Definition of the Status of the Human Lung Stem Cell Niches ex Vivo in Tissue Biopsies Performed in Patients With Emphysema and Interstitial Fibrosis

Scientific Background

Degenerative lung disorders are the result of inflammatory events that end in destruction of the normal pulmonary architecture. Two main patterns exist:

1. Chronic obstructive lung disease (COPD) is a major cause of morbidity and mortality. Until 2020 it is expected to become the third cause of mortality following cardiovascular disorders and cancer. Emphysema is the destructive form of COPD as the alveolated tissue disappears. This anatomic pattern is not always homogenous and functional impairment is not connected to the extent of the tissue destruction. Therapeutic elimination of the destroyed tissue may be achieved by surgery or by using various blocking techniques (Lung Volume Reduction).
2. Pulmonary fibrosis (PF) is characterized by abnormal repair of the respiratory epithelium. Various diseases may result in PF such as infections, autoimmune (connective tissue disorders), sarcoidosis, hypersensitivity pneumonitis, pneumoconiosis, histiocytosis X, lymphangioleiomyomatosis LAM etc. In many situations a cause effect relationship cannot be identified and the disease is idiopathic IPF.

During the last years cell based therapies using progenitor cells and various scaffolds became available in various medical fields. Lung regenerative medicine appears to be an alternative to transplantation in these end-stage diseases.

However, very complex cellular composition of the lung, comprising more than 40 different cell types, makes this objective challenging. The epithelial lining of the respiratory tract, composed of conducting and respiratory parts, varies along its proximo-distal axis. The conducting airways from the trachea to bronchioles of human lungs consist of pseudostratified epithelium, comprising equal proportions of basal cells, secretory cells, and ciliated cells, as well as some neuroendocrine cells. The smallest bronchioles, known as terminal and respiratory bronchioles, are lined with a simple columnar or cuboidal epithelium containing secretory and ciliated cells with fewer basal cells. The epithelia of these conducting airways form a tight barrier against the outside world and are specialized for the process of mucociliary clearance. The alveoli are lined by type 1 and 2 alveolar epithelial cells, called AT1 and AT2, respectively, hereafter. These cells are also specialized for barrier function and the extremely thin AEC1s share a basement membrane with the surrounding network of pulmonary capillaries to facilitate the diffusion of gases between the atmosphere and the circulation.

This general distribution of epithelial cell types is conserved between humans and model organisms such as rodents. However, there are notable differences. For example, the transition from a pseudostratified to columnar epithelium occurs more proximally in rodents, so only the trachea and mainstem bronchi are lined with a pseudostratified epithelium. Nearly all intralobar airways in mice are lined with a simple columnar or cuboidal epithelium with few basal cells. In mice, the abrupt transition from a conducting airway to the alveoli is known as a broncho-alveolar duct junction. In humans, terminal bronchioles give rise to respiratory bronchioles from which many alveolar ducts terminate ultimately in alveoli. Considering essential role of epithelial compartment in the lung much effort has been done to identify epithelial stem and progenitor cells responsible for regenerative and reparative functions.

Epithelial progenitors reside in unique microenvironment or niches, represented by vascular and mesenchymal cells, which are richly innervated. This architecture highly resembles HSM niches.

Considerable progress has been made in mice toward identifying the signals that regulate lung epithelial stem cell self-renewal and differentiation. These include Notch, Hippo/Yap, ROS/Nrf2, EGF, FGF, c-myb, and cytokines including IL-4, -13 and -6. Neighboring epithelial cells, stromal cells (including mesenchymal cells, fibroblasts, smooth muscle cells, and endothelium) and immune cells all represent potential sources for these factors. Distinct stem cell niches have been defined in adult mouse trachea and lung in the steady state and there is increasing evidence that in different pathological conditions stem cell niches are affected and altered.

Very little is known about stem cells and stem cell niches in human adult lung and the signals which regulate maintenance of the lung tissue homeostasis in health and disease.

Recently, the Reisner group at the Weizmann Institute has demonstrated that vacating the lung stem cell niches is a pre-requisite for successful transplantation of mouse or human lung progenitors (Nature Medicine 2015). To that end, they used an initial lung injury with naphthalene which triggered an immediate stimulation of endogenous progenitors. Thus within 48 hours the dividing progenitors could be effectively ablated by 6 Gy total body irradiation enabling effective engraftment of donor lung progenitors (Fig.1).

