Naresh Polisetti
  • Senior Scientist, University Klinik Freiburg Freiburg
Isolation and ex vivo Expansion of Limbal Mesenchymal Stromal Cells
Authors:  Naresh Polisetti, Lyne Sharaf, Thomas Reinhard and Günther Schlunck, date: 07/20/2022, view: 1426, Q&A: 0

Limbal mesenchymal stromal cells (LMSC), a cellular component of the limbal stem cell niche, have the capability of determining the fate of limbal epithelial progenitor cells (LEPC), which are responsible for the homeostasis of corneal epithelium. However, the isolation of these LMSC has proven to be difficult due to the small fraction of LMSC in the total limbal population, and primary cultures are always hampered by contamination with other cell types. We recently published the efficient isolation and functional characterization of LMSC from the human corneal limbus using CD90 as a selective marker. We observed that flow sorting yielded a pure population of LMSC with superior self-renewal capacity and transdifferentiation potential, and supported the maintenance of the LEPC phenotype. Here, we describe an optimized protocol for the isolation of LMSC from cadaveric corneal limbal tissue by combined collagenase digestion and flow sorting with expansion of LMSC on plastic.


Graphical abstract:




Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells
Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha 5-derived matrices support the efficient expansion of LEPC compared to tissue culture plates and other laminin isoforms by preserving stem/progenitor cell phenotype. Here, we describe an optimized protocol for the isolation of LEPC from cadaveric corneal limbal tissue by collagenase digestion and efficient expansion of LEPC using recombinant human laminin-511 E8 fragment (LN-511E8) as culture substrate.
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