**3. Isolation of sphere colonies from human Corneal Endothelium**

#### **3.1. Primary sphere-forming assay**

This study was conducted in accordance with the Declaration of Helsinki. Corneas were obtained from the Central Florida Lions Eye Tissue Bank and the Rocky Mountain Lions' Eye Bank at 4 to 10 days after death. The age of the donors was 41 to 78 years. The CE and Descemet's membrane were peeled away in a sheet from the periphery to the center of the inner surface of the cornea with fine forceps, as described previously (Sakai et al., 2002). To avoid the inclusion of posterior stromal tissue, we only used endothelium that was smoothly peeled off together with Descemet's membrane. The harvested CE was incubated at 37ºC for 3 hours in basal medium containing 0.02% collagenase (Sigma-Aldrich, St. Louis, MO). This was followed by incubation in 0.2% ethylenediaminetetraacetic acid (EDTA) at 37ºC for 5 minutes, and then dissociation into single cells by trituration with a fire-polished Pasteur pipette. The viability of the isolated CECs was >90%, as shown by trypan blue staining (Wako Pure Chemical Industries, Osaka, Japan). After addition of a trypsin inhibitor (Invitrogen-Gibco), the cells were resuspended in basal medium and the number of cells was counted (Coulter counter; Beckman-Coulter, Hialeah, FL). Neither cytokeratin-3 nor cytokeratin-12 expression was detected, indicating that the cells thus obtained were all CECs without contamination by other corneal cell types.

Despite the successful isolation and characterization of stem cells from various tissues, relatively few animal studies have been done to investigate the efficacy of stem cell transplan‐ tation. A three-dimensional carrier that maintains cell-to-cell interactions is indispensable for tissue engineering using stem cells, but the resulting structural complexity does not allow us

We have isolated precursors with the propensity to develop into corneal endothelial-like cells from the CE of human donor corneas (Yokoo et al., 2005). We have also demonstrat‐ ed that cultured human corneal endothelial cells (HCECs) and rabbit CE-derived precur‐ sors are an effective cell source for treating corneal endothelial defects in a rabbit model (Mimura 2005a, 2005b). Because the number of corneal endothelial cell (CEC) precursors that can be isolated from a native cornea is insufficient for corneal transplantation, establishment of a method for the mass production of precursor cells is required before

In this chapter, we introduce our recent work in the fields of regenerative medicine and tissue engineering for the CE using bipotential precursor cells. We isolated precursors with the propensity to develop into CECs from human CE, and we investigated the distribution and proliferative capacity of precursor cells derived from the central and peripheral regions of the cornea by the sphere-forming assay. We also tested the effect of injecting human corneal endothelial spheres anterior chamber (instead of full-thickness corneal transplantation) in a rabbit model of bullous keratopathy, a condition associated with corneal endothelial defects.

Neural crest cells, from which the CE is derived (Bahn et al., 1984; Johnston et al., 1979), migrate and differentiate in two waves during corneal development (Liu et al., 1998; Meier et al., 1982). In the first wave, the corneal epithelium is formed by periocular mesenchymal cells of neural crest origin and it synthesizes the primary stroma, after which neural crest cells migrate to the margin of the optic cup and then migrate between the lens and corneal epithelium to contribute to development of the CE and the trabecular meshwork. In the second wave, neural

to easily perform investigations of stem cell transplantation.

**2. Origin and development of the Corneal Endothelium**

crest cells invade the primary stroma and differentiate into corneal keratocytes.

**3. Isolation of sphere colonies from human Corneal Endothelium**

This study was conducted in accordance with the Declaration of Helsinki. Corneas were obtained from the Central Florida Lions Eye Tissue Bank and the Rocky Mountain Lions' Eye Bank at 4 to 10 days after death. The age of the donors was 41 to 78 years. The CE and Descemet's membrane were peeled away in a sheet from the periphery to the center of the inner surface of the cornea with fine forceps, as described previously (Sakai et al., 2002). To

CEC transplantation can be employed clinically.

