We are building a technology platform to address human disease at the level of adult or tissue-specific stem cells. Using this platform together with mouse genetics and human genomics, we have identified the stem cells that underlie lung regeneration following influenza infection, the cell-of-origin of highly lethal gastrointestinal cancers, and the regiospecific pattern of stem cells in human organs. We are adapting this platform to identify new therapeutic approaches for acute and chronic airway diseases as well as to target precursor lesions of highly lethal cancers. We anticipate that our "open-architecture" approach to adult stem cells will yield insights to a large spectrum of human diseases.
We are establishing ways to clone adult stem cells of regenerative epithelia from normal and diseased patients. Multiple pedigree lines derived from single cells are then established for each for multifaceted data collection and analysis. The advances made during this period have enabled projects investigating lung regeneration, precursors to highly lethal upper gastrointestinal cancers and putative intermediates in the development of ovarian cancer.
Stem Cells Underlying Lung Regeneration
Simultaneous projects on the cloning of human airway stem cells and our trials with H1N1 influenza infections in mice coincided with the cloning of the so-called lung stem cell (Kumar et al., Cell, 2011). Whereas there was good evidence that the upper airway stem cells were p63-postive basal cells, lung regeneration and a stem cell that might do this for the lower airways were unknown until we showed that sub-lethal H1N1 influenza infections in mice resulted in the loss of 60% of lung tissue that was restored to normal appearance in two months. This finding paralleled reports that survivors of acute respiratory distress syndrome (ARDS) recover considerable pulmonary function within 12 months despite apparently suffering massive lung damage. The lung regeneration process we observed in H1N1-infected mice was linked to the infiltration of damaged regions by a p63-expressing cell that was present in very small numbers in the absence of lung damage. We cloned this cell from mouse and human tissue and demonstrated that it was a novel epithelial stem cell that could form alveoli-like structures in vitro rather than upper airway stem cells that yielded upper airway epithelium. Key to all our analyses of these stem cells was the application of a "pedigree" analysis, including the generation of defined lines of stem cells derived from a single cell. Serial passaging of these pedigrees and their subsequent differentiation demonstrated the key stem cell characteristics of long-term self-renewal and multipotency of differentiation, both firsts in the area of adult stem cells. These studies also open the way for either cell-based therapies for severe chronic lung disease or the use of biologics that promote the activation of endogenous lung stem cells.
Chronic airway diseases including asthma, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis affect millions of individuals worldwide whose medical needs remain largely unmet. Our cloning of human, regiospecific stem cells of the upper and lower airways has set a conceptual and technical framework for understanding adult stem cells from epithelial origins. Upper airway diseases, including asthma and the aspects of COPD involving bronchi and bronchioles, have generally been considered the consequence of hyperactivation of the immune system. Epithelial remodeling is a common feature, however, and mouse models we have made to study airway epithelia suggest that these epithelia represent a largely understudied source of innate immune signaling and are perhaps a key driver of the chronic phase of both asthma and COPD. We are presently working together with scientific and clinical colleagues to explore this hypothesis. For lower airway disease, we intend to advance our findings that the distal airway stem cell underlies the massive alveoli regeneration following H1N1 influenza-induced ARDS. We are working to identify the kinetics of secreted factors associated with all stages of H1N1-ARDS in an effort to develop approaches that might enhance lung stem cell recruitment and differentiation to address both ARDS, which has a 40% death rate, and potentially distal lung involvement seen in COPD and pulmonary fibrosis, which are invariably fatal or debilitating.
Unusual Origin of Upper Gastrointestinal Cancers
Barrett's esophagus and gastric intestinal metaplasia are histologically similar precursors of esophageal and gastric adenocarcinoma respectively and appear decades before the onset of cancer. The origin of these metaplasias has been a major debate in gastroenterology, with most favoring, in the case of Barrett's, a "transcommitment" of esophageal stem cells due to chronic acid reflux. We developed a mouse model of Barrett's esophagus that produced Barrett's glands with expression profiles nearly identical to those of human patients but lacked esophageal stem cells altogether (Wang et al., Cell 2011). We tracked the origins of the Barrett's cells through retrospective developmental analyses and concluded that they arose from residual embryonic cells situated at the junction of the esophagus and stomach of all normal humans and mice. While constrained by the surrounding epithelia, damage to normal epithelia in the form of acid reflux essentially clears regions to permit the expansion and proliferation of these residual embryonic cells, all in the absence of activating mutations. Together these studies suggest a novel model for early tumorigenesis followed by some of the most aggressive and intractable cancers in humans.
