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PAX6 expression identifies progenitor cells for corneal keratocytes

Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

¹Correspondence: Eye and Ear Institute, Rm. 1009, 203 Lothrop St., Pittsburgh, PA 15213, USA. E-mail: jlfunder{at}pitt.edu

SPECIFIC AIM

The corneal stroma is populated by keratocytes, specialized cells responsible for biosynthesis of the unique transparent connective tissue of the cornea. Keratocytes have a limited ability to replicate without loss of their differentiated phenotype. Both in vivo and in vitro, dividing keratocytes become fibroblastic, secreting nontransparent fibrotic extracellular matrix. The hypothesis of this study was that cornea, like many other adult tissues, contains a population of progenitor cells capable of giving rise to differentiated keratocytes even after extensive proliferation. The data presented here identify and characterize a unique population of cells in adult corneal stroma capable of extensive proliferation in vitro and generation of keratocyte daughter cells. This population is characterized by expression of a number of genes associated with stem cells, as well as PAX6, a homeobox gene expressed during ocular development.

PRINCIPAL FINDINGS

1. Clonogenic cells in cornea
One characteristic of stem and progenitor cells from adult tissues is the ability to grow clonally. When freshly isolated bovine stromal cells were cultured in a reduced-serum medium supplemented with growth factors, previously reported to clone and maintain adult stem cells, 3% of the cells were found to grow clonally. A portion of these clones exhibited a dendritic morphology similar that of keratocytes and secreted proteoglycans containing keratan sulfate, a specialized stromal matrix component. Several clonal lines were selected for further analysis.

2. Properties of the clonal stromal cells
The clonal cells did not exhibit properties typical of keratocytes during growth, but when cultures where transferred to serum-free or low-mitogen medium, the cells assumed a phenotype very similar to that of primary keratocytes. As shown in Fig. 1 A, three clonal lines each secreted high molecular weight keratan sulfate proteoglycan, a unique molecular component of the corneal stroma and a marker of the keratocyte phenotype. Two other keratocyte molecular markers were expressed by the cloned cells: keratocan (Kera), a cornea-specific proteoglycan core protein, and aldehyde dehydrogenase 3A1 (ALDH), a housekeeping gene unusually abundant in keratocytes in vivo. The mRNA abundance of both of these genes was similar in clonal cells after 24 population doublings to mRNA of quiescent primary keratocytes (Fig. 1B ). In contrast, cultures of keratocytes induced to the fibroblastic phenotype by four passages in 10% fetal bovine serum had reduced expression of these genes by 50- to 200-fold. Keratocytes in vivo and in quiescent primary cultures in vitro exhibit a dendritic morphology. As shown in Fig. 1C , filamentous actin of primary keratocytes is principally localized in the dendritic processes and does not organize into stress fibers. Vinculin was diffusely distributed. Clonal cells from the stroma exhibited a strikingly similar morphology even after 50 cumulative population doublings (CPD) in culture (Fig. 1D ), whereas passage four keratocyte-derived fibroblasts displayed a classic fibroblastic cytoskeletal arrangement, with heavy actin stress fibers crossing the center of the cell anchored to vinculin-containing focal adhesions. Because of this ability to produce differentiated keratocytes after extensive expansion in vitro, the clonogenic cells appear to represent a population of progenitor cells, resident in the stroma but distinct from the majority of keratocytes.

View larger version (54K): [in this window] [in a new window]   Figure 1. Phenotype of cloned stromal cells. A) Proteoglycans from culture media of three selected stromal clones (CPD 22) were assayed for keratan sulfate-containing proteoglycans by immunoblotting after SDS-PAGE using monoclonal antibody J19 with (+) or without (–) pretreatment with keratanase. B) mRNA pools for keratocan (shaded) and ALDH (open) were determined by quantitative RT-PCR in primary keratocytes, clonal stromal cells (CPD 24), and passage-four fibroblasts. Values are normalized to keratocytes = 100. C) Primary keratocytes were immunostained for f-actin (green) and vinculin (red). D) Clonal stromal cells (CDP 50) were stained similarly to C. E) Fibroblasts, passage four, were stained similarly to panels C, D.

3. Spheroid formation
In the course of this study, we observed serum-free medium containing insulin and fibroblast growth factor 2 induced keratocytes in monolayer cultures to aggregate into spheroids 0.1–1 mm in diameter. These spheroids detach from the substratum and survive for extended periods in suspension. The clonal progenitor lines also exhibited this behavior. Analysis of the spheroids showed them to express high levels of keratocan mRNA. These balls of cells also showed positive immunostaining for keratocan and keratan sulfate. These results suggest that spheroids contain highly differentiated keratocytes. Spontaneous formation of spheroids by keratocytes is a behavior not observed in other attachment-dependent mesenchymal cells and consequently represents a further feature marking the clonal cells as keratocyte progenitors.

4. Replicative life span
Embryonic stem cells as well as some adult stem cells have been described with extended replicative life spans of up to 300 CPD in vitro. After continuous serial passage, we found three clonal cell lines all reached replicative senescence after 65–70 CPD. This lifespan is similar to that reported for normal somatic bovine cells; thus, the progenitor cells do not appear to possess the extended lifespan observed in some stem cells. Even at late passage, however, ALDH and keratocan were up-regulated when the cells were transferred into low mitogen medium. The ability to assume a dendritic morphology was similarly maintained until senescence. The clonal lines all exhibited a normal karyotype of 60 chromosomes.

