Alveolar Organoids Research Articles

A18 Culture of Immortalized Human Alveolar Epithelial Cells in 2D and 3D to Model Lung Adenocarcinoma Progression in Vitro

Lung cancer is the leading cause of cancer death in the United States. Lung adenocarcinoma (LUAD) is the most common histologic subtype, arising from epithelial cells of terminal respiratory units called alveoli. The overall 5-year survival of lung cancer remains low at 19%. There is an urgent need to understand early events in LUAD development, as well as to develop new 1st- , 2nd- , and 3rd-line targeted therapies. Human organoids are powerful research tools for the molecular and mechanical manipulation of genetically diverse cells without exposing human subjects to treatment. Establishment of normal cell lines from human alveolar epithelial cells (AECs) has remained challenging due to the difficulty of growing primary cells in long-term culture. Human alveolar organoids would provide a powerful tool to 1) study LUAD development, progression, and drug resistance; 2) screen for new therapeutics; and 3) study the effects of environmental exposures on AECs. Optimize growth and genetic conditions to derive human AEC lines from purified primary cells for the stepwise modeling of LUAD. We tested different immortalization strategies using primary purified AECs to determine which condition allowed cells to continue proliferating while retaining their epithelial phenotype in two-dimensional (2D) culture and their ability to form spheroids in three-dimensional (3D) culture. Using purified primary AECs from three deceased, deidentified human subjects, we found that the initial propagation of AECs in media containing Y-27632 and subsequent transduction with Simian virus 40 Large T antigen allowed cells to divide readily in 2D as a monolayer, while expressing epithelial marker E-cadherin but not mature lung genes. When placed in 3D Matrigel culture with fibroblasts, these cells form multilobulated structures expressing mature AEC markers, reminiscent of the peripheral lung. We are currently optimizing this system to allow stepwise modeling of LUAD.

A method of generating alveolar organoids using human pluripotent stem cells.

The lung consists of branched structures that are anatomically, developmentally and functionally divided into airway and alveolar regions. Each region contributes to lung-specific functions involving a defense system and gas exchange, and their dysfunction can cause fatal lung diseases. In the alveolar region, the cuboidal alveolar type 2 (AT2) cells account for 90% of the alveolar epithelial cells and serve as the tissue stem cells secreting pulmonary surfactant, and flattened alveolar type I (AT1) cells cover most of the alveolar surface directly contributing to gas exchange adjacent to capillary vessels. It has been difficult to culture alveolar epithelial cells in vitro, as the lineage-specific features of those cells are rapidly lost in a conventional two-dimensional culture setting. The culture of alveolar organoids (AOs) is an emerging technique that can help maintain the features of alveolar epithelial cells in vitro, and their application to human disease modeling is eagerly awaited. We herein describe our method of generating and culturing alveolar epithelial cells and AOs derived from human induced pluripotent stem cells (iPSCs). iPSCs derived from lung disease patients, including those with rare genetic diseases, will make help elucidate the disease mechanism and hopefully identify therapeutic targets.

In Vitro Disease Modeling of Hermansky-Pudlak Syndrome Type 2 Using Human Induced Pluripotent Stem Cell-Derived Alveolar Organoids

(Stem Cell Reports 12, 431–440; March 5, 2019) In the originally published version of our manuscript, the legend to Figure S3 in the Supplemental Information was accidentally removed during the proofing process. To address this, we have now replaced the Supplemental Information with the correct version. We apologize for the oversight and for any resulting confusion. In Vitro Disease Modeling of Hermansky-Pudlak Syndrome Type 2 Using Human Induced Pluripotent Stem Cell-Derived Alveolar OrganoidsKorogi et al.Stem Cell ReportsFebruary 14, 2019In BriefPulmonary surfactant secretion by alveolar type 2 (AT2) cells is crucial for alveolar homeostasis. Gotoh and colleagues generated Hermansky-Pudlak syndrome type 2 (HPS2) patient-derived iPSCs and their gene-corrected counterparts and differentiated them into alveolar organoids (AOs). In HPS2-AOs, aberrant lamellar bodies and impaired surfactant secretion were demonstrated. Human iPSC-derived AOs might be useful for investigating alveolar lung diseases. Full-Text PDF Open Access

Mark1 regulates distal airspace expansion through type I pneumocyte flattening in lung development.

During the later stages of lung development, two types of pneumocytes, cuboidal type II (AECII) and flattened type I (AECI) alveolar epithelial cells, form distal lung saccules. Here, we highlight how fibroblasts expressing MAP-microtubule affinity regulating kinase 1 (Mark1) are required for the terminal stages of pulmonary development, called lung sacculation. In Mark1-knockout (KO) mice, distal sacculation and AECI flattening are significantly impaired. Fetal epithelial cells generate alveolar organoids and differentiate into pneumocytes when co-cultured with fibroblasts. However, the size of organoids decreased and AECI flattening was impaired in the presence of Mark1 KO fibroblasts. In Mark1 KO fibroblasts themselves, cilia formation and the Hedgehog pathway were suppressed, resulting in the loss of type I collagen expression. The addition of type I collagen restored AECI flattening in organoids co-cultured with Mark1 KO fibroblasts and rescued the decreased size of organoids. Mathematical modeling of distal lung sacculation supports the view that AECI flattening is necessary for the proper formation of saccule-like structures. These results suggest that Mark1-mediated fibroblast activation induces AECI flattening and thereby regulates distal lung sacculation.