430 Regenerative Medicine and Tissue Engineering

**3.1. Primary sphere-forming assay**

Half of the cells were labeled with a fluorescent cell tracker (CM-DiI; C-7000; Molecular Probes, Eugene, OR), as described elsewhere (Mimura et al. 2004), to examine sphere formation by reaggregation. DiI-labeled cells and unlabeled cells were mixed and seeded at a density of 1 cell/μL (250 cells/cm2 ), 10 cells/μL (2,500 cells/cm2 ), 30 cells/μL (7,500 cells/cm2 ), or 50 cells/μL (12,500 cells/cm2 ) on 60-mm uncoated dishes containing 5 mL of medium for floating culture (Reynolds & Weiss, 1992, 1996) (Fig. 1B). No spheres were generated in the cultures with only 1 viable cell/μL, but numerous spheres were formed at 30 and 50 cells/μL, with some arising from reaggregation as indicated by DiI staining. Spheres were completely DiI-positive or DiInegative when culture was performed at 10 cells/μL (Fig. 1C), indicating that these spheres were derived from proliferation and not from reaggregation of the dissociated cells.

Incubation was done in a humidified incubator under an atmosphere of 5% CO2, with 40 ng/mL basic fibroblast growth factor (bFGF) and 20 ng/mL epidermal growth factor (EGF) being added to the medium every other day. To investigate whether the isolated cells were contaminated with corneal epithelial cells, expression of epithelial markers such as keratins K3 and K12 (Irvine et al., 1997; Moll et al., 1982) was assessed by the reverse transcription– polymerase chain reaction (RT-PCR) before the start of culture. Then primary culture was performed and the existence of fibroblast-like cells was investigated to assess contamination by stromal cells. CECs were isolated without contamination by corneal epithelial cells, as demonstrated by RT-PCR analysis of corneal epithelial markers (K3 and K12 genes), as well as the characteristic hexagonal shape of the cells in primary culture (data not shown). Almost complete disaggregation into single cells was achieved, since counting of single, double, and triple cells showed that 99% of all cells were single (Fig. 1B).

After incubation for 5 days, small floating spheres formed. These spheres grew larger after 10 days, while the nonproliferating cells died and were eliminated (Fig. 1D). After 10 days, we only counted cell clusters with a diameter of at least 50 μm, in order to distinguish growing spheres from dying ones. To verify that the increase of colony size was actually due to cell proliferation, we added the thymidine analogue BrdU to cultures at 24 hours before fixation. Then the spheres were stained with an FITC-conjugated anti-BrdU antibody (1:100; Roche Diagnostics, Basel, Switzerland) at room temperature (RT) for 60 minutes in the dark. We found that BrdU labeled most of the cells in each sphere on day 10 (Fig. 1E), indicating that the spheres contained proliferating cells. These results suggested that the sphere colonies arose from single isolated HCECs and that the sphere-forming cells possess the capacity to proliferate. When the number of spheres obtained was counted after 10 days of culture, we found that 257 ± 83 spheres (mean ± SD, n=8) were generated per dish (50,000 cells). In a typical case, 2.5 ± 104 cells were isolated from a 10-mm piece of corneal tissue, generating approximately 130 spheres after 10 days. These spheres had a diameter of 88.3 ± 15.9 μm (mean ± SD, n=35). The replating efficiency showed a dramatic decline between primary and secondary sphere colonies. When the primary spheres were trypsinized and incubated in serum-free floating culture, secondary colonies were generated (Fig. 1F) at a level of approximately 15 ± 1 (n=3) per dish of 10,000 cells. This suggests that HCECs have the capacity for self-renewal and formation of sphere colonies, but this capacity is limited.

