Secretion and organization of a cornea-like tissue in vitro by stem cells from human corneal stroma

Abstract

Purpose: To investigate the potential of human corneal stromal stem cells to assume a keratocyte phenotype and to organize extracellular matrix (ECM) in vitro similar to corneal stromal tissue.

Methods: Human corneal stromal stem cells (hCSSC) were isolated as side population cells by flow cytometry. Cloned hCSSC were cultured as free-floating pellets in serum-free media for 3 weeks. Gene expression was examined using gene array, quantitative RT-PCR, immunostaining, and immunoblotting. Transmission electron microscopy showed collagen fibril size and alignment.

Results: Pellet cultures of hCSSC in serum-free media upregulated the expression of keratocyte-specific genes and secreted substantial ECM containing characteristic stromal components: keratocan, keratan sulfate, collagen I, collagen V, and collagen VI. Abundant connexin 43 and cadherin 11 in pellets demonstrated cell-cell junctions typical of keratocytes in vivo. Electron microscopy of the pellet cultures revealed abundant fibrillar collagen, some of which was aligned in parallel arrays similar to those of stromal lamellae. Gene array identified expression in pellets of several genes highly expressed by keratocytes. Transcripts for these keratocyte genes -- FLJ30046, KERA, ALDH3A1, CXADR, PTGDS, PDK4, MTAC2D1, F13A1 -- were increased by as much as 100-fold in pellets compared with hCSSC. Simultaneously, expression of stem cell genes BMI1, KIT, NOTCH1, SIX2, PAX6, ABCG2, SPAG10, and OSIL was reduced by a similar factor in pellets compared with hCSSC.

Conclusions: Scaffolding-free pellet culture of hCSSC induces keratocyte gene expression patterns in these cells and secretion of an organized stroma-like ECM. These cells offer a novel potential for corneal bioengineering.

Figure 1

Three-dimensional cultures of human corneal stromal stem cells. (A) Formation of a free-floating pellet after centrifugation of 2 × 10⁵ hCSSC in a 15-mL conical polysty-rene tube (arrow). (B) Pellet has formed a smooth sphere after 1 week of culture. (C) Identification of viable cells after staining with calcein AM (green) and dead cells using propidium iodide (red, arrowhead) after 3 weeks of culture. (D) H&E staining of hCSSC cultured 3 weeks as a pellet. (E) Flattened cells near the periphery of an hCSSC pellet. (F) H&E of a pellet formed by fibroblasts cultured for 3 weeks. (G) Stained section of hCSSC cultured in a fibrin gel for 3 weeks. (H) Fibroblasts cultured in fibrin gel. Scale bars, 50 μm.

Figure 2

Keratocan and keratan sulfate expression in fibrin gel and pellet cultures. (A) Relative abundance of keratocan mRNA assayed by qRT-PCR. (B) Immunoblotting for keratocan. (C) Immunoblotting for keratan sulfate in proteoglycans isolated from culture media. hCSSC cultured in fibrin gel (hCSSC-Fibrin) and as pellets (hCSSC-Pellet) were compared with fibroblasts cultured in fibrin gel (FB-Fibrin) and as pellets (FB-Pellet). Error bars show SD of triplicate analyses. *Significant difference (P < 0.01) between fibroblasts and hCSSC in the same culture conditions.

Figure 3

Immunofluorescent staining of pellet ECM. Cryosections of 3-week pellets generated by hCSSC (A–D) and by fibroblasts (E–H) were stained for keratocan (A, E), keratan sulfate (B, F), collagen V (C, G), and collagen VI (D, H) (all green). Nuclei are counterstained in red. All are shown at the same magnification. Scale bar, 50 μm. (B, inset) Keratan sulfate (green) in the extracellular regions between cells counterstained with DiO (red). Scale bar, 5 μm.

Figure 4

Cadherin 11 and connexin 43 in pellet cultures. Cryosections of pellets formed from hCSSC (A, B) or from fibroblasts (C, D) were stained for connexin 43 (A, C) and for cadherin 11 (B, D). Immunostaining is shown in green, and nuclei are counterstained in red. Scale bars, 50 μm.

Figure 5

Transmission electron micrographs of the pellet cultures. (A, B) Morphology of hCSSC pellets. In the peripheral area, the collagen fibrils aligned in parallel arrays (arrows). (C, D) Similar sections from pellet cultures of fibroblasts. Scale bars, 1 μm (A, C); 300 nm (B, D).

Figure 6

Differential gene expression profiles of hCSSC and keratocytes. mRNA abundance relative to 18s ribosomal RNA was determined by qRT-PCR for a panel of 18 genes using cDNA from primary human keratocytes cultured in protein-free DME-F12 medium and from hCSSC in monolayer cultures in SCGM. Ratios of abundance of each transcript in the two cell types are expressed on a log scale.

Figure 7

Changes in gene expression by hCSSC in pellet culture. mRNA abundance was compared from hCSSC cultured for 3 weeks as a pellet and from hCSSC in monolayer cultures in SCGM, as described in Figure 6. Ratios of abundance of each transcript between the cells cultured under different conditions are expressed on a log scale. Keratocyte marker genes are upregulated and stem cell genes are downregulated in pellets.