Scientists Use Stem Cells to Grow Three-Dimensional Mini Lungs.

In research done in several laboratories, lung tissue was derived from flat cell culture systems or by growing cells on scaffolds made from donated organs.

Now in a new study published in the online journal eLife, a multi-institution team has defined a culture system for generating self-organizing human lung organoids, which are three-dimensional structures that mimic the structure and complexity of human lungs.

“These mini lungs can mimic the responses of real tissues and will be a good model to study how organs form, change with disease, and how they might respond to new drugs,” said study senior author Jason R. Spence, Ph.D., an assistant professor of internal medicine and cell and developmental biology at the University of Michigan Medical School.

Spence and his colleagues successfully grew structures that resembled both the large airways or bronchi and small lung sacs, known as alveoli.

These mini lung structures were developed in a cell culture system. Therefore, they lack several components of the human lung, including blood vessels, which are a critical component of gas exchange during breathing.

Despite that, these cultured organoids can serve as a unique research model system for researchers as they grind out basic science ideas that are turned into clinical innovations. These three-dimensional mini-lungs should be an excellent complement to research in liver laboratory animals.

Traditionally, the behavior of cells has been investigated in the laboratory in two-dimensional culture systems where cells are grown in thin layers on cell-culture dishes. Most cells in the body, however, exist in a three-dimensional environment as part of complex tissues and organs. Tissue engineered have been trying to re-create these environments in the laboratory by successfully generating small version of particular organs known as organoids, which serve as models of the stomach, brain, liver and human intestine. The advantage of growing three-dimensional structures of lung tissue, according to Dr. Spence, is that the organization of organoids bears greater similarity to the human lung.

To make these lung organoids, researchers at the U-M’s Spence Lab and colleagues from the University of California, San Francisco; Cincinnati Children’s Hospital Medical Center; Seattle Children’s Hospital and University of Washington, Seattle manipulated several of the cell signaling pathways that control the formation of organs.

First, stem cells were induced to form a type of tissue called endoderm, which is found in early embryos and gives rise to the lung, liver and several other internal organs. Second, the group activated two important development pathways (FGF and WNT signaling ) that are stimulate endoderm to form three-dimensional tissue. By inhibiting two other key development pathways at the same time (BMP and TGFβ signaling), the endoderm became tissue that resembles the early lung found in embryos.

In the laboratory, this early culture-derived lung-like tissue spontaneously formed three-dimensional spherical structures as it developed. Afterwards, they had to expand these structures and develop them into lung tissue. In order to do this, Spence and his colleagues and collaborators exposed the cells to additional proteins involved in lung development (FGF and Hedgehog).

After all this manipulation, the resulting lung organoids survived in the laboratory for over 100 days.

“We expected different cells types to form, but their organization into structures resembling human airways was a very exciting result,” said author Briana Dye, a graduate student in the U-M Department of Cell and Developmental Biology.

While this type of experiment is remarkable, this is only the beginning of lung tissue engineering.  These mini-lungs  will hopefully serve and new model systems for drug testing and researching genetic diseases that affect the lungs, such as cystic fibrosis, sarcoidosis, or inherited forms of emphysema.  It will be a while before scientists can make replacement lungs for human patients, but these experiments by Spence and others are a remarkable start.

Induced Pluripotent Stem Cells Make Lungs

Since my father died of disseminated lung cancer (squamous cell carcinoma), this report has particular meaning to me.

When a person dies, their lungs can be harvested and stripped of their cells. This leaves a so-called “lung scaffold” that can then be used to build new lungs by means of tissue engineering techniques. Lung scaffolds consist of a protein called collagen, and sugar-rich proteins called “proteoglycans” (say that fast five times) and a rubber band-like protein called elastin. Depending on how the lung scaffolds are made more or less of these components can remain in the lung scaffold (see TH Peterson, and others, Cells Tissues Organs. Feb 2012; 195(3): 222–231). The important thing is that the cells are gone and this greatly reduces the tendency for the lung scaffold to be rejected by someone else’s immune system.

Once a lung scaffold is generated from a whole lung, cells can be used to reconstitute the lung. The key is to use the right cell type or mix of cell types and to induce them to form mature lung tissue.

The laboratory of Harald Ott at Harvard University Medical School used a technique called “perfusion decellularization” to make lung scaffolds from the lungs of cadavers. Then he and his co-workers used lung progenitor cells that were derived from induced pluripotent stem cells (iPSCs). This study was published in The Annals of Thoracic Surgery, and it examined the ability of iPSCs to regenerate a functional pulmonary organ

Whole lungs from rat and human cadavers were stripped of their living material by means of constant-pressure perfusion with a strong detergent called sodium dodecyl sulfate (SDS; 0.1% if anyone is interested). Ott and his crew then sectioned some of the resulting lung scaffolds and left others intact, and then applied human iPSCs that had been differentiated into developing lung tissue.

Lung tissue develops from the front part of the developing gut. This tissue is called “endoderm,” since it is in the very innermost layer of the embryo.

Lung Development

Therefore, the iPSCs were differentiated into endoderm with a cocktail of growth factors (FGF, Wnt, Retinoic acid), and then further differentiated in the anterior endoderm (foregut; treated cells with Activin-A, followed by transforming growth factor-β inhibition), and then even further differentiated into anterior, ventral endoderm, which is the precise tissue from which lungs form. In order to be sure that this tissue is lung tissue, they must express a gene called NK2 homeobox 1 (Nkx2.1). If these cells express this gene, then they are certainly lung cells.

Ott and his group showed that their differentiate iPSCs strongly expressed Nkx2.1, and then seeded them on slices and whole lung scaffolds. Then Otts’s group maintained these tissues in a culture system that was meant to mimic physiological conditions.

Those cells cultured on decellularized lung slices divided robustly and committed to the lung lineage after 5 days. Within whole-lung scaffolds and under the physiological mimicking culture, cells upgraded their expression of Nkx2.1. When the culture-grown rat lungs were transplanted into rats, they were perfused and ventilated by host vasculature and airways.

Thus these decellularized lung scaffolds supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Furthermore, whole-organ scaffolds and a culture system that mimics physiological conditions, allows scientists to enable seeding a combination of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Transplantation of these laboratory-grown lungs seem to further maturation of these grafted lung tissues.