5. Stem cell gene expression by progenitor cells
We detected mRNA in clonal progenitor cells for several genes previously reported to be up-regulated in adult stem cells or in ocular progenitor cells. Using quantitative RT-PCR, we found that mRNA was significantly more abundant in progenitor cells than in keratocytes for CD73, CD90, CD166, SCF (stem cell factor), Bmi1, FHL1 (four-and-a half LIM), Notch1, Six3, Six2, PAX6, and ABCG2. This expression profile clearly distinguishes this population of cells from typical keratocytes. Protein expression of one of these genes, PAX6, was explored in more detail.

6. PAX6 expression in progenitor cells
PAX6 is a transcription factor essential for ocular development and is expressed in early ocular progenitor cells but not in adult keratocytes. PAX6 mRNA was highly up-regulated in the clonal progenitor cells. Immunostaining showed PAX6 protein to be abundant in nuclei of the clonal cells (Fig. 2 A) whereas primary keratocytes cultured under the same conditions had little PAX6 expression (Fig. 2B ). Similarly, keratocytes in intact corneas generally showed no PAX6 staining except for rare PAX6-positive cells observed throughout the stroma (Fig. 2C ). Flow cytometetric analysis carried out on freshly isolated stromal cells (Fig. 2D ) showed that 4% of these cells stain with antibodies for PAX6. These results demonstrate a small population of PAX6-positive cells to be present in stroma. Because PAX6 expression was found in all of the clonal progenitor lines, we believe it likely that the 3% of cells that grow clonally from stromal digests are representative of the 4% of the cells found to express PAX6 in the normal stroma in vivo.

View larger version (118K): [in this window] [in a new window]   Figure 2. PAX6 expression by stromal progenitor cells. A) PAX6 protein in cloned progenitor cells was immunostained (green) and counterstained for cytoplasmic myosin (red). B) PAX6 protein was not detected in primary keratocytes cultured under identical conditions. C) PAX6 was detected only in rare cells in intact corneal stroma (arrowhead). Keratocyte cell membranes were counterstained with the dye DiD (red). D) Freshly isolated stromal cells were fixed and stained for PAX6 protein, then analyzed by flow cytometry. The solid line shows results with a PAX6 primary antibody. The dashed line shows a nonimmune rabbit IgG primary. Fluorescence intensity is shown as a logarithmic scale. The arrow indicates a population of stained cells representing 4% of the total. White bars mark 50 µm.

CONCLUSIONS AND SIGNIFICANCE

We have identified a population of cells in adult corneal stroma that exhibits many aspects of progenitor cells for this specialized tissue. These cells, initially isolated by clonal growth, represent 3% or less of the cells released from the stroma by collagenase digestion. The clonal cells can be expanded for >60 population doublings. Throughout this replicative lifespan, transfer of the clonogenic cells into low mitogen induces the phenotypic characteristics of differentiated keratocytes. No other known cells, including fibroblasts derived from keratocytes, respond in a similar manner. The progenitor cells differ from keratocytes in the expression of a number of genes associated with adult stem cell populations (Bmi1, CD90, CD73, CD166, SCF, ABCG2, Notch1, FHL1) and also express high levels of PAX6, Six3, and Six2, homeobox genes expressed in embryonic ocular cells but not adult stem cells or adult keratocytes. The existence of such a population of cells has not been previously proposed or identified in the corneal stroma; however, their presence is consistent with recent identification of progenitor cells in a number of postmitotic adult tissues.

The presence of a population of stromal progenitor cells might explain patterns of tissue regeneration observed in the stroma. Corneas respond to acute wounds by activating nearby keratocytes, which assume a fibroblastic phenotype and secrete a nontransparent extracellular matrix. These corneal scars can last for decades, possibly because the fibroblasts formed in this manner do not revert to the keratocyte phenotype. In nonacute situations, such as corneal transplantation or grafting of acellular stromal tissue, several studies indicate that host cells over a period of several years invade and repopulate keratocytes in the graft without scar formation. Thus, a source of cells sufficient for replacement of the majority of adult keratocytes must be available. Because this regeneration occurs with no disruption of corneal function, it seems likely the replacement cells arise from progenitor cells of the host. Knowledge of the existence of stromal progenitor cells, and particularly of the PAX6 expression that serves as a marker, will allow determination of their roles in both wound healing and replacement in grafts. The newly described progenitor cells may eventually serve in cell-based therapy or as a starting material for bioengineered corneas.

View larger version (35K): [in this window] [in a new window]   Figure 3. Proposed origin and phenotype of progenitor cells in the adult corneal stroma. 1) Stem cells in the early embryo become neural precursor cells under the influence of Notch and BMI1 genes. 2) Neural crest cells expressing homeobox genes Six3, Six2, and PAX6 migrate to the periocular mesenchyme. 3) Periocular cells migrate into the primitive stroma. 4) The majority of new stromal cells differentiate to keratocytes, losing expression of the stem cell genes and gaining differentiated markers keratan sulfate (KS), keratocan (Kera), and aldehyde dehydrogenase 3A1 (ALDH). 5) A small fraction of the stromal cells maintain expression of the homeobox and neural progenitor genes but do not differentiate. 6) Upon stimulation to proliferate, keratocytes rapidly lose differentiated markers and become fibroblastic. 7) Progenitor cells can divide >50 times but, when returned to quiescence, assume a keratocyte phenotype.

FOOTNOTES

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