|New Endodermal Marker Uncovered|
Original article from STEM CELLS
Markers to identify endodermal stem and progenitor cells are scarce and the identification of new markers will be important for the understanding of embryonic morphogenesis and lineage determination in addition to tissue regeneration and repair (Slack and Zaret and Grompe). Definitive endoderm contributes to the gastrointestinal tract, liver and pancreas which all share a close ontogeny and common developmental regulation (Zaret, Oikawa et al and Sherwood et al). The epitope defined by the GCTM-5 monoclonal antibody was previously identified as a putative marker of hepatic progenitors and therefore may mark the progenitors of other endodermal tissues also (Stamp et al). Researchers from the group of Martin F. Pera sought to further define this epitope, and present data in Stem Cells demonstrating that the GCTM-5 epitope is specifically expressed on tissues derived from the definitive endoderm, in particular the fetal gut, liver, and pancreas, with increases in GCTM-5 expression observed during disease and transformation or tumourigenesis. Further they provide evidence that the GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP (Stamp, Braxton, Wu, Akopian and Hasegawa et al).
Initial studies analysed the prevalence of GCTM-5 immunoreactivity in endodermal tissues, and found immunopositive cells in tissues such as normal biliary and pancreatic ducts. GCTM-5+ cell populations were also observed in areas of tissue disease in ductal cells of the cirrhotic liver (Turner et al), and other disease states such as pancreatitis and dysplasia, neoplasia and metastasis of various endodermal tissues. GCTM-5 immunoreactivity was generally found on the apical surface of the epithelia in normal tissues, but this placement was disrupted in cancerous tissues, where a wider cell surface distribution was observed. It was also noted that while GCTM-5 marked some adult cells, for example the mucin-secreting cells of the adult colon, it more generally marked progenitor cell compartments, such as EpCAM+ and albumin+ hepatic stem cells. Analyses of single GCMT-5+ cells from the non-parenchymal fraction of adult liver found that they could be serially cultivated in media previously reported to support propagation of human liver progenitor cells (Schmelzer et al), and the colonies formed from these cells were positive for EpCAM, E-cadherin, and albumin.
Additional work went towards the biochemical characterization of the GCTM-5 antigen, previously characterised as an epitope on a ∼50 kDa protein present in cell lysates of primary biliary epithelial cells and differentiating populations of human embryonic stem cells (hESCs) (Stamp et al). The authors took advantage of an existing pancreatic adenocarcinoma cell line (CFPAC-1) which is known to express cell surface GCMT-5 and secretes/sheds the antigen into the cell culture medium. From this GCMT-5+ medium, the antigen was recovered by immunoprecipitation and affinity chromatography and after mass spectrometric analysis was identified as part of the mucin-like protein FCGBP, a protein encoded by a 16 kbp transcript that is transcribed to yield a 300 kDa protein with multiple Von Willebrand factor and mucin-like repeats (Harada et al). This was corroborated through commercially available antibodies against protein epitopes from either the C-terminal or N-terminal of FCGBP, giving protein bands of approximately 100 kDa and approximately 55 kDa respectively, which approximate roughly to the sizes of fragments of FCGBP protein isolated from intestinal mucous that are thought to be generated by a process of autocatalytic cleavage of the intact FCGBP molecule (Albert et al). However, neither of the antibodies recognised the native form of the GCTM-5 antigen in concentrated conditioned medium nor immunoprecipitated the high molecular weight form of the GCTM-5 antigen from CFPAC-1 cells. Additionally, liver bile duct cells were non-reactive, although transcripts for FCGBP were found in adult liver non-parenchymal fraction. However, an anti-FCGBP C-terminal antibody did react with the 100 kDa band seen in conditioned medium in GCTM-5 immunoprecipitates of CFPAC1 concentrated conditioned medium, suggesting that the relevant epitopes for the antibodies only became exposed in degraded fragments of the intact native protein.
Overall, the antibody for GCMT-5 analysed in this report will be useful for marking endodermal derivatives as well as identifying cells under certain disease states. Perhaps its most useful prospective use demonstrated within this paper will be in the isolation of known or putative progenitor cells of tissues such as biliary ductular reaction, colonic crypts, pancreatic tubular complex, and the oesophageal multilayered epithelium allowing detailed analysis of these cells in both normal development and in disease states.
Albert TK et al.
Harada et al.
Oikawa T et al.
Schmelzer E et al.
Sherwood RI et al.
Stamp L et al.
Stamp, Braxton, Wu, Akopian and Hasegawa et al.
Turner R et al.
Zaret KS, Grompe M.
STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.