Targeting Stem Cells of Precursors of Upper Gastrointestinal Cancers
Our earlier work laid the groundwork for revealing that the origins of Barrett's esophagus is not from a "transcommitment" of esophageal stem cells but rather from a minute population of residual embryonic cells existing at the gastroesophageal junction of all individuals. To further this work, we have been engaged in a close collaboration with an international team of surgical gastroenterologists and molecular geneticists to clone the stem cells from patients with Barrett's esophagus along with patient-matched stem cells of nearby esophagus and gastric epithelium. Our analysis has shown that we can readily identify patients with low and high-risk Barrett's at the level of CNV and exome sequencing of these stem cell populations. We have also identified cell surface markers specific to Barrett's stem cells and are generating monoclonal antibodies to these for diagnostic and therapeutic efforts. All of our efforts have as their goal the preemptive destruction of Barrett's before it converts to untreatable cancer.
Cellular Origin of HPV-induced Cervical Cancers
It has been long known that human papilloma virus (HPV)-linked cervical cancers arise at the junction of the squamous cervix and glandular uterine or endocervix, even though HPV broadly infects cells of the urogenital tract. Given the parallels with the Barrett's origin, we, and our long-time collaborator Christopher Crum of the Brigham and Women's Hospital (Boston) asked if the squamocolumnar junction of the cervix also contains unusual populations of cells and whether these too can be a source of neoplasia. Using markers that identify Barrett's glands, we were pleased that many recognize a discrete population of cells precisely at the cervical junction and that these markers are preserved in both squamous and adenocarcinoma of the cervix, suggesting that these tumors are derived only from this minute population of cells (Herfs et al, PNAS, 2012). As this cell population that gives rise to cervical cancers is only 10-30 cell diameters, our work suggests ways of preventing cervical cancer through cell ablation.
Early Detection of High-Grade Ovarian Cancer
High-grade ovarian cancer is insidious because it progresses and metastasizes to peritoneal surfaces without overt symptoms. This situation is driving the development of early detection and preemptive treatments of precancerous lesions. Disturbingly, the absence of compelling data for precursor lesions in the ovarian epithelium has stymied early detection strategies and represents a major conundrum in oncology. However, the origin of ovarian cancer has undergone a recent paradigm-shift with the work of Chris Crum and colleagues on ovaries and fallopian tubes removed proactively from BRCA patients. These studies have revealed the fallopian tube as the site of development of early cancers, and we are applying our stem cell cloning methods to identify precursors of high-grade ovarian cancer in the fallopian tube to aid finding early detection screen for this disease.
Basic Features of Long-Term Self-Renewal
Our increasingly robust datasets for adult stem cells, including those from a wide range of regenerative epithelia, have revealed the identities of tissue-specific transcription factors that are likely to control both differentiation as well as long-term self-renewal programs. A major goal going forward is to understand how these factors impart "stemness," or long-term self-renewal, to these tissue-specific adult stem cells, and the profile of transcription factors that determine the stable commitment of these cells to the differentiated cell types of their origins.
Co-Principal Investigator: Frank McKeon, Ph.D.
Herfs M, Yamamoto Y, Laury A, Wang X, Nucci MR, McLaughlin-Drubin ME, MŸnger K, Feldman S, McKeon FD, Xian W, Crum CP. 2012. A discrete population of squamocolumnar junction cells implicated in the pathogenesis of cervical cancer. Proc Natl Acad Sci U S A. 109: 10516-10521.
Quick CM, Ning G, Bijron J, Laury A, Wei TS, Chen EY, Vargas SO, Betensky RA, McKeon FD, Xian W, Crum CP. 2012. PAX2-null secretory cell outgrowths in the oviduct and their relationship to pelvic serous cancer. Mod Pathol 25: 449-455.
Crum CP, McKeon F, Xian W. 2012. The oviduct and ovarian cancer: causality, clinical implications, and targeted prevention. Clin Ob Gyn 55: 24-35.
Xian W, McKeon F. (2012). Adult stem cells underlying lung regeneration. Cell Cycle. 11: 887-94.
Xian W, Ho YK, Crum CP, McKeon F. 2012. Cellular Origin of BarrettÕs Esophagus: Controversy and Therapeutic Implications. Gastroenterology 142: 1424-1430.
Wang X, Ouyang H, Yamamoto Y, Kumar PA, Tay SW, Dagher R, Vincent M, Lu X, Ho YK, Crum CP, Xian W, McKeon F. 2011. Residual embryonic cells as precursors of a BarrettÕs-like metaplasia. Cell 145: 1023-1035.
Kumar PA, Hu Y, Yamamoto Y, Neo BN, Wei TS, Mu D, Sun Y, Lim SJ, Dagher R, Zielonka EM, Wang DY, Lim B, Chow VT, Crum CP, Xian W, McKeon F. 2011. Distal airway stem cells render alveoli in vitro and during lung regeneration following H1N1 influenza infection. Cell 147: 525-538.