**Figure 1.** Sphere formation from donor human corneal endothelium. (A) Anterior view of a human cornea and a dia‐ gram of the corneal epithelium and stroma. Stromal keratocytes were isolated from specimens obtained from both the peripheral cornea (7.5-10.0 mm in diameter) and the central cornea (7.5 mm in diameter). (B-F) Sphere formation by human corneal endothelial cells (HCECs). After disaggregation into single cells, HCECs were plated at a density of 10 viable cells/μL in basal medium (B). More than 99% of the cells were single cells on day 0. (C) Spheres were com‐ pletely DiI-positive or DiI-negative after culture at a density of 10 viable cells/μL. (D) The mean (±SD) sphere diameter was 88.3 ± 15.9 μm on day 10. (E) Each sphere colony was labeled with BrdU on day 10. (F) Secondary spheres gener‐ ated after the dissociation of primary spheres. The replating efficiency was much lower than that of the primary spheres. Scale bar=100 μm. (G) The number of primary spheres obtained was compared between the peripheral and central regions of the cornea. The number of sphere colonies obtained from the peripheral cornea (n=14) after 10 days of culture was significantly higher than that obtained from the central cornea (n=10) (unpaired *t*-test). This ex‐ periment was repeated 3 times using different donor corneas, and representative data are shown as the mean ± SD. \*P<0.0001. These figures were modified from Yokoo et al. (2005) and Yamagami (2007) with permission.

#### **3.2. Distribution of sphere colonies derived from human Corneal Endothelial cells**

isolated HCECs and that the sphere-forming cells possess the capacity to proliferate. When the number of spheres obtained was counted after 10 days of culture, we found that 257 ± 83 spheres (mean ± SD, n=8) were generated per dish (50,000 cells). In a typical case, 2.5 ± 104 cells were isolated from a 10-mm piece of corneal tissue, generating approximately 130 spheres after 10 days. These spheres had a diameter of 88.3 ± 15.9 μm (mean ± SD, n=35). The replating efficiency showed a dramatic decline between primary and secondary sphere colonies. When the primary spheres were trypsinized and incubated in serum-free floating culture, secondary colonies were generated (Fig. 1F) at a level of approximately 15 ± 1 (n=3) per dish of 10,000 cells. This suggests that HCECs have the capacity for self-renewal and formation of sphere

**Number of Primary Spheres / 5,000 cells**

**\***

**0 5 10 15 20**

**Figure 1.** Sphere formation from donor human corneal endothelium. (A) Anterior view of a human cornea and a dia‐ gram of the corneal epithelium and stroma. Stromal keratocytes were isolated from specimens obtained from both the peripheral cornea (7.5-10.0 mm in diameter) and the central cornea (7.5 mm in diameter). (B-F) Sphere formation by human corneal endothelial cells (HCECs). After disaggregation into single cells, HCECs were plated at a density of 10 viable cells/μL in basal medium (B). More than 99% of the cells were single cells on day 0. (C) Spheres were com‐ pletely DiI-positive or DiI-negative after culture at a density of 10 viable cells/μL. (D) The mean (±SD) sphere diameter was 88.3 ± 15.9 μm on day 10. (E) Each sphere colony was labeled with BrdU on day 10. (F) Secondary spheres gener‐ ated after the dissociation of primary spheres. The replating efficiency was much lower than that of the primary spheres. Scale bar=100 μm. (G) The number of primary spheres obtained was compared between the peripheral and central regions of the cornea. The number of sphere colonies obtained from the peripheral cornea (n=14) after 10 days of culture was significantly higher than that obtained from the central cornea (n=10) (unpaired *t*-test). This ex‐ periment was repeated 3 times using different donor corneas, and representative data are shown as the mean ± SD.

\*P<0.0001. These figures were modified from Yokoo et al. (2005) and Yamagami (2007) with permission.

colonies, but this capacity is limited.

