Lung Spheroidal Cells Superior to Mesenchymal Stem Cells When Treating Pulmonary Fibrosis in Mice


Lung diseases are no fun for anyone. The constant feeling of suffocation, withering weakness, and significant limitations on human activity are indicative of a loss of lung capacity. Chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis are among the top five causes of mortality, according to the World Health Organization (Cottin Eur Respir Rev 22:2632).  Regenerating damaged lungs, therefore, represent one of the Holy Grails of regenerative medicine.

Animal studies have used infusions of mesenchymal stem cells (MSCs) from isolated from human bone marrow, adipose tissue, placental tissue, or cord blood to treat animals with various types of lung disease (Moodley Y, et al.Am J Pathol 175:303313; Ortiz LA, et al. Proc Natl Acad Sci USA 104:1100211007; Ortiz LA, et al. Proc Natl Acad Sci USA 100:84078411). Also, a Phase I clinical trial has assessed the safety of fat-based MSCs as a treatment for lung damage in human patients. Because this study was only designed to test the safety of this procedure, little to nothing can be said of the efficacy of this test (Tzouvelekis A, J Transl Med. 2013 Jul 15;11:171).

Recently, several laboratories have identified resident stem cells in the lung and some researchers have even managed to isolate them and growth them in culture (Desai TJ, et al., (2014) Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature 507:190–194; Kajstura J, et al., (2011) Evidence for human lung stem cells. N Engl J Med 364:1795–1806; Kim CF, et al., (2005) Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121:823–835; Wansleeben C, et al. (2013) Stem cells of the adult lung: Their development and role in homeostasis, regeneration, and disease. Wiley Interdiscip Rev Dev Biol 2:131–148; Barkauskas CE, et al. (2013) Type 2 alveolar cells are stem cells in adult lung. J Clin Invest 123:3025–3036; Hogan BL, et al. (2014) Repair and regeneration of the respiratory system: Complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15:123–138). Could such cells work better than MSCs?

At this point it is difficult to say, since there are a fair amount known about isolating and growing MSCs in culture, but there is relatively little known about resident lung stem cell populations. However, a new paper might change that feature of the debate.

Ke Cheng and his colleagues from North Carolina State University have developed a rapid, reproducible, and scalable method to generate clinically applicable amounts of resident lung progenitor cells. This technique capitalizes on the “Spheroid method” used for tumor cells. Spheroids are three-dimensional structures that grow as small balls of cells in culture. Cheng and others applied the spheroid culture method to lung cells with great success.

Lung cells were acquired from lung biopsies taken from human patients. The tissues were appropriately minced, treated with enzymes to disintegrate the structural components, and then grown in culture. The cells that grew were eventually seeded onto a special culture system for growing spheroids. In this culture system, spheroids formed, consisting of internal clumps of lung progenitor cells surrounded by a shell of stroma-like cells. These cells expressed the cadre of genes you would expect them to. Cheng and his coworkers called these cells “lung spheroid cells” or LSCs.

Generation of lung spheroids and lung spheroid cells. (A): Schematic showing the protocol to grow lung spheroids and lung spheroid cells. (B-I): Edge of lung tissue explants with outgrowth cells becoming confluent and ready to harvest. (B-II): Lung spheroids formed from outgrowth cells in suspension culture. (B-III): Plated lung spheroids onto fibronectin-coated surfaces to generate lung spheroid cells. (B-IV): Expansion of LSCs in suspension cultures. (C): Cumulative doubling for LSCs from three different donors. (D): Immunocytochemistry on lung spheroids. Scale bars = 50 µm. Abbreviations: LSCs, lung spheroid cells; PF, pulmonary fibrosis; SCID, severe combined immunodeficiency.
Generation of lung spheroids and lung spheroid cells. (A): Schematic showing the protocol to grow lung spheroids and lung spheroid cells. (B-I): Edge of lung tissue explants with outgrowth cells becoming confluent and ready to harvest. (B-II): Lung spheroids formed from outgrowth cells in suspension culture. (B-III): Plated lung spheroids onto fibronectin-coated surfaces to generate lung spheroid cells. (B-IV): Expansion of LSCs in suspension cultures. (C): Cumulative doubling for LSCs from three different donors. (D): Immunocytochemistry on lung spheroids. Scale bars = 50 µm. Abbreviations: LSCs, lung spheroid cells; PF, pulmonary fibrosis; SCID, severe combined immunodeficiency.

When Cheng and his group implanted LSCs into Matrigel, the cell appropriately differentiated into lung-specific cell types and formed structures that greatly resembled the tiny air sacs in lungs, known as alveoli. These structures, based on their expression patterns of particular genes are where specific proteins were found in the cells, seemed for a mature lung structure. Interestingly, when the culture medium that had been used to grow the LSCs (conditioned media) was given to other cultured cells, it promoted survival or proliferation of human lung epithelial cells and tube formation of human endothelial cells on Matrigel.

