Multipotent Stem Cells in Human Corneal Stroma



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江边孤钓

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发表于 2009-3-5 10:48 |显示全部帖子

University of Pittsburgh Medical Center Eye Center, Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

Key Words. Cornea ? Keratocyte ? Keratocan ? Keratan sulfate ? ABCG2 ? PAX6 ? Side population ? Adult stem cells ? Progenitor cells ? Chondrogenesis( }2 ^2 U  ]+ r# q3 U" ~
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Correspondence: James L. Funderburgh, Ph.D., Department of Ophthalmology, University of Pittsburgh, 1009 Eye and Ear Institute, 203 Lothrop St., Pittsburgh, PA 15213-2588, USA. Telephone: 412-647-3853; Fax: 412-647-5880; e-mail: jlfunder@pitt.edu

ABSTRACT

As the outermost layer of the eye, the cornea both serves as a barrier and provides an optical function, transmitting and focusing incident light on the retina. The corneal stroma is a connective tissue making up 90% of the corneal thickness, with physical properties that provide the cornea its essential character. The stroma is composed of collagenous lamellae consisting of tightly packed collagen fibrils embedded in a hydrated matrix of glycoproteins and proteoglycans. The keratocytes are a population of quiescent, neural crest–derived cells, sandwiched between the lamellae, responsible for secretion of the unique stromal extracellular matrix. In response to acute injury, keratocytes become mitotic, adopt a fibroblastic phenotype, and move to the injured area . This fibroblastic activation results in deposition of new extracellular matrix  with a composition and light transmission properties different from that of the normal, uninjured stroma. Stromal scarring can also result from a number of chronic conditions in addition to acute wound healing. Scar tissue is long-lasting and can cause a permanent disruption of vision.

Keratoplasty is currently the only effective method providing recovery of vision after corneal blindness. Although donated corneal tissue currently meets the needs of most recipients in the U.S., worldwide 8 to 10 million individuals suffer from corneal blindness without access to therapy. Additionally, numerous individuals reject allogeneic corneal tissue, and the supply of donated corneas may soon be reduced by the increasing number of refractive surgeries. Because of these problems, there is significant interest in development of artificial and bioengineered corneas. Griffith et al.  have demonstrated that corneal equivalents generated from the three corneal cell layers mimic human corneas in key physical and physiological functions. These studies used immortalized cell lines transformed with retrovirus, consequently not suitable for transplantation. Focus has therefore turned to stem cells as a source of tissues for use in cell-based therapy and corneal tissue engineering.
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Stem cells have self-renewal ability as well as the capability for multilineage differentiation and in vivo functional reconstruction of several tissues . Embryonic stem cells derived from the inner cell mass of the blastocyst are pluripotent cells that can be propagated indefinitely in an undifferentiated state. Stem cells exist in many adult tissues as well. Compared with embryonic stem cells, tissue-specific stem cells have less self-renewal ability. However, recent studies suggest that tissue-specific stem cells can differentiate into lineages other than the tissue of origin . Adult stem cells occur in small numbers compared with the somatic cells of the tissue, and isolation can be difficult. Adult stem cells have the ability to efflux fluorescent dye Hoechst 33342, leading to reduced red and blue fluorescence in fluorescence-activated cell sorting (FACS) . These cells are referred to as "side population" (SP) cells. The SP cells are lost after treatment with verapamil, a drug that blocks action of the ATP-binding cassette transporter G family member known as ABCG2. This transporter protein has been identified as the Hoechst efflux pump  and as a specific marker for many kinds of stem cells such as hematopoietic , mesenchymal , muscle , neural , cardiac , islet , and keratinocyte  stem cells. Recently stem cells in the corneal epithelium have been shown to express ABCG2 and to separate as an SP ; however, no studies have identified SP cells or other forms of stems cells from the human corneal stroma, nor has generation of differentiated human keratocytes from stem cells been demonstrated.
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In this study, we identify an SP of cells from the human corneal stroma which exhibits many aspects of stem cells, including clonal growth in vitro, extended lifespan, and the ability to differentiate into several different cells types, including keratocytes of the cornea.

