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To investigate the stemness of limbal epithelial stem cell sheets in relation to the donor’s age. Human limbal explants from cadaveric donors were set on human amniotic membrane scaffolds with the xeno-free medium. We evaluated limbal epithelial sheet size, expression of stem/progenitor cell markers, and colony formation efficiency from donors of different age groups (age ≤ 45, age 45–65, and age > 65). Expression of the proliferation marker Ki67, stem/progenitor cell markers p63α and ABCG2, cornea specific marker PANCK, and differentiation marker CK12 were evaluated. To determine the effect of donor age on the storage period of limbal explant sheets, the limbal explant outgrowth sheets were stored in 4 °C for 2 days and analyzed for JC-1, p63α, and PANCK with FACS on each day. From days 6 to 12, the outgrowth area of the limbal epithelial stem cell sheet was significantly larger in the age ≤ 45 groups (296 ± 54.7 mm2, day 9) compared to the other two age groups [age 45–65 group (278 ± 62.6 mm2), age > 65 group (257 ± 44.0 mm2), day 9] (p 65) showed lower expression of stem/progenitor cell markers p63α and ABCG2 and decreased CFE compared to the other two groups. There were significantly more p63α+ cells in outgrowth cells in the age ≤ 45 group (18.2 ± 3.6%) compared to the age > 65 group (14.1 ± 4.6%; p 65 groups (25.7 ± 7.1%, p low). Cells from the age ≤ 45 group showed a higher clonogenic capacity than those from the other two age groups (45 65 group, positive cells of p63α on D0, 1, and 2 were significantly lower compared to those in the age ≤ 45 group on the storage period (p

Background Human embryonic stem cells (hESC) offer a renewable source of a wide range of cell types for use in research and cell-based therapies to treat disease. Inspection of protein markers provides important information about the current state of the cells and data for subsequent manipulations. However, hESC must be routinely analyzed at the genomic level to guard against deleterious changes during extensive propagation, expansion, and manipulation in vitro. Results We found that short tandem repeat (STR) analysis, human leukocyte antigen (HLA) typing, single nucleotide polymorphism (SNP) genomic analysis, mitochondrial DNA sequencing, and gene expression analysis by microarray can be used to fully describe any hESC culture in terms of its identity, stability, and undifferentiated state. Conclusion Here we describe, using molecular biology alone, a comprehensive characterization of 17 different hESC lines. The use of amplified nucleic acids means that for the first time full characterization of hESC lines can be performed with little time investment and a minimum of material. The information thus gained will facilitate comparison of lines and replication of results between laboratories.

Background In order to compare the gene expression profiles of human embryonic stem cell (hESC) lines and their differentiated progeny and to monitor feeder contaminations, we have examined gene expression in seven hESC lines and human fibroblast feeder cells using Illumina® bead arrays that contain probes for 24,131 transcript probes. Results A total of 48 different samples (including duplicates) grown in multiple laboratories under different conditions were analyzed and pairwise comparisons were performed in all groups. Hierarchical clustering showed that blinded duplicates were correctly identified as the closest related samples. hESC lines clustered together irrespective of the laboratory in which they were maintained. hESCs could be readily distinguished from embryoid bodies (EB) differentiated from them and the karyotypically abnormal hESC line BG01V. The embryonal carcinoma (EC) line NTera2 is a useful model for evaluating characteristics of hESCs. Expression of subsets of individual genes was validated by comparing with published databases, MPSS (Massively Parallel Signature Sequencing) libraries, and parallel analysis by microarray and RT-PCR. Conclusion we show that Illumina's bead array platform is a reliable, reproducible and robust method for developing base global profiles of cells and identifying similarities and differences in large number of samples.