In vitro differentiation and paracrine assays of lung spheroid cells. (A): LSCs grown on Matrigel and displaying alveoli-like structures (inset). (B): LSCs grown on Matrigel expressed aquaporin 5 (red). (C): Human lung epithelial cells cultured in control media and LSC-CM and stained for live (green)/dead (red) assay. (D): HUVEC tube formation assay on Matrigel surface in control or conditioned media from LSCs. Data are presented as mean ± SD. All experiments were run in triplicate, unless noted otherwise. Scale bars = 50 µm. ∗, p < .05 compared with the control media group. (E): Representative antibody array images showing the proteins presenting in the CM from LSCs and NHDF cells. Abbreviations: AQ5, aquaporin 5; BDNF, brain-derived neurotrophic factor; CM, conditioned media; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GRO, growth-regulated protein; HGF, hepatocyte growth factor; HUVEC, human umbilical vein endothelial cell; IGFBP2, insulin-like growth factor binding protein 2; IL, interleukin; LSCs, lung spheroid cells; POS, positive; NHDF, normal human dermal fibroblast cell.
In vitro differentiation and paracrine assays of lung spheroid cells. (A): LSCs grown on Matrigel and displaying alveoli-like structures (inset). (B): LSCs grown on Matrigel expressed aquaporin 5 (red). (C): Human lung epithelial cells cultured in control media and LSC-CM and stained for live (green)/dead (red) assay. (D): HUVEC tube formation assay on Matrigel surface in control or conditioned media from LSCs. Data are presented as mean ± SD. All experiments were run in triplicate, unless noted otherwise. Scale bars = 50 µm. ∗, p < .05 compared with the control media group. (E): Representative antibody array images showing the proteins presenting in the CM from LSCs and NHDF cells. Abbreviations: AQ5, aquaporin 5; BDNF, brain-derived neurotrophic factor; CM, conditioned media; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GRO, growth-regulated protein; HGF, hepatocyte growth factor; HUVEC, human umbilical vein endothelial cell; IGFBP2, insulin-like growth factor binding protein 2; IL, interleukin; LSCs, lung spheroid cells; POS, positive; NHDF, normal human dermal fibroblast cell.

Now the $64,000 question is this: “How do LSCs stack up against MSCs at treating lung damage?”

To address this question, Cheng and others used a mouse model of pulmonary fibrosis. To induce pulmonary fibrosis in mice, Severe Combined Immune Deficient (SCID) mice were given intratracheal washes of the anticancer drug bleomycin, which induces a condition in mice that, to some degree, resembles human pulmonary fibrosis. Different groups of mice with this lung damage were given either intravenous infusions of LSCs, MSCs or saline as a control.

The administered LSCs reduced the amount of cell death and scarring observed in the lungs. LSCs also increased the formation of new blood vessels and decreased the expression of pro-fibrotic genes. While the infusion of MSCs did improve the lung tissue in these mice, the LSCs were clearly superior.

Therapeutic benefits of human LSCs in mice with bleomycin-induced pulmonary fibrosis. (A): Schematic showing the design of the mouse studies. (B): Macroscopic views of explanted lungs 14 days after LSC or saline treatment. H&E staining (C) and Masson’s trichrome staining (D) were performed on the lungs. (E): Quantitation of fibrous thickening by Ashcroft score from the H&E staining images (n = 6–7 animals per group). (F): Quantitation of tissue infiltrates from the H&E staining images (n = 6–7 animals per group). Data are presented as mean ± SD. Scale bars = 100 µm. ∗, p < .05 compared with the sham group; #, p < .05 compared with the Bleo + saline group. Abbreviations: Bleo, bleomycin; H&E, hematoxylin and eosin; LSCs, lung spheroid cells.
Therapeutic benefits of human LSCs in mice with bleomycin-induced pulmonary fibrosis. (A): Schematic showing the design of the mouse studies. (B): Macroscopic views of explanted lungs 14 days after LSC or saline treatment. H&E staining (C) and Masson’s trichrome staining (D) were performed on the lungs. (E): Quantitation of fibrous thickening by Ashcroft score from the H&E staining images (n = 6–7 animals per group). (F): Quantitation of tissue infiltrates from the H&E staining images (n = 6–7 animals per group). Data are presented as mean ± SD. Scale bars = 100 µm. ∗, p < .05 compared with the sham group; #, p < .05 compared with the Bleo + saline group. Abbreviations: Bleo, bleomycin; H&E, hematoxylin and eosin; LSCs, lung spheroid cells.

Can LSCs provide the kind regenerative “OOMPH” damaged lungs need? The culture system that was developed by Cheng and others can produce large quantities of cells from even small biopsies. This makes the procedure suitable and efficacious for clinical situations in which a patient is receiving infusions of their own cells or someone else’s cells. An added advantage to this system in the absence of any need for cell sorting, which is expensive, and requires highly-trained technicians who operate large, expensive machines. Also, none of the mice treated in this study showed any signs of tumors, which underscores the clinical safety of LSCs.

Cultured MSCs might also provide an excellent model system to study lung pathologies. Making LSCs from patients with cystic fibrosis, inherited versions of emphysema, or other pulmonary diseases could provide an accessible and effective model system for drug testing and pathological studies.

This work was published in the journal Stem Cells and Translational Medicine.

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Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).