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.

Scientists Grow Small Chunks of Brain Tissue From Induced Pluripotent Stem Cells

Induced pluripotent stem cells are made from adult cells by means of genetic engineering techniques that introduce into the cells a combination for four different genes that drive the cells to de-differentiate into a cell that has many of the characteristics of embryonic stem cells without the destruction of embryos.

A new study from the laboratory of Juergen Knoblich at the Institute of Molecular Biotechnology in Vienna has mixed induced pluripotent stem cells (iPSCs) to form structures of the human brain. He largely left the cells alone to allow them to form the brain tissue, but he also placed them in a spinning bioreactor that constantly circulates the culture medium and provides nutrients and oxygen to the cells. One other growth factor he supplied to the cells was retinoic acid, which is made by the meninges that surround our brains. All of this and the cells not only divided, differentiated and assembled, but they formed brain structures that had all the connections of a normal brain. These brain-like chunks of tissue are called “mini-brains” and the recent edition of the journal Nature reports their creation.

“It’s a seminal study to making a brain in a dish,” says Clive Svendsen, a neurobiologist at the University of California, Los Angeles. Svendsen was not involved in this study, but wishes he was. Of this study, Svendsen exclaimed, “That’s phenomenal” A fully formed artificial brain is still years and years away, but the pea-sized neural clumps developed in Knoblich’s laboratory could prove useful for researching human neurological diseases.

Researchers have previously used pluripotent human stem cells to grow structures that resemble the developing eye (Eiraku, M. et al. Nature 472, 51–56 (2011), and even tissue layers similar to the cerebral cortex of the brain (Eiraku, M. et al. Cell Stem Cell 3, 519–532 (2008). However, this latest advance has seen bigger and more complex neural-tissue clumps by first growing the stem cells on a synthetic gel that resembled natural connective tissues found in the brain and elsewhere in the body. After growing them on the synthetic gel, Knoblich and his colleagues transferred the cells to a spinning bioreactor that infuses the cells with nutrients and oxygen.

“The big surprise was that it worked,” said Knoblich. The clump formed structures that resembled the brains of fetuses in the ninth week of development.

Under a microscope, the blobs contained discrete brain regions that seemed to interact with one another. However, the overall arrangement of the different proto-brain areas varied randomly across tissue samples. These structures were not recognizable physiological structures.

A cross-section of a brain-like clump of neural cells derived from human stem cells.
A cross-section of a brain-like clump of neural cells derived from human stem cells.

“The entire structure is not like one brain,” says Knoblich, who added that normal brain maturation in an intact embryo is probably guided by growth signals from other parts of the body. The tissue balls also lacked blood vessels, which could be one reason that their size was limited to 3–4 millimeters in diameter, even after growing for 10 months or more.

Despite these limitations, Knoblich and his collaborators used this system to model key aspects of microcephaly, which is a condition that causes extremely stunted brain growth and cognitive impairment. Microcephaly and other neurodevelopmental disorders are difficult to replicate in rodents because the brains of rodents develop differently than those of humans.

Knoblich and others found that tissue chunks cultured from stem cells derived from the skin of a single human with microcephaly did not grow as large as clumps grown from stem cells derived from a healthy person. When they traced this effect, they discovered that it was due to the premature differentiation of neural stem cells inside the microcephalic tissue chunks, which depleted the population of progenitor cells that fuels normal brain growth.

The findings largely confirm prevailing theories about microcephaly, says Arnold Kriegstein, a developmental neurobiologist at the University of California, San Francisco. But, he adds, the study also demonstrates the potential for using human-stem-cell-derived tissues to model other disorders, if cell growth can be controlled more reliably.

“This whole approach is really in its early stages,” says Kriegstein. “The jury may still be out in terms of how robust this is.”

Tumor Suppressor Gene is Required For Neural Stem Cells to Differentiate into Mature Neurons

Cancer cells form when healthy cells accumulate mutations that either inactivate tumor suppressor genes or activate proto-oncogenes. Tumor suppressor genes work inside cells to put the brakes on cell proliferation. Proto-oncogenes work to drive cell proliferation. Loss-of-function mutations in tumor suppressor genes remove controls on cell proliferation, which causes cells to divide uncontrollably. Conversely activating mutations in proto-oncogenes removes the controls on the activity of proto-oncogenes, converting them into oncogenes and driving the cell to divide uncontrollably. If a cell accumulates enough of these mutations, they can grow in such an uncontrollable fashion that they start to gain extra chromosomes or pieces of chromosomes, which contributes to the genetic abnormality of the cell. Accumulation of more mutations allows the cell to break free from the original tumorous mass and spread to other tissues.

There are over 35 identified tumor suppressor genes and one of these, CHD5, has another role besides controlling cell proliferation. Researchers at Karolinska Institutet in Stockholm, Swede, in collaboration with other laboratories at Trinity College in Dublin and BRIC in Copenhagen has established a vital role for CHD5 in normal nervous development.

Once stem cells approach the final phase of differentiation into neurons, the CHD5 protein is made at high levels. CHD5 reshapes the chromatin structure into which DNA is packaged in cells, and in doing so, it facilitates or obstructs the expression of other genes.

Ulrika Nyman, postdoc researcher in Johan Holmberg’s laboratory, said that when they switched of CHD5 expression in stem cells from mouse embryos at the time when the brain develops, the CHD5-less stem cells were unable to turn off those genes that are usually expressed in other tissues, and equally unable to turn on those genes necessary for making mature neurons. Thus these CHD5-less stem cells were trapped in a nether-state between stem cells and neurons.

CHD5 function in stem cell differentiationretinoic

The gene that encodes the CHD5 protein is found on chromosome 1 (1p36) and it is lost in several different cancers, in particular neuroblastomas, a disease found mainly in children and is thought to arise during the development of the peripheral nervous system.

Neuroblastomas that lack this part of chromosome 1 that contains the CHD5 gene are usually more aggressive and more rapidly fatal.

Treatment with retinoic acid forces immature nerve cells and some neuroblastomas to mature into specialized nerve cells. However, when workers from Holmberg’s laboratory prevented neuroblastomas from turning up their expression of CHD5, they no longer responded to retinoic acid treatment.

Holmberg explained, “In the absence of CHD5, neural tumor cells cannot mature into harmless neurons, but continue to divide, making the tumor more malignant and much harder to treat. We now hope to be able to restore the ability to upregulate CHD5 in aggressive tumor cells and make them mature into harmless nerve cells.”