432 Regenerative Medicine and Tissue Engineering

**G**

**Group**

**Center (<7.5mm, n=10)**

**Periphery (7.5-10mm, n=14)**

HCECs were obtained from the central cornea (up to 7.5 mm from the center) and the peripheral cornea (from 7.5 to 10 mm) (Fig. 1A). As a result, the number of primary sphere colonies per 5,000 cells (mean ± SD) was significantly higher when peripheral HCECs were used (13.6 ± 3.5 spheres/5,000 cells) than when central HCECs were used (3.3 ± 1.6 spheres/ 5,000 cells) (Fig. 1G). The rate of sphere formation by HCECs from the peripheral cornea was approximately 4 times that for HCECs from the central cornea in repeated experi‐ ments (data not shown).

It has generally been accepted that human CE does not proliferate after birth, but our findings and some previous reports suggest that the CE may undergo slow proliferation *in vivo*. In 2003, Amann et al. demonstrated that paracentral and peripheral HCECs exist at a higher density than central HCECs by specular microscopy and histological observation of donor corneas. The presence of slowly proliferating HCEC precursors in the peripheral cornea could explain this higher cell density at the periphery. Otherwise, the cell density should be uniform throughout the corneal endothelium, because it tends to equalize over time. Another sugges‐ tive point is the outcome of Sato's method of anterior–posterior refractive surgery that involves making multiple peripheral and midperipheral incisions in the endothelium and stromal layer from the anterior chamber to treat myopia (Kanai et al., 1982; Kawano et al., 2003). This type of radial keratotomy performed via the anterior chamber leads to a decrease of HCECs many years later, possibly as a result of the corneal incisions causing more rapid cell loss than would occur with normal aging (Kanai et al., 1982; Kawano et al., 2003). It is possible that direct damage to HCEC precursors slows their proliferation, so that replacement of CECs decreases. The third point to consider is the outcome of corneal transplantation for various conditions associated with damage to the cornea, such as bullous keratopathy, keratoconus, and corneal leukoma. In hosts who retain their peripheral CE, such as patients with keratoconus, the grafts survive for much longer than in hosts with loss of the peripheral endothelium, such as patients with bullous keratopathy (Boisjoly et al., 1993; Williams et al., 1992; Yamagami et al., 1996). Keratoconus patients are typically younger than those with bullous keratopathy, so it could be suggested that their peripheral endothelium has greater proliferative potential because of this age difference, but differentiation of CEC precursors from the host cornea augmenting viable cells from the graft may be another reason for the longer survival of grafts after transplantation for keratoconus compared with bullous keratopathy. Therefore, when fullthickness corneal transplantation is done, a larger graft may be preferred for eyes with bullous keratopathy because it can supply more HCEC precursors, whereas a smaller graft may allow the optimum use of host-derived HCEC precursors in patients with keratoconus.