Abstract PR11: Investigating lung cancer cells-of-origin using three-dimensional organoid cultures

Abstract There are many distinct epithelial cell types in the adult lung that could give rise to non-small cell lung cancer. These cell types include tracheal basal stem cells, tracheal club cells, distal lung bronchioalveolar stem cells (BASCs), and distal lung alveolar type 2 cells (AT2). My laboratory is using mouse models to test directly which lung stem and progenitor cells are most fit to serve as cells-of-origin of lung adenocarcinoma and lung squamous cell carcinoma. Previously, we demonstrated that tracheal club cells and BASCs are the likely cells-of-origin for KRASG12D; Lkb1-null adenosquamous tumors. The same strategy is now being used to test which cells can give rise to adenocarcinomas using the LSL:EGFRT90M/L858R (LSL=lox-stop-lox) mouse strain, or squamous cell carcinoma using the Lkb1flox/flox; Ptenflox/flox mouse strain. Briefly, tracheas and lungs from Cre-naïve mice are enzymatically dissociated, and fluorescence activated cell sorting (FACS) is used to enrich for four distinct populations: NGFR+/Sca1+ tracheal basal cells, NGFR-/Sca1+ tracheal club cells, Sca1+ distal lung BASCs, and Sca1- distal lung AT2 cells. Each population is then incubated with either adeno-GFP control virus or adeno-Cre virus to induce EGFR T790M/L858R expression or delete both alleles of Lkb1 and Pten. Three dimensional air-liquid-interface cultures are then used to expand the populations as organoids. For EGFR organoids, both basal and club cells give rise to larger, faster-growing bronchiolar organoids after EGFR is induced. AT2 cells similarly give rise to much larger alveolar organoids after EGFR induction, and BASCs change from giving rise to both bronchiolar and alveolar organoids to nearly exclusively alveolar. For Lkb1/Pten organoids, all four populations can tolerate deletion of the two genes, but it is only the basal cell and club cell organoids that progress to a squamous morphology in vitro. While both BASC and AT2 organoids continue to grow, they appear phenotypically normal. Transplant assays to assess malignancy and RNA-seq and ChIP-seq studies to explore gene transcription and epigenetic changes are under way. These experiments will shed light on the cells of the adult lung that give rise to genotype-specific tumors, and will aid us to better classify lung cancer heterogeneity from a cell-of-origin perspective. Citation Format: Kwok-Kin Wong, Carla Kim, Christine Fillmore Brainson. Investigating lung cancer cells-of-origin using three-dimensional organoid cultures [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR11.

Long-term expansion of alveolar stem cells derived from human iPS cells in organoids.

The stable expansion of tissue-specific stem cells in vitro has contributed to research on several organs. Alveolar epithelial type II (AT2) cells function as tissue stem cells in the lung, but robust models for studying human AT2 cells are lacking. Here we report a method for the efficient generation and long-term expansion of alveolar organoids (AOs) harboring SFTPC+ alveolar stem cells derived from human induced pluripotent stem cells (hiPSCs). hiPSC-derived SFTPC+ cells self-renewed, with transcriptomes and morphology consistent with those of AT2 cells, and were able to differentiate into alveolar epithelial type I (AT1)-like cells. Single-cell RNA-seq of SFTPC+ cells and their progenitors demonstrated that their differentiation process and cellular heterogeneity resembled those of developing AT2 cells in vivo. AOs were applicable to drug toxicology studies recapitulating AT2-cell-specific phenotypes. Our methods can help scientists overcome the limitations of current approaches to the modeling of human alveoli and should be useful for disease modeling and regenerative medicine.

CD44high alveolar type II cells show stem cell properties during steady-state alveolar homeostasis.

The alveolar epithelium is composed of type I cells covering most of the gas-blood exchange surface and type II cells secreting surfactant that lowers surface tension of alveoli to prevent alveolar collapse. Here, we have identified a subgroup of type II cells expressing a higher level of cell surface molecule CD44 (CD44high type II cells) that composed ~3% of total type II cells in 5-10-wk-old mice. These cells were preferentially apposed to lung capillaries. They displayed a higher proliferation rate and augmented differentiation capacity into type I cells and the ability to form alveolar organoids compared with CD44low type II cells. Moreover, in aged mice, 18-24 mo old, the percentage of CD44high type II cells among all type II cells was increased, but these cells showed decreased progenitor properties. Thus CD44high type II cells likely represent a type II cell subpopulation important for constitutive regulation of alveolar homeostasis.

Prolactin and epidermal growth factor regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.

The epithelial cell-specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non-milk-producing MEC were isolated as spherical end bud-like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01-10 micrograms/ml) and EGF (1-100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21-day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland.