Human adipose-derived stem cells support the growth of limbal stem/progenitor cells

<div><p>The most efficient method to expand limbal stem cells (LSCs) <i>in vitro</i> for clinical transplantation is to culture single LSCs directly on growth-arrested mouse fibroblast 3T3 cells. To reduce possible xenobiotic contamination from 3T3s, primary human adipose-derived stem cells (ASCs) were examined as feeder cells to support the expansion of LSCs <i>in vitro</i>. To optimize the ASC-supported culture, freshly isolated limbal epithelial cells in the form of single cells (SC-ASC) or cell clusters (CC-ASC) were cultured using three different methods: LSCs seeded directly on feeder cells, a 3-dimensional (3D) culture system and a 3D culture system with fibrin (fibrin 3D). The expanded LSCs were examined at the end of a 2-week culture. The standard 3T3 culture served as control. Expansion of SC-ASC showed limited proliferation and exhibited differentiated morphology. CC-ASC generated epithelial cells with undifferentiated morphology in all culture methods, among which CC-ASC in 3D culture supported the highest cell doubling (cells doubled 9.0 times compared to cells doubled 4.9 times in control) while maintained the percentage of putative limbal stem/progenitor cells compared to the control. There were few cell-cell contacts between cultured LSCs and ASCs in 3D CC-ASC. In conclusion, ASCs support the growth of LSCs in the form of cell clusters but not in single cells. 3D CC-ASC could serve as a substitute for the standard 3T3 culture to expand LSCs.</p></div

Protein expression of selected limbal transcripts in human ocular tissue.

<p>A) Minimal expression of PITX2 was observed in the cornea. B) Distinct PITX2 cytoplasmic expression was present in pockets within the basal layer and suprabasal layer of the limbal epithelium. Insert highlights both PITX2 cytoplasmic and nuclear localization in the basal epithelial cells. Thin arrows indicate cells with PITX2 nuclear localization. C) Minimal expression of PITX2 was also observed in the conjunctiva. D) Weak expression of FZD7 was detected in the basal layer of the cornea. E) Highly localized FZD7 expression was observed at the basal layer of the limbal epithelium. F) We observed low expression in the suprabasal and superficial layers of the conjunctiva for FZD7. G) Minimal expression of TNC was observed in the cornea, while distinct expression was present in the subepithelial stroma along the limbus (H). (I) We detected minor expression of TNC in the suprabasal and superficial layers of the conjunctiva. Thick arrows represent examples of superficial epithelial cells and arrowheads represent examples of basal epithelial cells in the limbus. Scale bar, 50 µm.</p

Network map of preferentially expressed signature limbal genes.

<p>The IPA network map highlights the upregulation of genes from the TGF-β pathway and extracellular matrix processes. Fibronectin-1 is centrally connected to a number of upregulated limbal genes.</p

Preferential Biological Processes in the Human Limbus by Differential Gene Profiling

<div><p>Corneal epithelial stem cells or limbal stem cells (LSCs) are responsible for the maintenance of the corneal epithelium in humans. The exact location of LSCs is still under debate, but the increasing need for identifying the biological processes in the limbus, where LSCs are located, is of great importance in the regulation of LSCs. In our current study we identified 146 preferentially expressed genes in the human limbus in direct comparison to that in the cornea and conjunctiva. The expression of newly identified limbal transcripts endomucin, fibromodulin, paired-like homeodomain 2 (PITX2) and axin-2 were validated using qRT-PCR. Further protein analysis on the newly identified limbal transcripts showed protein localization of PITX2 in the basal and suprabasal layer of the limbal epithelium and very low expression in the cornea and conjunctiva. Two other limbal transcripts, frizzled-7 and tenascin-C, were expressed in the basal epithelial layer of the limbus. Gene ontology and network analysis of the overexpressed limbal genes revealed cell-cell adhesion, Wnt and TGF-β/BMP signaling components among other developmental processes in the limbus. These results could aid in a better understanding of the regulatory elements in the LSC microenvironment.</p></div

Comparison of mRNA expression levels between microarray and qRT-PCR of selected transcripts.

