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.

Reversing Lung Diseases By Directing Stem Cell Differentiation


Lung diseases can scar the respiratory tissues necessary for oxygen exchange. Without proper oxygen exchange, our cells lack the means to make the energy they so desperately need, and they begin to shut down or even die. Lung diseases such as asthma, emphysema, chronic obstructive pulmonary disease and others can permanently diminish lung capacity, life expectancy and activity levels.

Fortunately, a preclinical study in laboratory animals has suggested a new strategy for treating lung diseases. Carla Kim and Joo-Hyeon Lee of the Stem Cell Research Program at Boston Children’s have described a new lung-specific pathway that is activated by lung injury and directs a resident stem cell population in the lung to proliferate and differentiate into lung-specific cell types.

When Kim and Lee enhanced this pathway in mice, they observed increase production of the cells that line the alveolar sacs where gas exchange occurs. Alveolar cells are irreversibly damaged in emphysema and pulmonary fibrosis.

Inhibition of this same pathway increased stem cell-mediated production of airway epithelial cells, which line the passages that conduct air to the alveolar sacs and are damaged in asthma and bronchiolitis obliterans.

For their experiments, Kim and Lee used a novel culture system called a 3D culture system that mimics the milieu of the lung. This culture system showed that a single bronchioalveolar stem cell could differentiate into both alveolar and bronchiolar epithelial cells. By adding a protein called TSP-1 (thrombospondin-1), the stem cells differentiated into alveolar cells.

Next, Kim and Lee utilized a mouse model of pulmonary fibrosis. However, when they cultured the small endothelial cells that line the many small blood vessels in the lung, which naturally produce TSP-1, and directly injected the culture fluid of these cells into the mice, the noticed these injections reverse the lung damage.

When they used lung endothelial cells that do not produce TSP-1 in 3D cultures, lung-specific stem cells produce more airway cells. in mice that were engineered to not express TSP-1, airway repair was enhanced after lung injury.

Lung Stem Cell Repair of Lung Damage

Lee explained his results in this way: “When the lung cells are injured, there seems to be a cross talk between the damaged cells, the lung endothelial cells and the stem cells.”

Kim added: “We think that lung endothelial cells produce a lot of repair factors besides TSP-1. We want to find all these molecules, which could provide additional therapeutic targets.”

Even though this work is preclinical in nature, it represents a remarkable way to address the lung damage that debilitates so many people. Hopefully this work is easily translatable to human patients and clinical trials will be in the future. Before that, more confirmation of the role of TSP-1 is required.

Human Amniotic Epithelial Cells – Remarkable Possibilities for a Small Price


My apologies to my readers for my inactivity. Many deadlines make for less blogging. Nevertheless, I hope to get back to a more regular blogging schedule once things quiet down a bit.

Today’s entry is about a fascinating group of cells found in the extraembryonic membranes of the fetus known as the amnion. The amniotic sac is a thin, transparent pair of membranes that is actually rather tough. This sac holds the fetus until shortly before birth. In inner membrane of the amnion sac contains the amniotic fluid and fetus and the outer membrane, the chorion, surrounds the amnion and is part of the placenta.

The amniotic membrane contains a remarkable cell type known as amniotic epithelial cells or hAECs (the “h” is for human). Upon isolation after birth, the amnion membrane and manually separated from the chorion membrane and washed in a saline (salt) solution in order to remove all the blood. Then the epithelial cells are liberated from the basement membrane upon which they sit by a product called TrypZean. TrypZean is a recombinant trypsin, which is very clean and devoid of animal products. Trypsin is one of the enzymes in your digestive system that degrades proteins. By expressing the human trypsin gene in bacteria and purifying the protein, Sigma-Aldrich corporation can sell it for a profit to scientists for various procedures.

A single amnion membrane can yield in the vicinity of 120 million viable hAECs, which can be maintained in serum-free culture conditions. After being grown for some time, hAECs will have normal chromosome compositions and will also maintain chromosomes that have nice, long ends (telomeres). This indicates that the cells are healthy and dying while they grow in culture (see Murphy et al., Current Protocols in Stem Cell Biology, 2010; Chapter 1: Unit 1E.6). .

In culture,. hAECs do not grow like weeds. Mesenchymal stem cells (MSCs) tend to grow better than their hAEC brethren, but hAECs possess a remarkable ability to differentiate into a wide variety of different cell types. Sivakami Ilancheran in the laboratory of Martin Pera at the University of Monash in Clayton, Australia showed that hAECs were able to differentiate into heart muscle, skeletal muscle, bone, fat cells, pancreatic cells, liver, and at least two kinds of nerve cells. Also, when injected into mice, hAECs never formed tumors (Ilancheran et al., Biology of Reproduction 77 (2007): 577-88). Murphy and others have also shown that hAECs can be isolated after collection and stored for clinical therapies.

Given that hAECs are accessible, what are they good for? When it comes to regenerative medicine, preclinical studies with hAECs have produced very solid results that may pave the way for other studies.

HAECs can differentiate into lung cells and this feature makes them an attractive candidate for lung diseases. Lung diseases cause inflammation of the lung and scarring that decreases overall lung capacity. Cystic fibrosis, acute respiratory distress syndrome, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary edema, and pulmonary hypertension are all lung diseases that could potentially be treated with hAECs.

In animal models of lung disease, particular chemicals are given to the animal that damage the lung. The wounded lung tissue initiates inflammation that brings white blood cells into the lung that augment the lung damage, which results in lung scarring. If hAECs are given to mice whose lungs have been damaged by the anti-cancer drug bleomycin, the signs of inflammation and the genes normally expressed during inflammation fade away. There is also less scarring in the lungs and the functional recovery of these animals is significantly better than those animals that do not receive hAECs (Murphy et al., Cell Transplantation 2011 20(6): 909-23). In fact, hAECs can differentiate into lung cells and integrate into lung tissue. The significance of this is not lost on respiratory specialists who treat patients with cystic fibrosis. Cystic fibrosis patients lack a functional copy of a ion transport protein and poor ion transport cause the production of thick, sticky mucous that clogs up the lung pathways and causes patients to suffocate to death. However, hAECs can differentiate into lung cells that express this ion transporter. Therefore, hAECs could be a potential treatment for cystic fibrosis. Clearly hAECs have great potential for tissue engineering applications with lung disease.

Lungs are not the only organ that hAECs can help heal. These cells can also differentiate into pancreatic insulin-making cells. In the laboratory, Wei and coworkers succeeded in stimulating hAECs to secrete insulin and express the main sugar transport protein found in pancreatic insulin-secreting cells (Wei et al., Cell Transplantation 2003 12(5): 545-552). When transplanted into diabetic mice, hAECs normalize their blood sugar levels and their weights returned to normal. This shows that hAECs might represent a major breakthrough in the management of diabetes.

Clearly these cells, which come from a tissue that is normally thrown out after birth, are brimming with possibilities for regenerative medicine. Hopefully more research will produce even more possibilities.