Pluripotent Stem Cells Used to Make a Functional Thyroid


The thyroid gland sits over the main cartilage of the larynx and produces thyroid hormone (thyroxine); a hormone that regulates the basal metabolic rate. When the thyroid slows down and fails to make sufficient amounts of thyroid hormone, the result is a condition called hypothyroidism. The symptoms of hypothyroidism are fatigue, weakness, weight gain or increased difficulty losing weight, coarse, dry hair, dry, rough pale skin, hair loss, cold intolerance, muscle cramps and frequent muscle aches, constipation, depression, irritability, memory loss, abnormal menstrual cycles and decreased libido.

If someone has any evidence of a thyroid tumor, then the thyroid is removed, and the patient must take oral thyroid hormone. Because getting the dose right can be difficult, we might ask, “Can we replace the thyroid with stem cell treatments?”

Human pluripotent stem cells can differentiate into balls of cells that are mini-organs called “organoids.”  Unfortunately, if left to themselves, the formation of these organoids is rather haphazard and the cells tend to differentiate into a whole host of different cell types. This is not fatal, however, since the differentiation of these stem cells can be orchestrated by using growth factors or certain culture conditions. Can we use such innovations to make a minithyroid?

Darrell Kotton and his group at Boston University School of Medicine Pulmonary, Allergy, Sleep and Critical Care Medicine have spent their time tweaking the conditions to drive human pluripotent stem cells to form thyroid cells. A new study of theirs that appears in the journal Cell Stem Cell details how the use of two growth factors, BMP4 and FGF2 can drive pluripotent stem cells to commit to thyroid cell fates.

In order to make thyroid cells from embryonic stem cells (ESCs), Kotton and his group had to make endodermal progenitors from them first. Fortunately, a study from Kotton’s own laboratory that was published in 2012 employed a technique used in several other papers that grew ESCs in a serum-free medium with a growth factor called activin. Christodoulou, C., and others (J. Clin. Invest. 121, 2313–2325) showed that over 80 percent of the ESCs grown under these conditions differentiated into endodermal progenitors.  When Kotton and his colleagues cultured these endodermal progenitors in BMP4 and FGF2, some of them differentiated into thyroid progenitor cells. Interestingly, this mechanism by which thyroid-specific cell fates are specified is conserved in creatures as disparate as frogs and mice.

To make mature thyroid cells from these progenitors, a three-dimensional culture system was used in combination with thyroid stimulation hormone and dexamethasone. Under these conditions, the cells formed spherical thyroid follicles that secreted thyroid hormone. To perfect their protocols, Kotton’s group used mouse ESCs, but they additionally showed that this same strategy can make mature thyroid cells from human induced pluripotent stem cells (iPSCs).

Thyroid specification

The appearance of cells in a culture system that look like mature thyroid follicles and express many of the same iodine-metabolizing enzymes as mature thyroid cells is exciting, but can such cells stand in for thyroid tissue in an animal that lacks sufficient thyroid tissue?

Kotton’s laboratory took this to the next step by transplanting their cultured thyroid follicles into laboratory mice that lacked a functional thyroid. These transplants were not inserted into the neck of the animal, but instead were place underneath the kidney, which is area rich in blood vessels. Interestingly, the implanted thyroid “organoids” or little organs did not fall apart upon transplantation. Instead they retained their characteristic structure. More interestingly, these organoids kept expressing iodine-metabolizing enzymes and made thyroid hormone. The synthesis and release of thyroid hormone was also regulated by the hypothalamic hormone thyroid stimulating hormone (TSH). TSH is made and released in response to insufficient thyroid hormone levels. The thyroid responds to TSH by making a releasing more thyroid hormone, which causes a feed-back inhibition of the release of TSH. The fact that these implanted organoids were properly regulated by TSH bespeaks of the maturity of these cells. Also, significantly, none of the laboratory animals showed any signs that the implanted cells had formed any tumors.

