Faculty Bibliography

Neil Theise speaks to Georgia Patey, Commissioning Editor: Neil Theise is a diagnostic liver pathologist, adult stem cell researcher and complexity theorist in New York City, where he is a Professor of Pathology at the Mount Sinai Beth Israel Medical Center of Icahn School of Medicine at Mount Sinai. He received his medical degree from Columbia University College of Physicians and Surgeons, where he also received his training in Anatomic Pathology. Subspecialty training was pursued in gastrointestinal (NYU), liver (Royal Free Hospital) and liver transplant (Mount Sinai, NYC) pathology. His earliest research focus was on defining the premalignant dysplastic nodules in human chronic liver disease. He revised understandings of human liver microanatomy, which in turn, led directly to identification of possible liver stem cell niches and the marrow-to-liver regeneration pathway. He is considered a pioneer of multiorgan adult stem cell plasticity. His publications on these topics in model systems and human liver stem cells have been highlighted on a record five covers of Hepatology.

A large number of cancer stem cells (CSCs) have been isolated and identified; however, none has been cultured in an unlimited manner in vitro without losing tumorigenicity and multipotency. In this study, we successfully clonogenically cultured a newly identified CD34+ liver CSC (LCSC) on feeder cells up to 22 passages (to date) without losing CSC property. Cloned CD34+ LCSC formed a round packed morphology and it could also be cryopreserved and recultured. Stem cell markers, CD34, CD117, and SOX2; normal liver stem cell markers, alpha fetoprotein, CK19, CK18, and OV6; putative CSC markers, CD44, CD133, EpCAM, and CD90; as well as CD31 were expressed in cloned CD34+ LCSC. SOX2 was the major factor in maintaining this LCSC before colonization, and interestingly, OCT4, SOX2, NAONG, Klf4, c-Myc, and Lin28 were upregulated in association with symmetric self-renewal for colony growth of CD34+ LCSC on feeder cells. Gene expression patterns of in vitro differentiation were consistent with our in vivo finding; furthermore, the tumorigenicity of cloned CD34+ LCSC was not different from uncloned CD34+ LCSC sorted from parental PLC. These results show that our cloned CD34+ LCSC maintained CSC property, including self-renewal, bipotency, and tumorigenicity after long-term culture, demonstrating that this LCSC can be cultured in an unlimited manner in vitro. Thus, establishing pure population of CSCs isolated from the patients will provide an opportunity to explore the mechanisms of tumorigenesis and cancer development, and to identify unique biomarkers presenting potential indicators of drug efficacy against CSCs for establishment of a novel strategy for cancer therapy.

Recent WHO classification for combined hepatocellular-cholangiocarcinoma and recognized stem cell subtypes has increased attention to such tumors; however, the resulting burst of reporting and research indicates that this classification, while provocative, is incomplete for description of the full array of primary liver carcinomas with biphenotypic (hepatobiliary) differentiation. We review the history of such lesions and consider the wider array of such tumors previously described. Mixed hepatobiliary phenotypes and immunophenotypes are found in individual tumors at the tissue level - with architectural and cytologic features supportive of both differentiation states - and at the cellular level, with individual cells that display cytology of one cell type, but immunophenotypically showing mixed expression. Pathobiologic and clinical questions to be answered by future research are suggested.

CD34+ stem cells play an important role during liver development and regeneration. Thus, we hypothesized that some human liver carcinomas (HLCs) might be derived from transformed CD34+ stem cells. Here we determined that a population of CD34+ cells isolated from PLC/PRF/5 hepatoma cells (PLC) appears to function as liver cancer stem cells (LCSC) by forming human liver carcinomas (HLC) in immunodeficient mice with as few as 100 cells. Moreover, the CD34+ PLC subpopulation cells had an advantage over CD34- PLCs at initiating tumors. Three types of HLCs were generated from CD34+ PLC: hepatocellular carcinomas, HCC; cholangiocarcinomas, CC; and combined hepatocellular cholangiocarcinomas, CHC. Tumors formed in mice transplanted with 12 subpopulations and 6 progeny subpopulations of CD34+ PLC cells. Interestingly, progenies with certain surface antigens (CD133, or CD44, or CD90, or EPCAM) predominantly yielded HCCs. CD34+PLCs that also expressed OV6 and their progeny OV6+ cells produced primarily CHC and CC.This represents the first experiment to demonstrate that the OV6+ antigen is associated with human CHC and CC. CD34+PLCs that also expressed CD31 and their progeny CD31+ cells formed CHCs. Gene expression patterns and tumor cell populations from all xenografts exhibited diverse patterns, indicating that tumor-initiating cells (TIC) with distinct antigenic profiles contribute to cancer cell heterogeneity. Therefore, we identified CD34+ PLC cells functioning as LCSCs generating three types of HLCs. Eighteen subpopulations from one origin had the capacity independently to initiate tumors, thus functioning as TICs. This finding has broad implications for better understanding of the multistep model of tumor initiation and progression. Our finding also indicates that CD34+ PLCs that also express OV6 or CD31 result in types of HLCs. This is the first report that PLC/PRF/5 subpopulations expressing CD34 in combination with particular antigens defines categories of HLCs, implicating a diversity of origins for HLC.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is frequently and highly expressed on carcinomas, tumor-initiating cells, selected tissue progenitors, embryonic and adult stem cells. During liver development, EpCAM demonstrates a dynamic expression since it can be detected in fetal liver, including cells of the parenchyma, whereas mature hepatocytes are devoid of EpCAM. Liver regeneration is associated with a population of EpCAM-positive cells within ductular reactions, which gradually lose the expression of EpCAM along with maturation into hepatocytes. EpCAM can be switched on and off through a wide panel of strategies to fine-tune EpCAM-dependent functional and differentiative traits. EpCAM-associated functions relate to cell-cell adhesion, proliferation, maintenance of a pluripotent state, regulation of differentiation, migration and invasion. These functions can be conferred either by the full-length protein and/or EpCAM-derived fragments, which are generated upon regulated intramembrane proteolysis. Control by EpCAM, therefore, not only occurs in the presence or lack of full-length EpCAM at cellular membranes, but also in varying rates of the formation of EpCAM-derived fragments that have their own regulatory properties, and in changes in the association of EpCAM with interaction partners. Thus, spatiotemporal localization of EpCAM in immature liver progenitors, transit-amplifying cells and mature liver cells will decisively impact on the regulation of EpCAM functions and might be one of the triggers that contribute to the adaptive processes in stem/progenitor cell lineages. This review will summarize EpCAM-related molecular events and how they relate to hepatobiliary differentiation and regeneration.