MATERIALS AND METHODS
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ABCG2-Expressing Cells in the Corneal Stroma

The ATP-binding cassette transporter ABCG2 has been identified as a specific marker for stem cells in a number of adult tissues . We hypothesized that similar to other adult tissues, stem cells in the corneal stroma would express the ABCG2 protein. Immunofluorescent staining of frozen human corneal sections, using a polyclonal antibody to extracellular domains of the ABCG2 protein, identified positive cells in human corneal stroma. These cells were rare but most often observed in the peripheral region of the stroma posterior to the limbal feature known as the Palisades of Vogt, in which the epithelium exhibits extensive folding (Figs. 1A, 1B). These results confirmed recent reports  that epithelial cells in this region express ABCG2 protein (Fig. 1B, triangles). A number of cells in the anterior stroma in this region also expressed ABCG2 (Fig. 1B, arrows). A second monoclonal antibody against ABCG2, Clone BXP21, identified a similar population of cells in the anterior stroma (Fig. 1C, arrows). This antibody also stained rare cells in more central regions of the stroma in which epithelial staining was absent (Fig. 1D).
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Figure 1. ABCG2 protein localization in human cornea. (A): Transverse frozen sections were obtained from the limbal and central regions of unfixed human corneas as indicated by white lines in the photograph. (B): ABCG2 protein (green) was detected in epithelial and stromal cells of limbal sections, using the antibody described by Litman et al. . Cells were counterstained with To-Pro-3 (red). Arrows show stained stromal cells and triangles indicate stained epithelial cells. Inset shows magnification of two cells designated by arrows. (C): ABCG2 protein could also be also demonstrated in limbal sections, using monoclonal antibody BXP21 (green). (D): ABCG2 was detected in occasional stromal cells in central cornea (arrow), using antibody BXP21 as in (C). Solid white bars = 50 μM.
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The ABCG2 transporter protein acts to remove Hoechst dye from cells, thus allowing isolation of cells in which ABCG2 is active by use of FACS . To get enough human stromal cells for FACS analysis, primary cultures were expanded in a culture medium (SCGM) modified from Jiang et al.  in which stem cells can proliferate without loss of differentiation potential. In passage four, stromal cells stained with Hoechst 33342 contained an SP of 1.3% of the cells in which both red and blue Hoechst fluorescence were reduced (Fig. 2A, arrow). Preincubation with verapamil, an inhibitor of ABCG2 function (Fig. 2B), virtually eliminated the SP cells. The isolated SP cells were cultured in SCGM and then were subcultured and cloned.8 d- \5 p0 M3 p
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Figure 2. Flow cytometric isolation of side population from cultured human corneal stromal cells. (A): Passage-four stromal cells stained with Hoechst 33342 were analyzed using 350-nm excitation with blue (635 nm) and red (488 nm) emission. Cells showing reduction of both blue and red fluorescence (side population cells) were collected as defined by the box outlined on the left (arrow). (B): An analysis similar to (A) but with a preincubation in 50 μM verapamil before incubation with Hoechst 33342.
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At passage 18, the cloned SP cells continued to express mRNA for ABCG2, as did noncultured primary stromal cells. SP cells transferred into a variety of media in which they exhibited differentiated characteristics showed a significant (p % c) \; `3 c8 m% g, T4 j
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Figure 3. Expression of ABCG2 in cultured side population cells. (A): mRNA for ABCG2 was detected in stromal cells by quantitative reverse transcription–polymerase chain reaction. Side population cells, selected as in Figure 2 and maintained in culture for 18 serial passages in SCGM, were compared with freshly isolated uncultured stromal cells (P0) and with side population cells after conditioning 1–3 weeks with KDM, NDM, CDM, and 10% FBS. After passaging four times in FBS, cells were transferred back to SCGM (FBS-SCGM). (B): ABCG2 protein expression in SCGM was detected by immunostaining cultured cells and by immunoblotting (inset). (C): ABCG2 detected in KDM as in (B). Error bars show SD of triplicate analyses. Asterisks indicate significant difference (p - s2 j" ?7 u% w
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PAX6 Expression in ABCG2-Positive Stromal Cells
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PAX6 is a homeobox transcription factor expressed in embryonic ocular precursor cells and epithelial cells but absent in adult keratocytes . Immunostaining for PAX6 showed rare stromal cells expressing this protein (Figs. 4A, 4B). These PAX6-positive stromal cells also stained for ABCG2 (Fig. 4C). We found that the cultured SP cells maintained a strong nuclear staining for PAX6 (Fig. 4D). Transcripts for PAX6 could be amplified from RNA from corneal epithelial cells and from noncultured stromal cells (Fig. 4E) and, as expected, PAX6 mRNA was present in SCGM-cultured SP cells. PAX6, however, was absent in SP cells cultured in KDM or in fibroblastic cells (Fig. 4E). To control for epithelial cell contamination in the stromal and SP cell preparations, we examined the same RNA samples for the presence of keratin 12, an epithelial-specific gene product. As shown in Figure 4F, keratin 12 mRNA was detected only in epithelial cells but not in the unfractionated stromal cells or in any of the SP cell populations. These experiments show the SP cells to have a specific phenotype, stable through subculture, cloning, and more than 20 serial passages. The absence of keratin 12 marker in the unfractionated cells from which the SP cells are derived demonstrates this population to have been isolated without contamination by epithelial cells.