#### **3.3. Characterization of primary spheres derived from human Corneal Endothelium**

Immunocytochemical analysis of 10-day spheres was performed as follows. The spheres were fixed with methanol (Wako Pure Chemical Industries) in phosphate-buffered saline (PBS) for 10 minutes, washed in PBS, and incubated for 30 minutes with 3% bovine serum albumin (BSA) in PBS containing 0.3% Triton X20 (BSA/PBST) to block nonspecific staining. Then, the spheres were incubated for 2 hours at RT with the following specific primary antibodies diluted in BSA/PBST: mouse anti-vimentin monoclonal antibody (mAb) (1:300; Dako, Glostrup, Den‐ mark), mouse anti-nestin mAb (1:200; BD PharMingen, San Diego, CA), rabbit anti-p75 neurotrophin receptor (p75 NTR) polyclonal antibody (pAb) (1:200; Promega Corp., Tokyo, Japan), mouse anti-neurofilament 145 mAb (NFM, 1:400; Chemicon, Temecula, CA), rabbit anti 3-tublin pAb (1:2000; Covance Research Products, Denver, PA), rabbit anti-glial fibrillary acidic protein (GFAP) pAb (1:400; Dako), mouse anti-O4 mAb (1:10; Chemicon), rabbit antiperipherin pAb (1:100; Chemicon), and mouse anti-α-smooth muscle actin (α-SMA) mAb (1:200; Sigma-Aldrich). As a control, mouse IgG (1:1000; Sigma-Aldrich) or normal rabbit serum (1:1000; Dako) was used instead of the primary antibody. After the spheres were washed in PBS, incubation was done for 1 hour at RT with the appropriate secondary antibody diluted in BSA/PBST. The secondary antibodies were fluorescent-labeled goat anti-mouse IgG (Alexa Fluor 488, 1:200; Molecular Probes) and fluorescent-labeled goat anti-rabbit IgG (Alexa Fluor 594, 1:400; Molecular Probes). Nuclei were counterstained with Hoechst 33342 (1:2000; Molecular Probes). After another wash in PBS, the spheres were examined under a laser scanning confocal microscope (Fluoview; Olympus, Tokyo, Japan). When anti-O4 or antip75NTR mAb was used, the permeabilization step was omitted.

Figure 2A shows a bright-field image of a typical sphere colony. Spheres derived from HCECs were not stained by nonimmune mouse IgG (Fig. 2D) or normal rabbit serum (Fig. 2G). Nestin has been used as a marker for the detection of immature neural progenitor cells in multipo‐ tential sphere colonies derived from the brain (Gage, 2000), skin (Toma et al., 2001), inner ear (Li et al., 2003), retina (Tropepe et al., 2000), corneal epithelium (Mimura et al., 2010a; Yokoo et al., 2008), corneal stroma (Amano et al., 2006; Mimura 2008a, 2008b; Uchida et al., 2005; Yamagami et al., 2007), and CE (Amano et al., 2006; Mimura 2005a, 2005b, 2005c, 2007, 2010b; Yokoo et al., 2005, Yamagami, 2006, 2007). Expression of α-SMA (a marker of mesenchymal myofibroblasts) and expression of p75 NTR (a marker of neural crest stem cells) was also investigated by immunocytochemistry because HCECs are derived from the neural crest. Cells in the spheres showed immunoreactivity for nestin (Fig. 2B) and for α-SMA (Fig. 2C), but not for p75 NTR (data not shown). Next, the spheres were immunostained for various neural markers. As a result, spheres were found to be positive for an immature neuronal marker (β3 tubulin, Fig. 2E) and an astroglial marker (GFAP, Fig. 2F), but not a mature neuronal marker (NFM), an oligodendroglial marker (O4), or a peripheral nerve neuronal marker (peripherin; data not shown). These findings indicated that spheres isolated from human donor CE contain bipotential precursors that are capable of undergoing differentiation into mesenchymal cells and neuronal cells.

#### **3.4. Secondary sphere formation**

To further evaluate the proliferative capacity of HCECs, cells from the primary spheres were passaged under the same conditions as those used for the initial sphere culture. On day 10, primary spheres were treated with 0.05% trypsin/0.02% EDTA and dissociated into single cells, which were added to 24-well culture plates at a density of 10 cells/μL in medium containing primary culture supernatant. These cells were then incubated for a further 10 days in basal medium.

Secondary spheres were generated from the dissociated primary spheres, but the yield of secondary sphere colonies was lower than after primary culture. Although self-renewal potential was indicated by the ability of cells from individual primary spheres to form secondary spheres, this potential was limited, as evidenced by the failure of sphere formation at the third passage. These results indicated that the precursor cells had a limited proliferative capacity. Photographs of representative secondary spheres are shown in Figure 1F. days in basal medium. Secondary spheres were generated from the dissociated primary spheres, but the yield of secondary sphere colonies was lower than after primary culture. Although self-renewal potential was indicated by the ability of cells from individual primary spheres to form secondary spheres, this potential was limited, as evidenced by the failure of sphere formation at the third passage. These results indicated that the precursor cells had a limited proliferative capacity. Photographs of representative secondary spheres are shown in Figure 1F.

