs that migrated onto the corneas were detected under a fluorescence microscope, and the area occupied by fluorescent cells migrating from the spheres was measured with the NIH image program (n=10).

Figure 5A shows cells migrating from DiI-labeled spheres on days 1-7. The mean area covered by migrating cells per sphere reached 1.2 ± 0.2 mm2 on day 7 (Fig. 5B).

adequate transport activity.

Changes of the potential difference (Fig. 4B) and short circuit current (Fig. 4C) were compared between human donor corneas without epithelium and HCEC-coated collagen sheets constructed with cells from spheres. The average potential difference and short circuit current of the HCEC-coated sheets ranged from 81% to 100% at 1, 5, and 10 minutes,

Fig. 4. Morphology (A) and transport activity (B, C) of cells from cultured HCEC spheres, modified from Mimura et al. (2005b) with permission. (A) Confluent cells cultured in DMEM containing 10% FBS show the characteristic hexagonal shape of corneal endothelial cells. Changes of the potential difference (B) and short circuit current (C) for human donor corneas without epithelium and HCEC-coated collagen sheets (mean SD). The mean potential difference and short circuit current after 1, 5, and 10 minutes ranged from 81% to 100% for the HCEC-coated sheets, similar to the results for human corneas denuded of epithelium, indicating that the HCEC-like cells generated in culture had adequate transport **Figure 4.** Morphology (A) and transport activity (B, C) of cells from cultured HCEC spheres, modified from Mimura et al. (2005b) with permission. (A) Confluent cells cultured in DMEM containing 10% FBS show the characteristic hexago‐ nal shape of corneal endothelial cells. Changes of the potential difference (B) and short circuit current (C) for human donor corneas without epithelium and HCEC-coated collagen sheets (mean ± SD). The mean potential difference and short circuit current after 1, 5, and 10 minutes ranged from 81% to 100% for the HCEC-coated sheets, similar to the results for human corneas denuded of epithelium, indicating that the HCEC-like cells generated in culture had ade‐ quate transport activity. When Na+ - K+ ATPase inhibitor ouabain was added to the chamber, the potential difference decreased to 0 mV and the short circuit current declined to 0 μA in all cases.

**Figure 5.** Migration of sphere-derived cells during culture for 7 days, modified from Mimura et al. (2005b) with per‐ mission. DiI-labeled spheres were seeded onto the denuded Descemet's membranes of rabbit corneas and cultured for 1 week in a humidified incubator. (A) Representative photographs of cell migration around an adherent DiI-la‐ beled sphere. Scale bar=100 μm. (B) Mean area occupied by cells migrating from the spheres on each day (n=10).
