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.

Human Fat Contains Multilineage Differentiating Stress Enduring Cells With Great Potential for Regenerative Medicine


A collaboration between American and Japanese scientists has discovered and characterized a new stem cell population from human fat that do not cause tumors and can differentiate into derivatives from ectoderm, mesoderm, and endoderm.

Multilineage Differentiating Stress-Enduring or Muse cells are found in bone marrow and the lower layers of the skin (dermis). Muse cells are a subpopulation of mesenchymal stem cells, and even express a few mesenchymal stem cell-specific genes (e.g., CD105, a cell-surface protein specific to mesenchymal stem cells). However, Muse cells also express cell surface proteins normally found in embryonic stem cells (e.g., stage-specific embryonic antigen-3, SSEA-3). Additionally, Muse cells have the ability to self-renew, and differentiate into cell types from all three embryonic germ layers, ectoderm (which forms skin and brain), mesoderm, (which forms muscle, bone, kidneys, gonads, heart, blood vessels, adrenal glands, and connective tissue), and endoderm (which forms the gastrointestinal tract and its associated tissues). Finally, Muse cells can home to damaged sites and spontaneously differentiate into tissue-specific cells as dictated by the microenvironment in which the cells find themselves.

A new publication by Fumitaka Ogura and others from Tohoku University Graduate School of Medicine in Sendai, Japan and Saleh Heneidi from the Medical College of Georgia (Augusta, Georgia), and Gregorio Chazenbalk from the David Geffen School of Medicine at UCLA has shown that Muse cells also exist in human fat.

The source of cells came from two places: commercially available fat tissue and freshly collected fat from human subjects, collected by means of liposuction. After growing these cells in culture, the mesenchymal stem cells and Muse cells grew steadily over the 3 weeks. Then the Dezawa research group used fluorescence-activated cell sorting (FACS) to isolate from all these cells those cells that express SSEA-3 on their cell surfaces.

FACS uses antibodies conjugated to dyes that can bind to specific cell proteins. Once the antibodies bind to cells, the cells are sluiced through a small orifice while they are illuminated by the laser. The laser activates the dyes if the cell fluoresces, one door opens and the other closes. The cell goes to one test tube. If the cell does not fluoresce, then the door stay shut and another door opens and the cell goes into a different test tube.  In this way, cells with a particular cell-surface protein are isolated from other cells that do not have that cell-surface protein.

Fluorescent-Activated Cell Sorting
Fluorescent-Activated Cell Sorting

In addition to expression SSEA-3, the fat-based Muse cells expressed other mesenchymal stem cell-specific cell-surface proteins (CD29, CD90), but they did not express proteins usually thought to be diagnostic for fat-based mesenchymal stem cells (MSCs) such as CD34 and CD146.  Muse cells also expressed pluripotency genes (Nanog, Oct3/4, PAR4, Sox2, and Tra-1-81).  The Muse cells grew in small clusters and some cell expressed ectodermal-specific genes (neurofilament, MAP2), others expressed mesodermal-specific genes (smooth muscle actin, NKX2) and endodermal-specific genes (alpha-fetoprotein, GATA6).  These data suggested that the cultured Muse cells were poised to form either ectoderm, mesodermal, or endodermal derivatives.

When transplanted into mice with non-functional immune systems, the Muse cells never formed any tumors or disrupted the normal structure of the nearly tissues.  When placed in differentiating media, fat-derived Muse cells differentiated into cells with neuron-like morphology that expressed neuron-specific genes (Tuj-1), liver cells, and fat.  When compared with Muse cells from bone marrow or skin, the fat-derived Muse cells were better at making bone, fat, and muscle, but not as good as bone marrow Muse cells at making neuronal cell types, but not as good at making glial cells.  Many of these assays were based on gene expression experiments and not more rigorous tests.  Therefore, the results of these experiments might be doubtful until they are corroborated by more rigorous experiments.

These cells are expandable and apparently rather safe to use.  More work needs to be done in order to fully understand the full regenerative capacity of these cells and protocols for handling them must also be developed.  However, hopefully pre-clinical experiments in rodents will give way to larger animal experiments.  If these are successful, then maybe human trials come next.  Here’s to hoping.

Embryonic Stem Used to Make A Thyroid from Scratch


Scientists from the Universite´ Libre de Bruxelles, Belgium in collaboration with scientists from the Lillehei Heart Institute at the University of Minnesota, University of Chicago, and Ghent University, in Merelbeke, Belgium have differentiated engineered mouse embryonic stem cells into thyroid cells that make thyroid hormone, organize themselves into a thyroid, and even rescue thyroid deficient mice.

In a paper published in the international journal Nature, lead author Francesco Antonica and her colleagues used mouse embryonic stem cells for these experiments. Antonica and others engineered these cells to express two transcription factors; NKX2-1 and PAX8. They used a trick to engineer these cells so that they would only express these genes if they were treated with the drug doxycycline. A variety of experiments showed that the genetic manipulation of the cells did not affect their pluripotency.