containing primary culture supernatant. These cells were then incubated for a further 10

Fig. 2. Immunocytochemistry (A-H) and RT-PCR analysis (I) of sphere colonies and their progeny. (A) Bright-field image of a typical sphere colony. (B) Immunostaining of the entire sphere on day 10 identifies cells expressing nestin, a marker of immature cells. (C-G) Spheres show immunostaining for a mesenchymal myofibroblast marker (-SMA, C), an immature neuronal marker (3-tubulin, E), and an astroglial cell marker (GFAP, F), indicating that both mesenchymal and neuronal differentiation have occurred. Sphere colonies derived from HCECs are not stained by nonimmunized mouse IgG (D) or normal rabbit serum (G). Differentiated cells derived from primary spheres are double immunostained by nestin and 3-tubulin, indicating that the colonies contain immature (undifferentiated) cells. (I) RT-PCR analysis of cells from spheres and their progeny. GAPDH gene expression is detected in the sphere colonies and their progeny (30 cycles), but not when reverse transcription is omitted. Nestin, -SMA, 3-tubulin, and GFAP genes are **Figure 2.** Immunocytochemistry (A-H) and RT-PCR analysis (I) of sphere colonies and their progeny. (A) Bright-field im‐ age of a typical sphere colony. (B) Immunostaining of the entire sphere on day 10 identifies cells expressing nestin, a marker of immature cells. (C-G) Spheres show immunostaining for a mesenchymal myofibroblast marker (α-SMA, C), an immature neuronal marker (β3-tubulin, E), and an astroglial cell marker (GFAP, F), indicating that both mesenchy‐ mal and neuronal differentiation have occurred. Sphere colonies derived from HCECs are not stained by nonimmu‐ nized mouse IgG (D) or normal rabbit serum (G). Differentiated cells derived from primary spheres are double immunostained by nestin and β3-tubulin, indicating that the colonies contain immature (undifferentiated) cells. (I) RT-PCR analysis of cells from spheres and their progeny. GAPDH gene expression is detected in the sphere colonies and their progeny (30 cycles), but not when reverse transcription is omitted. Nestin, α-SMA, β3-tubulin, and GFAP genes are detected in both spheres and their progeny, but not when total RNA is processed without reverse transcription (35 cycles). Scale bars=100μ m (A-G) or 200μ m (H). Figures are modified from Yokoo et al. (2005) with permission.

detected in both spheres and their progeny, but not when total RNA is processed without

#### reverse transcription (35 cycles). Scale bars=100 m (A-G) or 200 m (H). Figures are modified from Yokoo et al. (2005) with permission. **3.5. Differentiation of sphere colonies**

BSA/PBST: mouse anti-vimentin monoclonal antibody (mAb) (1:300; Dako, Glostrup, Den‐ mark), mouse anti-nestin mAb (1:200; BD PharMingen, San Diego, CA), rabbit anti-p75 neurotrophin receptor (p75 NTR) polyclonal antibody (pAb) (1:200; Promega Corp., Tokyo, Japan), mouse anti-neurofilament 145 mAb (NFM, 1:400; Chemicon, Temecula, CA), rabbit anti 3-tublin pAb (1:2000; Covance Research Products, Denver, PA), rabbit anti-glial fibrillary acidic protein (GFAP) pAb (1:400; Dako), mouse anti-O4 mAb (1:10; Chemicon), rabbit antiperipherin pAb (1:100; Chemicon), and mouse anti-α-smooth muscle actin (α-SMA) mAb (1:200; Sigma-Aldrich). As a control, mouse IgG (1:1000; Sigma-Aldrich) or normal rabbit serum (1:1000; Dako) was used instead of the primary antibody. After the spheres were washed in PBS, incubation was done for 1 hour at RT with the appropriate secondary antibody diluted in BSA/PBST. The secondary antibodies were fluorescent-labeled goat anti-mouse IgG (Alexa Fluor 488, 1:200; Molecular Probes) and fluorescent-labeled goat anti-rabbit IgG (Alexa Fluor 594, 1:400; Molecular Probes). Nuclei were counterstained with Hoechst 33342 (1:2000; Molecular Probes). After another wash in PBS, the spheres were examined under a laser scanning confocal microscope (Fluoview; Olympus, Tokyo, Japan). When anti-O4 or anti-

