Abstract
BackgroundLimbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives.MethodsWe compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA.ResultsXSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells.ConclusionsThe combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy.Graphical abstract
Highlights
Limbal stem cells (LSC) sustain the corneal integrity and homeostasis
XSHEM produced cells with LSC morphology and higher viability We compared the culture features and the morphology of LSC when cultured with CnT07 and XSHEM medium
LSC in XSHEM and in CnT07 were positive for p63 and negative for corneal differentiation markers We evaluated the expression of ΔNp63α, other characteristic stem cells, and corneal markers such as Bmi1, ABCG2, CK15, CK19, PAX6, CK12, and CK3, by quantitative polymerase chain reaction (qPCR) and immunofluorescence (Fig. 2)
Summary
Limbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. The transparency and the integrity of the cornea are maintained by a subset of stem cells located at the epithelial basal layer of the limbus, an anatomic circumferential area that separates the transparent cornea from the conjunctiva [1] These stem cells are called limbal stem cells (LSC), and they are defined by their small size, high nucleus-to-cytoplasm ratio [1, 2], and positivity for the putative stemness marker ΔNp63α [2, 3] as well as negativity for corneal epithelial differentiation markers. The cells are detached and seeded on a biocompatible carrier for transplantation [8, 9] This approach is more advantageous than explant systems, since it reduces the risk of contamination of the culture by other limbal cells (such as stromal fibroblasts) [10] and increases the amount of cells that can be obtained due to higher proliferation rates [11,12,13]. The cell suspension cultures are an optimized option since they are more enriched in stem cell progenies than explant culture methods [10, 12, 13] leading to improved outcomes [14]
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