hUC cells expressed pluripotent markers OCT NANOG KLF and
hUC Immunology Compound Library expressed pluripotent markers, OCT4, NANOG, KLF4 and SOX2, but expression levels of all, except KLF4 were significantly lower than human ESCs (Fig. S3). Nevertheless, our results were in agreement with a previous report showing expression of pluripotency markers in cells from peri-natal sources (Nekanti et al., 2010). The results indicated that expression of these markers was site-dependent, as highest levels of OCT4 and NANOG were found in CPJ-hUC cells. These cells were also found to be the most proliferative when compared to CT- and WJ-hUC cells. However, despite their common characteristics, hUC cells derived from different sites of hUC differed in their colony forming efficiency, regulation of cell cycle, and proliferation capabilities. Furthermore, comparative analysis of the isolated hUC cells showed that passaging gradually affected their properties. The cell size, CFE, and growth rate was reduced in case of CT- and WJ-hUC cells upon passaging compared to the CPJ-hUC cells. Passaging also affected the expression of pluripotent markers as well as certain cell surface markers. Comparison of LP and HP hUC cells showed not only reduction in expression of pluripotency and cell surface markers, but increased the cell size, suggesting that the cells are undergoing gradual changes upon passaging. This hypothesis was strengthened by the fact that HP hUC cells had higher expression of CD105, which is known to be expressed in differentiating cells (Campbell and Pei, 2012; Habip Akbulut et al., 2012; Jiang et al., 2010). Previous studies (Cheng et al., 2011; Zaim et al., 2012) have suggested that long-term passaging of stem/progenitor cells from different tissues causes senescence and the patterns of senescence differed between tissue sources and are age-dependent. Our analysis of HP hUC cells for negative Gal staining (see Fig. S4) and lack of upregulation of senescence markers (data not shown), such as p53 and p21 did not show that cells were undergoing senescence. Our findings are similar to an earlier report (Hass et al., 2011) which showed that the cells derived from neonatal tissues displayed no sign of cellular senescence over long-term culture. Nevertheless, additional studies on the type and cause of growth arrest in these cells should help develop long-term culturing techniques. Further analysis of HP hUC cells showed that they expressed elevated levels of chondrogenic markers such as SOX9 and COL2, indicating that they were undergoing differentiation by expressing specific proteins found in chondrocytes. These findings were consistent with the microarray analysis showing the increased expression of COL12A1 in HP hUC cells. In fact, COL12A1 is reported to be associated with the COL2 and COL1 in the production of the extracellular matrix suggesting that HP hUC cells were undergoing chondrogenic differentiation (Dharmavaram et al., 1998; Gregory et al., 2001; Narcisi et al., 2015; Taylor et al., 2015). In addition, the genome wide transcriptome analysis also revealed that there were various genes involved in cell proliferation and differentiation that were differentially expressed between the hUC cells derived from different sites of the same hUC. Out of these, two genes (ANXA3 and DDX43) were expressed at significantly higher levels in CPJ- compared to CT- and WJ-hUC cells. These genes have been extensively studied as biomarkers in the prognosis of various kinds of cancer cell lines (Ambrosini et al., 2014; Zhai et al., 2014). The upregulation of these genes correlates with increased cell proliferation and self-renewal. In addition, two genes (HOXA6 and HOXD10) were expressed at lower levels in CPJ- compared with CT- and WJ-hUC cells. Both of these genes have been implicated in the regulation of cell differentiation (Osborne et al., 1998; Walters et al., 1997). Their lower expression could be responsible for longer-term proliferation as observed in case of CPJ-hUC cells. Interestingly, one gene (MAEL) was found to be expressed at high levels in CPJ-hUC cells. MEAL has been reported to have an essential role in spermatogenesis and transposon repression (Yuan et al., 2014). Investigation of the potential role of MAEL in hUC cells should be of interest.