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

 -  lung emphysema, lung interstitial fibrosis, lung cancer patients

 Exclusion Criteria:

 -  lung infectious diseases (bronchiectasis, lung abscess, pneumonia)

 -  pulmonary edema