Kotton and his coworkers were also able to used human ESCs and human iPSCs to make thyroid organoids. Human iPSCs-derived thyroid organoids were made from human patients with normal thyroid function and from hypothyroid children who carry a loss-of-function mutation in the NKX2-1 gene. This show that Kotton’s system can be used as a model to study inherited thyroid deficiencies. However, there is even more excitement that this system or something similar to it might be useful to safely treat thyroid loss in patients who have lost their thyroid as a result of cancer, or injury.

Society for Neuroscience Conference – More to Report


A very interesting poster at the SfN meeting described experiments with the antihypertensive medicine Telmisartan and its ability to protect brain cells from dying from an overdose of neurotransmitters.

During a stroke, dead or dying neurons tend to dump enormous quantities of neurotransmitters into their surrounding environment, and these excessive concentrations of neurotransmitters are deleterious for the surrounding neurons. This phenomenon is called “excitoxicity,” and it is an important killer of neurons in a stroke.

In this poster, a Chinese scientist used Telmisartan to pre-treat cultured neurons that were then given large quantities of the neurotransmitter glutamate. The drug protected the neurons from dying from the excessive concentrations of glutamate. Telmisartan also profected cells by binding to the AT[1A] receptor, and activating the PPAR[gamma] transcription factor. While these results may sound cryptic, PPAR[gamma] is a target for a group of anti-diabetic drugs called the triglitazones. By activating this transcription factor, telmisartan rescued these cultured neurons from certain death, and Dr. Wang (the poster presenter) suggested that Telmisartan could potentially be prescribed to delay the effects of stroke are even Alzheimer’s disease.

I also attended a series of short oral presentations at this meeting, and one symposium included modeling diseases with induced pluripotent stem cells. That was a fascinating symposium and I felt like a kid in a candy store. One Japanese researchers discussed his successes at using induced pluripotent stem cells (iPSCs) to make brain “organics.” These organoids contain multiple organ-specific cell types, recapitulate some function of the organ, and share at least some of the cellular organization of the organ. Brain organoids were made by deriving iPSCs from cells taken from human volunteers, which were ten grown in embryonic stem cell medium for one week to expand the cells. Then the cells were for about another week in Neural Induction Medium, and then shaken for four more weeks. The cells self-organized into minibrains that exhibited cortical organization with the layered structure of a brain that expressed many of the same genes as the layers of a developing brain. These minibrains also showed glutamate-induced calcium mobilization. Thus these minibrains qualify as a brain organoid.

Next, he used this same procedure to make minibrains from iPSCs derived from patients with fragile X syndrome, which, besides Down Syndrome, is the leading cause of mental retardation, globally speaking. Minibrains from these Fragile X Syndrome patients formed and looked normal. However, they showed abnormal connections between neurons. This tremendous model system can provide ways to study neurological diseases at very detailed levels.

The next talk was by Haruhisa Inoue from Kyoto University who examined the use of iPSCs as a way to treat neurological diseases. In particular, Dr. Inoue was interested in Amyotrophic Lateral Sclerosis or ALS. In the case of ALS, a cells called astrocytes are the problem. The astrocytes generate a foul environment that causes the neurons in the spinal cord to die off.

Dr. Inoue used iPSC technology to derive mature astrocytes from non-ALS and ALS patients. The two sets of astrocytes showed profound functional differences. When he transplanted normal astrocytes into the spinal cords of ALS mice, her also discovered that the mice showed rather significant functional improvements. Thus, Dr. Inoue thinks that transplantation of astrocytes made from iPSCs derived from the cells of healthy volunteers might provide an excellent way to delay or even reverse the effects of ALS.

Digestive Cells Converted into Insulin-Secreting Cells


By switching off a single gene, Columbia Medical Center scientists have converted cells from the digestive tract into insulin-secreting cells. This suggests that drug treatments might be able to convert gut cells into insulin-secreting cells.

