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

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

Characterization of primary ASCs.

<p>The expression of CD90, CD105, CD31, CD34, adiponectin, and osteocalcin was examined by immunocytochemistry in the primary ASCs at passage 4–6. Scale bar = 100 μm.</p

Few cell-cell contacts between the epithelial cells and the ASC feeder cells in 3D CC-ASC culture.

<p>(A) Representative images showing an empty pore, a pore with possible cell-cell contact, a pore with no cell-cell contact but with cell extension from the epithelial cells, and a pore with no cell-cell contact but with cell extension from the ASC feeder cells, respectively. (B) An image showing the cell-cell contact between LSCs and ASC feeder cells using electron microscopy. White arrow indicates the interface of cell-cell contact. (C) Percentages of pores with empty content, cell-cell contact, no contact but with cell extension from epithelial cells, and no contact but with cell extension from ASC feeder cells in the 3D CC-ASC culture. Epi: cultured epithelial cells. Scale bar = 3 μm.</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

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