To obtain large quantities of cells for clinical applications, further studies are needed to explore efficient methods for the expansion of neurosphere-forming cells
June 7, 2021
To obtain large quantities of cells for clinical applications, further studies are needed to explore efficient methods for the expansion of neurosphere-forming cells. immunohistochemical analysis of UC tissue was performed. Results Immunohistochemical analysis revealed that p75NTR+ cells were specifically localized to the subendothelial area of the UA and umbilical vein. The p75NTR+ cells co-expressed PDGFR, CD90, CD146, and NG2, phenotypic markers of MSCs and pericytes. Isolated UA cells possessed the potential to form neurospheres that further differentiated into neuronal and glial cell lineages. Genetic lineage tracing analysis showed that EGFP+ neural crest-derived cells were detected in the subendothelial area of UA with p75NTR immunoreactivity. Conclusions These results show that UA tissue harbors p75NTR+ pericyte-like cells in the subendothelial area that have the capacity to form neurospheres and the potential for neurogenic differentiation. The lineage tracing data suggests the p75NTR+ cells Doxifluridine are putatively derived from the neural crest. characterization of MSC markers expression in the UC tissue. Our data showed that expression of the MSC marker CD90 was observed throughout the vessel walls including the subendothelial area Doxifluridine in both UA and UV (Fig.?2a, see VW in UA, UV, Enlarge). The expression in vessel wall smooth muscle cells were confirmed by immunostaining for ASMA (Fig.?2a, UA-VW). The expression was also detected in the perivascular region of WJ, but not in other areas of WJ and amnion (Fig.?2a, see PV in UA, UV, Enlarge). The other MSC marker CD105 was widely expressed in the UA and UV including endothelium, subendothelial area, and vascular wall (Fig.?2b, UA, UV, Enlarge, UA-VW). The expression was also detected in WJ including the perivascular region and amnion (Fig.?2b, WJ). The pericyte marker CD146 was strongly expressed in the subendothelial area and vessel wall in Rabbit Polyclonal to RALY both UA and UV and weakly expressed in the perivascular region of WJ (Fig.?2c). Expression of the other pericyte marker PDGFR was markedly observed in the subendothelial area of UA and UV, and in WJ including the perivascular region and subamnion (Fig.?2d). Similar results were obtained from five independent UC samples. The expression profile of these markers in each region is summarized in Table?1. These results indicated that MSC and pericyte marker-expressing cells are relatively rich in the subendothelial area of umbilical vessels and the perivascular region of WJ. Open in a separate window Fig.?2 Localization of mesenchymal stromal cell markers in umbilical cords. Umbilical cord samples were stained for CD90 (a), CD105 (b), CD146 (c), and PDGFR (d) (red). Vascular endothelial cells, vessel wall smooth muscle cells and nuclei were stained for von Willebrand factor (vWF, green), -smooth muscle actin (ASMA, green), and DAPI (blue), respectively. Asterisks indicate the vascular lumen. Dotted line indicates the border of the vascular wall (VW) and perivascular area (PV) of Wharton’s jelly (WJ). Other abbreviations as in Fig.?1. Scale bar: 100?m. Table?1 The expression of MSC markers and their localization in umbilical cord tissue by immunohistochemistry. in fresh fragments of WJ [19,20]. Interestingly, our data showed that there were at least two types of p75NTR+ cells with different expression levels and different morphologies. That both types of p75NTR+ cells expressed PDGFR, CD90, CD146, and NG2, suggests that the cells have a phenotypic feature of pericytes. Thus, it is supposed that UA tissue contains several different types of pericyte-like cells that express p75NTR. To support this supposition, the presence of at least three different subsets of pericytes have been shown in human UA tissue . In diverse human tissues, perivascular cells, especially pericytes, give rise to multipotent MSCs . Previous studies found that umbilical vessel-derived cultured MSCs showed high potential for proliferation and multilineage differentiation [, , ]. Our data confirmed that the isolated UA cells could proliferate on plastic dishes and exhibit CFU-F-forming ability. Interestingly, our data showed that UA-derived MSCs did not express p75NTR. This result is in agreement with a previous report . Because it is possible that a p75NTR? cell fraction contributed CFU-Fs, further studies are needed to clarify whether the p75NTR+ cells possess clonogenic and multi-lineage differentiation potential as MSCs. In the neurosphere culture condition, we found that the UA cells form neurospheres that can further differentiate into neuronal and glial cell lineages. Most of the neurosphere-forming cells expressed p75NTR, suggesting that the neurospheres originated from subendothelial p75NTR+ cells. To our knowledge, this is the first study to report that neurospheres can be generated from freshly isolated UA cell fraction, Doxifluridine although Doxifluridine previous studies showed that UC-derived cultured MSCs exhibit neurosphere formation capacity [8,25,26]. Our data showed that the neurospheres could be generated from the bulk of UA cells but not from freshly isolated p75NTR+ cells. These findings suggested that UA cells other than p75NTR+ cells support the development of neurospheres. Genetic lineage tracing analysis using.