Figure 2A shows a bright-field image of a typical sphere colony. Spheres derived from HCECs were not stained by nonimmune mouse IgG (Fig. 2D) or normal rabbit serum (Fig. 2G). Nestin has been used as a marker for the detection of immature neural progenitor cells in multipo‐ tential sphere colonies derived from the brain (Gage, 2000), skin (Toma et al., 2001), inner ear (Li et al., 2003), retina (Tropepe et al., 2000), corneal epithelium (Mimura et al., 2010a; Yokoo et al., 2008), corneal stroma (Amano et al., 2006; Mimura 2008a, 2008b; Uchida et al., 2005; Yamagami et al., 2007), and CE (Amano et al., 2006; Mimura 2005a, 2005b, 2005c, 2007, 2010b; Yokoo et al., 2005, Yamagami, 2006, 2007). Expression of α-SMA (a marker of mesenchymal myofibroblasts) and expression of p75 NTR (a marker of neural crest stem cells) was also investigated by immunocytochemistry because HCECs are derived from the neural crest. Cells in the spheres showed immunoreactivity for nestin (Fig. 2B) and for α-SMA (Fig. 2C), but not for p75 NTR (data not shown). Next, the spheres were immunostained for various neural markers. As a result, spheres were found to be positive for an immature neuronal marker (β3 tubulin, Fig. 2E) and an astroglial marker (GFAP, Fig. 2F), but not a mature neuronal marker (NFM), an oligodendroglial marker (O4), or a peripheral nerve neuronal marker (peripherin; data not shown). These findings indicated that spheres isolated from human donor CE contain bipotential precursors that are capable of undergoing differentiation into mesenchymal cells

To further evaluate the proliferative capacity of HCECs, cells from the primary spheres were passaged under the same conditions as those used for the initial sphere culture. On day 10, primary spheres were treated with 0.05% trypsin/0.02% EDTA and dissociated into single cells, which were added to 24-well culture plates at a density of 10 cells/μL in medium containing primary culture supernatant. These cells were then incubated for a further 10 days in basal

p75NTR mAb was used, the permeabilization step was omitted.

and neuronal cells.

medium.

**3.4. Secondary sphere formation**

434 Regenerative Medicine and Tissue Engineering

Individual primary spheres (day 10) were transferred to 13 mm glass coverslips coated with 50 μg/ml poly-L-lysine (PLL) and 10 μg/ml fibronectin (BD Biosciences, Billerica, MA) in separate wells, as described previously (Reynolds & Weiss, 1992). To promote differentiation, 1% fatal bovine serum (FBS) was added to the basal medium, and culture was continued for another 7 days. Immunocytochemical examination of spheres and their progeny was per‐ formed after 7 days of adherent culture on glass coverslips.

To investigate whether sphere progeny possessed the characteristics of mesenchymal or neural cells, single spheres (day 10) were transferred onto PLL/laminin-coated glass coverslips in medium containing 1% or 15% FBS or onto bovine ECM-coated culture plates in medium containing 15% FBS. Spheres remained adherent to the PLL/laminin-coated glass coverslips, 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 spheres for α-SMA, p75NTR, NFM, peripherin, GFAP, or O4.

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 in HCECs from primary culture.

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 the multipotential stem cells.