After genetically engineering their mouse embryonic stem cells, they grew half of them without doxycycline and the other half in the presence of doxycycline. Three days after growing cells on doxycycline, the cells expressed high levels of Pax8 and NKX2-1, and also showed high levels of expression of thyroid-specific genes such as thyroid-stimulating hormone (TSH) receptor (Tshr), the sodium/iodide symporter NIS (Slc5a5) and thyroglobulin (Tg), as well as Foxe1, which is yet another key transcription factor for thyroid development. In contrast, the cultures without doxycycline showed no such changes in gene expression.

These cells, however, did not stop there. 22 days after being grown on doxycycline, the cells rounded up and formed clusters that exactly resemble those found in a living thyroid gland. The resemblance to thyroid glands, however, was not superficial. Thyroid-specific proteins were detected in these clusters. Those cells formed a circle that surrounded a space and it also showed proper localization of thyroid-specific proteins. There is a protein found on the bottom of the thyroid celll called NIS, which stands for sodium/iodide symporter. This proteins transports two sodium cations (Na+) for each iodide anion (I–) into thyroid cells. The uptake of iodide into follicular cells of the thyroid gland is the first step in the synthesis of thyroid hormone. Another protein found at the bottom of thyroid cells called E-cadherin helps the cells stick to each other.

Thyroid hormone production

These ESC-derived thyroid cells also express E-cadherin at the bottom of the cell. Also, at the other end of the cell (the apical end) thyroid cells express a protein called zona occludens 1 (ZO-1). These ESC-derived thyroid cells also express ZO-1 at the top of the cell. Finally, thyroglobulin, which is the precursor version of thyroid hormone was also expressed in these cells and was also found in the space at the center of the cell clusters – just like in a thyroid gland.

a, Schematic diagram of the thyroid gland organized in follicles. b, Immunostaining of NIS in adult thyroid tissue. c–f, Immunofluorescence at day 22 of thyroid follicles derived from ESCs on ectopic expression of Nkx2-1 and Pax8 for NKX2-1 and NIS (c), NKX2-1 and E-cadherin (E-cad.) (d), NKX2-1 and ZO-1 (e) and NKX2-1 and TG (f). g, Immunodetection of TG-I in the luminal compartment of NKX2-1-positive follicles. h–j, Iodide-organification assay in cells differentiated after Dox induction of Nkx2-1-Pax8 (h), Nkx2-1 (i) and Pax8 (j). Histograms show the organification percentage of iodine-125 at day 22 in cells differentiated without Dox and rhTSH (left column), in the presence of Dox only (centre column) and on Dox and rhTSH treatment (right column). Data are mean ± s.e.m. (n = 3). Tukey’s multiple comparison test was used for statistical analysis. ***P < 0.001. Scale bars, 200 μm (b) and 20 μm (c–g). PBI, protein-bound 125I.
a, Schematic diagram of the thyroid gland organized in follicles. b, Immunostaining of NIS in adult thyroid tissue. c–f, Immunofluorescence at day 22 of thyroid follicles derived from ESCs on ectopic expression of Nkx2-1 and Pax8 for NKX2-1 and NIS (c), NKX2-1 and E-cadherin (E-cad.) (d), NKX2-1 and ZO-1 (e) and NKX2-1 and TG (f). g, Immunodetection of TG-I in the luminal compartment of NKX2-1-positive follicles. h–j, Iodide-organification assay in cells differentiated after Dox induction of Nkx2-1-Pax8 (h), Nkx2-1 (i) and Pax8 (j). Histograms show the organification percentage of iodine-125 at day 22 in cells differentiated without Dox and rhTSH (left column), in the presence of Dox only (centre column) and on Dox and rhTSH treatment (right column). Data are mean ± s.e.m. (n = 3). Tukey’s multiple comparison test was used for statistical analysis. ***P < 0.001. Scale bars, 200 μm (b) and 20 μm (c–g). PBI, protein-bound 125I.

So, it looks like a thyroid, it makes the same genes as a thyroid, but is it a thyroid functionally speaking? To answer this question, Antonica and colleagues took normal mice and feed them radioactive iodine. This destroys the thyroid and they were able to confirm that these mice were devoid of thyroid activity and showed the symptoms of hypothyroidism. Then they grafted their ESC-derived thryoid cells into the kidney capsule of the hypothyroid mice. The grafts took and tissue examination showed that the grafts looked like thyroid tissue and also expressed thyroid-specific proteins. Therefore, transplantation of the ESC-derived thyroid tissue does not change its characteristics.

Amazingly, 8 or 9 hypothyroid mice that had received the grafts recovered full thyroid function. Those hypothyroid mice transplanted with ESCs that were not grown in the presence of doxycycline showed no signs of recovery from hypothyroidism.

These experiments show that ESCs can be differentiated into thyroid follicles that can serve as an excellent model for thyroid physiology and development. Such a model system can also be used to test thyroid drugs and model thyroid diseases. Additionally, such cells can also potentially be used to treat thyroid diseases. If this technology can be recapitulated with human pluripotent stem cells – particularly with induced pluripotent stem cells and might be patient specific, then a ready-made treatment for patients who have lost their thyroids as a result of surgery is potentially at hand.