121
Views
0
CrossRef citations to date
0
Altmetric
Research Article

Evaluation of methods for cultivating limbal mesenchymal stromal cells

, , , &
Pages 936-947 | Received 18 Jan 2012, Accepted 03 Apr 2012, Published online: 15 May 2012
 

Abstract

Background aims. Mesenchymal stromal cells (MSC) with similar properties to bone marrow-derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We have evaluated methods for culturing human limbal MSC (L-MSC). Methods. Four basic strategies were compared: serum-supplemented medium (10% fetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor and fibroblast growth factor 2, or one of two commercial serum-free media, defined keratinocyte serum-free medium (Invitrogen) and MesenCult-XF® (Stem Cell Technologies). The resulting cultures were examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141 and CD271), immunocytochemistry (alpha-smooth muscle actin; α-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis) and co-culture experiments with human limbal epithelial (HLE) cells. Results. While all techniques supported the establishment of cultures to varying degrees, sustained growth and serial propagation were only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70–80% CD34 CD45 CD90 + CD73 + CD105 + , approximately 25% α-sma + and displayed multipotency. Cultures established in MesenCult-XF were > 95% CD34 CD45 CD90 + CD73 + CD105 + , 40% CD141 + , rarely expressed α-sma, and displayed multipotency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ∆Np63, along with the corneal differentiation marker cytokeratin 3. Conclusions. MesenCult-XF is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.

Acknowledgments

This work was supported in part by a grant from the National Health & Medical Research Council, Australia, with supplementary funding from the Discipline of Medical Sciences (Queensland University of Technology, Brisbane, Australia) and the Prevent Blindness Foundation, Queensland, Australia (supported through Viertel's Vision program). LJB was supported by an ANZ Trustees Scholarship in Medical Research. CFH and KA were supported by a University of Queensland Research scholarship (CFH) and by grants from the Australian Stem Cell Foundation and Mater Medical Research Institute. We would like to thank Dr Karsten Schrobback and Dr Matthew Cook for their assistance with the mesodermal differentiation staining. Many thanks also to the Australian Red Cross, Kelvin Grove, Queensland, Australia for the irradiation of our HLS samples.

Declaration of interest: The authors report no conflicts of interest.

Reprints and Corporate Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

To request a reprint or corporate permissions for this article, please click on the relevant link below:

Academic Permissions

Please note: Selecting permissions does not provide access to the full text of the article, please see our help page How do I view content?

Obtain permissions instantly via Rightslink by clicking on the button below:

If you are unable to obtain permissions via Rightslink, please complete and submit this Permissions form. For more information, please visit our Permissions help page.