Fig. 1: Engraftment and functional repair of injured lungs by mouse embryonic lung cells. Following lung injury with NA and conditioning with 6Gy TBI, C57BL/6 adult mice were transplanted with syngeneic E16 stage embryonic lung cells from GFP+ donors. (a,b) Representative two-photon microscopy extended focus images of the lungs of transplanted mice 6 weeks after transplantation, showing entire scan depth from top to bottom of chimeric lung, without (a: z-stack 88μm), and with (b) co-staining of blood vessels with Quantum dots (red) (bar=90μm). (c) Two-photon extended focus image, showing entire scan depth from top to bottom of chimeric lung (z-stack 96μm) 16 weeks post-transplantation. (d) Two-photon microscopy of non-transplanted C57BL mouse lung showing background (bar=90μm). (e,f,g) Representative images of chimeric lungs stained with anti-GFP (green) and anti-AQP-5 (red), indicating incorporation of donor-derived type I alveocytes into the gas-exchange surface. (h,i,j) Chimeric lung stained with anti-GFP (green), anti-Sp-C antibodies (blue), demonstrating donor-derived surfactant producing type II alveocytes (bars=20μm). All the individual images shown above are representative of n=10 mice pooled from 3 independent experiments. (m,n) Lung function measurements 6 weeks after transplantation. (k,l) Staining of chimeric lung for CFTR (red) and (GFP), demonstrating CFTR positive donor cells. (m) Lung baseline compliance. Comparison of control intact mice vs mice with lung injury (Student's t-test, P<0.001), and of mice with lung injury vs mice transplanted after injury (Student's t-test, P=0.008). (n) Tissue damping. Comparison of control mice vs mice with lung injury (Student's t-test, P=0.015), and of mice with lung injury vs mice transplanted after injury (Student's t-test, P=0.021). Values are means ± SEM of 10-15 (n=15 control, n=10 injured and n=10 treated) mice in each group pooled from two independent experiments However, while this conditioning was required for enabling effective engraftment of donor lung progenitors in normal mice, we envision that in patients with different lung diseases, the lung niches could be already partially depleted and therefore transplantation might require less severe conditioning.

Objective To characterize stem cell compartments in their niches in different clinical situations (non-diseased compared to emphysematous and fibrotic pulmonary tissue) and to assess their proliferative and developmental properties in vitro.

To further implement lung organoid culture system in the drug screening and development of patient personalized medicine.

Methods Biopsies of lung tissue will be obtained by surgical procedures: open lung biopsies. This procedure is routinely performed for histological diagnosis of fibrotic diseases. Patients with emphysema and end stage fibrotic disease are at higher risk to develop lung cancer. A large proportion of surgically treated lung cancer patients have emphysema and fibrotic disease as histologic background. Lobectomy and pneumonectomy are frequently performed as state of the art procedures in lung cancer surgical management. Part of the resected lung tissue contain emphysematous and fibrotic changes or make look macroscopically and microscopically normal. These "non-cancerous" areas will be sampled and preserved at the Sheba Tissue Bank for evaluation together with samples from fibrotic and emphysematous areas. The biopsies will be analyzed by immunohistology, FACS and 3D organoids (after the removal of fibroblasts) at the immunology laboratory at Weizmann Institute.

All Samples will be collected following informed consent signatures of patients both on the designated informed consent form for this protocol and the tissue bank form. We may have as many as 3-4 patients/ week. Collected samples will be resected by experienced personnel either from the Thoracic Surgery Department or the Pathology Department (as part of their job description in the Tissue Bank). Fresh samples will be transported to the Weizmann institute for FACS and IHC.

As very limited knowledge exists on human lung tissues it will be mandatory to test lung tissues from healthy and diseased lungs. Initially we suggest testing 10-15 normal lung tissues and 15-20 diseased samples during the period of 2 years. As mentioned before the normal lung tissues will be obtained as "non-cancerous" areas from patients undergoing lobectomies/pneumonectomies.