<p>All 9 transcripts were preferentially expressed in the limbus and minimally expressed in the cornea and conjunctiva. White bars represent the microarray results and black bars represent the qRT-PCR results. Similar expression patterns were observed between the microarray and qRT-PCR.</p

Relative mRNA levels of the putative stem cell markers and maturation marker in cultured LEC cell clusters with ASCs as evaluated by qRT-PCR.

<p>The expression of each marker was normalized to the expression of housekeeping gene GAPDH and the value of the control group was designated 1. *: p<0.05 in comparison with results of the control group. Ctl: control. CC-ASC: cell clusters of LECs cultured directly on ASCs. 3D CC-ASC: cell clusters of LECs cultured on ASCs using the 3D method. Fibrin 3D CC-ASC: cell clusters of LECs cultured on ASCs using the fibrin 3D method.</p

Cell clusters of limbal epithelial cells cultured on ASCs.

<p>(A) Cell morphology of cultured LSCs. The image for fibrin 3D CC-ASC was from the 33% culture which supported epithelial expansion. (B) Cell doubling of limbal epithelial cells. *: p<0.05 in comparison with results of control, CC-ASC, and fibrin 3D CC-ASC cultures. Ctl: control. CC-ASC: cell clusters of LECs cultured directly on ASCs. 3D CC-ASC: cell clusters of LECs cultured on ASCs using the 3D method. Fibrin 3D CC-ASC: cell clusters of LECs cultured on ASCs using the fibrin 3D method. Scale bar = 100 μm.</p

Single-cell suspension of limbal epithelial cells cultured on ASCs.

<p>(A) Cell morphology of cultured LSCs. The images for SC-ASC and 3D SC-ASC were from the 33% culture which supported epithelial expansion. (B) Cell morphology of cultured LSCs. The images for SC-ASC and 3D SC-ASC were from the 33% culture which supported epithelial expansion. (C) Cell doubling of limbal epithelial cells. *: p<0.05 in comparison with results of control. Ctl: control. SC-ASC: single cell suspension of LECs cultured directly on ASCs. 3D SC-ASC: single cell suspension of LECs cultured on ASCs using the 3D method. Scale bar = 100 μm.</p

Expression of p63α in cultured LEC cell clusters with ASCs evaluated by immunocytochemistry.

<p>(A) Representative images showing the expression of p63α in cultured LECs. (B) The percentages of p63α-bright cells in cultured LECs. (C) The absolute numbers of p63α-bright cells in cultured LECs. The absolute number of p63α-bright cells = the percentage of p63α-bright cells x (number of cells harvested/number of cells seeded). *: p<0.05 in comparison with results of control. Ctl: control. CC-ASC: cell clusters of LECs cultured directly on ASCs. 3D CC-ASC: cell clusters of LECs cultured on ASCs using the 3D method. Fibrin 3D CC-ASC: cell clusters of LECs cultured on ASCs using the fibrin 3D method. Scale bar = 100 μm.</p

Expression of K14 and K12 in cultured LEC cell clusters with ASCs evaluated by immunocytochemistry.

<p>(A) Representative images showing the expression of K14 in cultured LECs. (B) The percentages of K14⁺ cells in cultured LECs. (C) The absolute numbers of K14⁺ cells in cultured LECs. (D) Representative images showing the expression of K12 in cultured LECs. (E) The percentages of K12⁺ cells in cultured LECs. (F) The absolute numbers of K12⁺ cells in cultured LECs. The absolute number of K14⁺ or K12⁺ cells = the percentage of K14⁺ or K12⁺ cells x (number of cells harvested/number of cells seeded). *: p<0.05 in comparison with results of control. Ctl: control. CC-ASC: cell clusters of LECs cultured directly on ASCs. 3D CC-ASC: cell clusters of LECs cultured on ASCs using the 3D method. Fibrin 3D CC-ASC: cell clusters of LECs cultured on ASCs using the fibrin 3D method. Scale bar = 100 μm.</p