Figure 4. PAX6 expression in stromal cells. (A): PAX6 protein (green) was detected in nuclei of corneal epithelial cells but rarely in stromal cells. Counterstain was DiD. (B): Magnification of the inset of (A) showing a single PAX6-positive stromal cell (arrow). (C): PAX6 (green) colocalized with ABCG2 staining (red) in stromal cells. (D): SP cells in SCGM were stained for PAX6 protein (green) and counterstained with cytoplasmic myosin (red). (E): Reverse transcription–polymerase chain reaction showed PAX6 mRNA present in freshly isolated stromal cells (P0) and epithelial cells (Epi) as well as SP cells cultured in SCGM, but not in SP cells cultured in KDM or FBS. (F): mRNA for keratin 12 was detected in freshly isolated epithelial cells but not in freshly isolated stromal cells (P0) or SP cells under any test conditions. Bars = 30 μM. Abbreviations: FBS, fetal bovine serum; KDM, keratocyte differentiation medium; NDM, neural differentiation medium; SCGM, stem cell growth medium; SP, side population.

Keratocyte Differentiation# Y& |3 }  U1 x3 h

In acute wound healing, keratocytes rapidly become fibroblastic. Similarly, keratocytes in vitro irreversibly lose their characteristic phenotype. We found that the human stromal SP cells maintained the ability to assume several well-identified aspects of the keratocyte phenotype. When transferred to a serum-free medium containing FGF2 (KDM) for 1 week or more, cloned SP cells upregulated expression of mRNA for keratocan, a proteoglycan core protein that in adults has expression uniquely localized in the corneal stroma (Fig. 5A). Uncultured stromal cells, primarily keratocytes, express this gene but SP cells in SCGM do not. SP cells differentiated to fibroblasts in 10% FBS did not express keratocan either. The expression of keratocan protein was readily detected by immunostaining of the SP cells after incubation in KDM (Fig. 5C). Keratocan could also be isolated from culture media by ion exchange chromatography as a proteoglycan. The keratocan protein was detected in this proteoglycan fraction after treatment with keratanase and Western blotting (Fig. 5D). Keratan sulfate, a glycosaminoglycan unique in abundance in the corneal stroma, was also detected in the culture media of the SP cells in KDM using immunoblotting (Fig. 5D) as was an increased abundance of ALDH, a protein present in elevated amounts in keratocytes in vivo.4 x4 I4 b; Y: {3 }& c( @/ a# I' D2 f) E
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Figure 5. Expression of keratocyte-specific genes by SP cells. (A): Keratocan mRNA was amplified by reverse transcription–polymerase chain reaction in uncultured primary stromal cells (P0) and passage-18 SP cells in SCGM, KDM, or FBS medium as described in Materials and Methods. (B, C): Keratocan protein was detected by immunostaining in SP cells cultured in SCGM (B) or after transfer to KDM (C). (D): Keratocan protein, keratan sulfate, and ALDH were detected by immunoblotting of extracts of passage-18 SP cells cultured in SCGM or after transfer to KDM. Abbreviations: ALDH, aldehyde dehydrogenase; FBS, fetal bovine serum; KDM, keratocyte differentiation medium; NDM, neural differentiation medium; SCGM, stem cell growth medium; SP, side population.3 g5 F& e1 d% G( t& Z- I. ?
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Differentiation to Nonocular Cells

