Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

The Cellular Origin of Barrett's Esophagus and Its Stem Cells.

The incidence of esophageal adenocarcinoma is rapidly increasing in Western countries. This is despite the introduction of sophisticated endoscopic techniques and our ability to readily monitor the presumed precursor lesion known as Barrett's esophagus. Preemptive approaches, including radiofrequency ablation (RFA), and photodynamic therapy (PDT) for Barrett's esophagus and dysplasia are achieving dramatic initial results. Although the long-term efficacy of these nonspecific ablative therapies is awaiting longitudinal studies, reports of recurrences are increasing. More targeted therapies, particularly directed at the stem cells of Barrett's esophagus, demand knowing the origin of this intestinal metaplasia (IM). The prevailing concept holds that Barrett's esophagus arises from the "transcommitment" of esophageal stem cells to produce an intestine-like epithelium. An alternative explanation derives from the discovery of a discrete population of residual embryonic cells (RECs) existing at the gastroesophageal junction in normal individuals that expands and colonizes regions of the esophagus denuded by chronic reflux. These RECs form IM within days of esophageal injury, suggesting a novel mechanism of tumorigenesis.A corollary of this work is that the Barrett's stem cell is distinct from that of the squamous epithelium and, once identified, will form the basis of new preemptive strategies for addressing Barrett's and its related neoplasia.

Cloning of ground-state intestinal stem cells from endoscopic biopsy samples.

'Adult' or 'somatic' stem cells harbor an intrinsic ability to regenerate tissues. Heterogeneity of such stem cells along the gastrointestinal tract yields the known segmental specificity of this organ and may contribute to the pathology of certain enteric conditions. Here we detail technology for the generation of 'libraries' of clonogenic cells from 1-mm-diamter endoscopic biopsy samples from the human gastrointestinal tract. Each of the 150-300 independent clones in a typical stem cell library can be clonally expanded to billions of cells in a few weeks while maintaining genomic stability and the ability to undergo multipotent differentiation to the specific epithelia from which the sample originated. The key to this methodology is the intrinsic immortality of normal intestinal stem cells (ISCs) and culture systems that maintain them as highly immature, ground-state ISCs marked by a single-cell clonogenicity of 70% and a corresponding 250-fold proliferative advantage over spheroid technologies. Clonal approaches such as this enhance the resolution of molecular genetics, make genome editing easier, and may be useful in regenerative medicine, unravelling heterogeneity in disease, and facilitating drug discovery.

Unlimited expansion of intestinal stem cells from a wide range of ages.

The recent technical advance in cloning and culturing ground-state intestinal stem cells (ISC) provides us an opportunity of accurate assessment of age-related impact on the function of highly proliferative intestinal stem cells. Our ability of indefinitely and robustly expanding single-stem-cell derived pedigrees in vitro allows us to study intestinal stem cells at the clonal level. Interestingly, comparable number of ISC clones was yielded from 1mm endoscopic biopsy of all donors despite the age. They were passaged in vitro as pedigrees and expanded to 1 billion cells in approximately sixty days without changes in stemness demonstrated by clonogenicity and multipotency. Therefore, our study shows that ISCs from a wide range of ages can be cloned and expanded to unlimited number in vitro with similar efficiency and stability. These patient-derived ISCs harbor intrinsic immortality and are ideal for autologous transplantation, supporting the promise of adult-stem-cell based personalized medicine.

Biobanking Organoids or Ground-State Stem Cells?

Autologous transplantation of human epidermal stem cells cultured in Green's method is one of the first examples of utilizing adult stem cells in regenerative medicine. Using the same method, we cloned p63-expressing distal airway stem cells and showed their essential role in lung regeneration in a mouse model of acute respiratory distress syndrome. However, adult stem cells of columnar epithelial tissues had until recently evaded all attempts at cloning. To address this issue, we developed a novel technology that enabled cloning ground-state stem cells of the columnar epithelium. The adaption of this technology to clone stem cells of cancer precursors furthered our understanding of the dynamics of processes such as clonal evolution and dominance in Barrett's esophagus, as well as for testing platforms for chemical screening. Taken together, the properties of these ground-state stem cells, including unlimited propagation, genomic stability, and regio-specificity, make them ideal for regenerative medicine, disease modeling and drug discovery.

Ground-State Stem Cells: A Novel Approach for Adult Stem Cell Research.

A robust and reliable culture system of adult stem cells is essential for applying the cutting-edge technologies of drug screening, gene editing, and genomics to stem cell research necessary for breakthroughs in this field. In addition, personalized regenerative medicine based on autologous transplantation requires our ability to clone and expand the numbers of these stem cells in vitro. In comparison to the 3D "organoid" culture system that shows limited ability to propagate stem cells as the majority of cells are differentiated or transit amplifying cells, ground-state stem cell culture system is a novel technology that permits long-lived adult stem cells to maintain immaturity, self-renewal capacity, multi-potency and genomic stability despite long-term culturing in a 2D system. The robustness, reliability and easy-to-use features of this new technology bypass the deficiencies of 3D organoid culture systems and provided unlimited stem cell sources for research, therapeutic use, and drug discovery.

Mutational spectrum of Barrett's stem cells suggests paths to initiation of a precancerous lesion.

The precancerous lesion known as Barrett's oesophagus can evolve to oesophageal adenocarcinoma in decades-long processes of regenerative growth. Here we report the isolation and propagation of distinct, patient-matched stem cells of Barrett's, gastric and oesophageal epithelia that yield divergent tumour types following in vitro transformation and xenografting. Genomic analyses reveal a broad mutational spectrum unique to Barrett's stem cells that likely reflects their risk for oncogenesis. Remarkably, 25% of cases show no cancer-related genomic changes, suggesting that Barrett's initiates without driver mutations. Most cases, however, sustain patterns of deletions almost identical to adenocarcinoma though tumour-associated gene amplifications were absent. Notably, those suspected of low-grade dysplasia have p53 mutations or undergo amplifications of proto-oncogenes and receptor tyrosine kinases, implicating these events in lethal transitions. Our findings suggest paths for the initiation and progression of Barrett's and define a discrete stem cell underlying its regenerative growth whose eradication could prevent oesophageal adenocarcinoma.

Regulation of transcription in cancer.

The purpose of this article is to provide an overview on the regulation of transcription in cancer cells. We describe here standard mechanisms of transcription in eukaryotic cells, an influence of common promoter polymorphisms contributing to malignant progression and DNA methylation as significant aspect of gene regulation. We also described transcription factors mechanism of action, and how their alteration can result in cancer.