Senior author Domenico Accili said this of this work: “People have been talking about turning one cell into another for a long time, but until now we hadn’t gotten to the point of creating a fully functional insulin-producing cell by the manipulation of a single target.”

Accili’s work suggests that lost pancreatic beta cells might be replaced by retraining existing cells rather than transplanting new insulin-secreting cells. For nearly two decades, scientists have been trying to differentiate a wide variety of stem cells into pancreatic beta cells to treat type 1 diabetes. In type 1 diabetes, the patient’s insulin-producing beta cells are destroyed, usually by the patient’s own immune system. The patient becomes dependent on insulin shots in order to survive.

Without insulin, cells have no signal to take up sugar and metabolize it. Also muscles and the liver do not take up amino acids and make protein, and the body tends to waste away, ravaged by high blood sugar levels that progressively and relentlessly damage it without the means to repair this damage.

Insulin-producing beta cells can be made in the lab from several different types of stem cells, but the resulting beta cells often do not possess all the properties of naturally occurring beta cells.

This led Accili and others to attempt to transform existing cells into insulin-secreting beta cells. In previous work, Accili and others demonstrated that mouse intestinal cells could be converted into insulin-secreting cells (see Talchai C, et al., Nat Genet. 2012 44(4):406-12), This recent paper demonstrates that a similar technique also works in human intestinal cells.

The gene of interest, FOXO1, is indeed present in human gut endocrine progenitor and serotonin-producing cells. In order to determine in FOXO1 inhibition could induce the formation of insulin-secreting cells, Accili and others used human induced pluripotent stem cells (iPSCs) and small “gut organoids,” which are small balls of gut tissue that grow in culture.

Inhibition of FOXO1 by either introducing a mutant version of the gene that encoded a protein that soaked up all the wild-type protein or by using viruses that forced the expression of a small RNA that prevented the expression of the FOXO1 gene caused loss of FOXO1 activity. FOXO1 inhibition promoted the generation of insulin-positive cells within the gut organoids that express all the genes and proteins normally found in mature pancreatic β-cells. These transdifferentiated cells also released “C-peptide,” which is a byproduct of insulin production, in response to drugs that drive insulin secretion (insulin secretagogues). Furthermore, these cultured insulin-secreting cells and survive when transplanted into mice where they continue to secrete insulin in response to increased blood sugar concentrations.

The findings of Accili and his colleagues provide some evidence that gut-targeted FOXO1 inhibition or transplantation of cultured gut organoids made from iPSCs could serve as a source of insulin-producing cells to treat human diabetes.

This is a remarkable piece of research, but there is one thing that troubles me about it. If the patient’s immune system has been sensitized to beta cells, making new beta cells will simply give the immune system something else to attack. It seems to me that retraining to immune system needs to be done first before replacement of the beta cells can ever hope to succeed.

Isolation of Pancreatic Stem Cells


There has been a robust debate as to whether or not the pancreas has a stem cell population. Several studies suggested that the pancreatic duct cells could differentiate into hormone-secreting pancreatic cells. Unfortunately, when the cells of the pancreatic duct are marked, they clearly never contribute to regeneration of the pancreas. According to an article that appeared in the journal Developmental Cell by Oren Ziv, Benjamin Glaser, and Yuval Dor entitled “The Plastic Pancreas,” tying off the pancreatic duct kills off the acinar cells, but it leads to a large increase in the number of hormone-secreting beta cells. Something seems to be contributing cells to the adult pancreas. However when lineage studies tried to confirm that the pancreatic duct cells formed the new cells, it failed to find any connection between the new cells in the pancreas and the duct.

Pancreas

Recent experiments from Chris Wright’s lab suggest that the acinar cells are a population of progenitor cells that divide and differentiate into different kinds of pancreatic cell types after injury to the pancreas. A similar result was observed in work by Desai and others. If that’s not odd enough for you, another set of experiments from Pedro Herrera research group has shown once all the insulin-secreting beta cells are killed off, the adjacent glucagon-secreting cells transdifferentiate into insulin-secreting beta cells. Therefore, something interesting is afoot in the pancreas.