One well-documented aspect of adult stem cells is the ability to differentiate into a number of different cell types. We examined this property in the cloned SP cells by culturing them as a pellet in medium found to induce chondrogenesis in adult stem cells . Under these conditions, mRNA and protein for several cartilage-specific extracellular matrix molecules could be detected (Fig. 6). mRNAs for collagen II, aggrecan, and cartilage oligomatrix protein (COMP) were detected in the chondrogenic conditions but not in the SP precursor, keratocyte, or fibroblastic cells (Fig. 6A). Collagen II and COMP protein expression was confirmed using immunoblotting (Fig. 6B). After several weeks, a significant amount of extracellular matrix was deposited in the pellet cultures. The matrix deposited under chondrogenic conditions stained strongly with toluidine blue, a characteristic resulting from proteoglycan accumulation typical of cartilage (Figs. 7B, 7D). Pellet cultures in SCGM failed to deposit a similar matrix (Figs. 7A, 7C).7 w& c% k5 M! e9 z# ?+ T/ G3 [
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Figure 6. Induction of chondrocyte-specific genes in side population cells. (A): mRNAs for collagen type II, COMP, and aggrecan were detected by RT-PCR in side population cells under culture conditions described in Figure 3. (B): Protein expression of collagen II and COMP were demonstrated by immunoblotting under the same culture conditions. Abbreviations: CDM, chondrocyte differentiation medium; COMP, cartilage oligomatrix protein; FBS, fetal bovine serum; KDM, keratocyte differentiation medium; RT-PCR, reverse transcription–polymerase chain reaction.- g- U% u4 Y/ X8 v6 K; v* r% a

Figure 7. Induction of cartilage matrix in pellet culture. Side population cells (2 x 105) were cultured as pellets in (A, C) SCGM or (B, C) CDM for 3 weeks as described in Materials and Methods. Sections of the pellets were stained using toluidine blue to detect proteoglycan staining typical of cartilage. In addition, (C, D) were counterstained with hematoxylin to visualize cell nuclei. Two magnifications are shown. Bars = 100 μ (A, B), 50 μ (C, D). Abbreviations: CDM, chondrocyte differentiation medium; SCGM, stem cell growth medium.7 S( m; H6 x! F, v/ e# K
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When the cloned SP cells were incubated with high levels of FGF2 and retinoic acid in a medium reported to induce neurogenesis, mRNA upregulation of both GFAP and neurofilament protein was observed (Fig. 8A). ANOVA analysis found a statistically significant (p 0 n, F; g) G" Q) i- f( l
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Figure 8. Induction of neural gene expression in SP cells. (A): mRNA pools for NF and GFAP were quantified by reverse transcription–polymerase chain reaction in SP cells exposed to NDM, SCGM, FBS, or NDM after culture in FBS (FBS-NDM) as described in Materials and Methods. Error bars show SD of triplicate analyses. Asterisks indicate significant (p

DISCUSSION

The authors would like to thank Dr. Hongmei Shen for her help in cell sorting and Cindy Stone for her help in the histology. This work was supported by National Institutes of Health grants EY13806, EY09368, and P30-EY08098, Research to Prevent Blindness, and Eye and Ear Foundation of PGH. J.L.F. is a Jules and Doris Stein Research to Prevent Blindness Professor.

DISCLOSURES$ R* J5 k2 h! X7 [* d6 W
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The authors indicate no potential conflicts of interest.
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