All these experiments were done with rodents. Whether or not they are transferable to human remains uncertain. Nevertheless, a fascinating paper in EMBO Journal from Hans Clevers lab at the Hubrecht Institute, Utrecht, Netherlands haws succeeded in culturing pancreatic precursor cells.

Here’s how they did it. Clevers and his crew took the pancreatic duct of mice and partially tied it off. In order to stem cells from the digestive tract to grow, they must upregulate a signaling pathway called the “Wnt” pathway. The Wnt pathway is quiet in the pancreas, but one the pancreas is injured, the Wnt pathway swings into gear and the cells begin to divide.

When Clevers and company dropped pancreatic duct tissue into culture, Wnt signaling activity soared and the cells grew into a mini-organ (organoid) that resembled and tiny pancreas in a culture dish. In fact, a single cell taken from the pancreatic duct could be cultured into an organoid.

Establishment of the pancreas organoids from adult pancreatic ducts. (A) Scheme representing the isolation method of the pancreatic ducts and the establishment of the pancreatic organoid culture. The pancreatic ducts were isolated from adult mouse pancreas after digestion, handpicked manually and embedded in matrigel. Twenty-four hours after, the pancreatic ducts closed and generated cystic structures. After several days in culture, the cystic structures started folding and budding. (B) Representative serial DIC images of a pancreatic organoid culture growing at the indicated time points. Magnifications: × 10 (days 0, 2, 4, 6, and 8) and × 4 (day 10 onwards). (C) Growth curves of pancreas cultures originated from isolated pancreatic ducts cultured as described in Materials and methods. Note that the cultures followed an exponential growth curve within each time window analysed. Graphs illustrate the number of cells counted per well at each passage from passages P1–P3 (left), P5–P7 (middle) and P10–P12 (right). The doubling time (hours) is indicated in each graph. Data represent mean±s.e.m., n=2. (D) Representative DIC images of XGAL staining in WT (left), Axin2-LacZ (middle) and Lgr5-LacZ (right) derived pancreas organoids.
Establishment of the pancreas organoids from adult pancreatic ducts. (A) Scheme representing the isolation method of the pancreatic ducts and the establishment of the pancreatic organoid culture. The pancreatic ducts were isolated from adult mouse pancreas after digestion, handpicked manually and embedded in matrigel. Twenty-four hours after, the pancreatic ducts closed and generated cystic structures. After several days in culture, the cystic structures started folding and budding.  (B) Representative serial DIC images of a pancreatic organoid culture growing at the indicated time points. Magnifications: × 10 (days 0, 2, 4, 6, and 8) and × 4 (day 10 onwards). (C) Growth curves of pancreas cultures originated from isolated pancreatic ducts cultured as described in Materials and methods. Note that the cultures followed an exponential growth curve within each time window analysed. Graphs illustrate the number of cells counted per well at each passage from passages P1–P3 (left), P5–P7 (middle) and P10–P12 (right). The doubling time (hours) is indicated in each graph. Data represent mean±s.e.m., n=2. (D) Representative DIC images of XGAL staining in WT (left), Axin2-LacZ (middle) and Lgr5-LacZ (right) derived pancreas organoids.

This experiment shows that there are techniques for growing unlimited quantities of pancreatic cells.  The therapeutic possibilities of this technology is tremendous.  In Clever’s own words, “We have found a way to activate the Wnt pathway to produce an unlimited expansion of pancreatic stem cells isolated from mice.  By changing the growth conditions we can select two different fates for the stem cells and generate large numbers of either hormone-producing beta cells or pancreatic duct cells.”

Can this work with human pancreatic duct cells?  That is the $64,000 question.   Clevers and his groups will almost certainly try to answer this questions next.  If Clevers and his crew can get this to work, then the possibilities are vast indeed.