An Entire Organ Grown Inside an Animal


For the first time, scientists from Scotland have reported that an entire, functional organ has been grown from scratch inside a laboratory animal. A Scottish research group successfully transplanted a small quantity of cells into a laboratory mouse that grew and developed into a functional thymus.

These findings were published in the journal Nature Cell Biology, and might open the door to new alternatives to organ transplantation. This research certainly shows great promise, but is still years away from clinical trials and reproducible human therapies.

If you are wondering what the thymus is, it serves as an integral part of the immune system. The thymus is located just above and slightly over the heart and produces a vital component of the immune system, called T-cells, which fight infections and regulate the immune response.

The thymus
The thymus

thymus location

 

A research team from the Medical Research Council Centre for Regenerative Medicine at the University of Edinburgh began this experiment with mouse embryonic fibroblasts.  These fibroblasts are found in the skin and connective tissue of the embryo.  These mouse embryonic fibroblasts were genetically engineered to expressed the FOXN1 gene, which encodes a transcription factor known as the “forkhead box N1″ protein.  The forkhead box N1 protein binds to DNA and activates the expression of genes necessary to make thymic epithelial cells.  Mice that do not have a functional copy of the FOXN1 gene a “nude” mice.  They are nude because they have no hair and have no thymus.  

Once engineered to express FOXN1, the fibroblasts began to differentiate into thymic epithelial cells.  The Scottish team mixed these genetically engineered fibroblasts with some other support cells and transplanted them into laboratory mice where they summarily formed a fully functional thymus.  Structurally the animal-grown thymus contained the two main regions – the cortex and medulla – and it also produced T-cells.

Prof Clare Blackburn, who was part of the research team, said it was “tremendously exciting” when the team realized what they had accomplished.  Blackburn told the BBC: “This was a complete surprise to us, that we were really being able to generate a fully functional and fully organised organ starting with reprogrammed cells in really a very straightforward way.  This is a very exciting advance and it’s also very tantalising in terms of the wider field of regenerative medicine.”

Such a procedure could benefit patients who need a bone marrow transplant and children who are born without a functioning thymus.  Likewise because our immune response diminishes as we age and out thymus shrivels, such a procedure might boost the waning immune system of aged patients.   could all benefit from such a procedure.

However, there are a number of problems to solve before this procedure can cross the bridge from animal studies to hospital therapies.  First of all, the recipient of these implants were nude mice that had no thymus and could not reject transplanted tissue.  Also, the use of embryonic fibroblasts would cause a robust immune response against them.  Some other cell type must be found for this procedure that grows robustly and does not cause transplantation rejection.

Researchers also need to be sure that the transplant cells do not pose a cancer risk by growing uncontrollably.  Prof Robin Lovell-Badge, from the National Institute for Medical Research, said: “This appears to be an excellent study.  This is an important achievement both for demonstrating how to make an organ, albeit a relatively simple one, and because of the critical role of the thymus in developing a proper functioning immune system.  However… the methods are unlikely to be easy to translate to human patients.”

This experiment is a testimony of just how far the field of regenerative medicine has come.  Already patients with lab-grown blood vessels, windpipes and bladders have benefited from advances in regenerative medicine. These tissue engineered structures have been made by “seeding” a patient’s cells into a scaffold which is then implanted.  The thymus in this case only required one injection of a cluster of cells.  While it is doubtful that other organs will be this easy to grow, it is an encouraging start.

Also, this experiment utilized “direct reprogramming” that did not require taking cells through the embryonic stage.  Instead one-gene reprogramming directed the cells to make thymus epithelium cells.  This almost certainly promises to be a much safer way to make cells for regenerative treatments.

Dr Paolo de Coppi, who pioneers regenerative therapies at Great Ormond Street Hospital, said: “Research such as this demonstrates that organ engineering could, in the future, be a substitute for transplantation.  Engineering of relatively simple organs has already been adopted for a small number of patients and it is possible that within the next five years more complex organs will be engineered for patients using specialised cells derived from stem cells in a similar way as outlined in this paper.  It remains to be seen whether, in the long-term, cells generated using direct reprogramming will be able to maintain their specialised form and avoid problems such as tumour formation.”

Preventing the Rejection of Embryonic Stem Cell Derivatives – Take Two


Yesterday I blogged about the paper from Yang Xu’s group that used genetically engineered embryonic stem cells to make adult cell types that were not rejected by the immune systems of mice with humanized immune systems. I would like to say a bit more about this paper before I leave it be.

First of all, Xu and his colleagues engineered the cells to express the cell-surface protein PD-L1, which stands for programmed cell death ligand 1 (also known as CD274), and another protein called CTLA4-Ig. The combination of these two proteins tends to make these cells invisible to the immune system for all practical intents and purposes.

PD-L1, however, is used by tumor cells to evade detection by the immune system. For example, increased expression of PD-L1 is highly correlated with the aggressiveness of the cancer. One particular experiment examined 196 tumor specimens that had been extracted from patients with renal cell carcinoma (kidney tumors). In these tumor samples, high expression of PD-L1 was positively associated with increased tumor aggressiveness and a those patients that had higher expression of PD-L1 have a 4.5-fold increased risk of death (see Thompson RH, et al., Proc Natl Acad Sci USA 101 (49): 17174–9). In patients with cancer of the ovaries, those tumors with higher PD-L1 expression had a significantly poorer prognosis than those with lower PD-L1 expression. The more PD-L1 these tumors expressed, the fewer tumor-hunting T cells (CD8+ T cells) were present (see Hamanishi J, and others, Proc Natl Acad Sci USA 104 (9): 3360–5).

So the Xu paper proposes that we introduce genetically engineered cells, which are already at risk for mutations in the first place, into the body, that constitutively express PD-L1, a protein known to be highly expressed in the most aggressive and lethal tumors. Does this sound like a good idea?

With respect to CTLA4-Ig, this is a cell-bound version of a drug that has been approved as an anti-transplantation rejection drug called Belatacept (Nulojix), made by Bristol-Myers-Squibb. Since this is a cell-bound version of this protein, it will almost certainly not have the systemic effects of Belatacept, and if the cells manage to release a certain amount of soluble CTLA4-Ig, it is likely to be very little and have no biological effect.

Therefore, this strategy, while interesting, does come with its own share of risks and caveats.

Preventing Rejection of Embryonic Stem Cell-Based Tissues


Embryonic stem cells (ESCs) are derived from human embryos. Because they are pluripotent, or have the capacity to make any adult cell type, ESCs are thought to hold great promise for cell therapy as a source of differentiated cell types.

One main drawback to the use of ESCs in regenerative medicine is the rejection of ESC-derived cells by the immune system of the patient. Transplantation of ESC-derived tissues would require the patient to take powerful anti-rejection drugs, which tend to have a boatload of severe side effects.

However, a paper reports a strategy to circumvent rejection of ESC-derived cells. If these strategies prove workable, then they might clear the way to the use of ESCs in regenerative medicine.

The first paper comes from the journal Cell Stem Cell, by Zhili Rong, and others (Volume 14, Issue 1, 121-130, 2 January 2014). In this paper, Rong and his colleagues from the laboratory of Yang Xu at UC San Diego and their Chinese collaborators used mice whose immune systems had been reconstituted with a functional human immune system. These humanized mice mount a robust immune response against ESCs and any cells derived from ESCs.

In their next few experiments, Xu and others genetically engineered human ESCs to routinely express two proteins called CTLA4-Ig and PD-L1. Now this gets a little complicated, but stay with me. The protein known as CTLA4-Ig monkeys with particular cells of the immune system called T cells, and prevents those T cells from mounting an immune response against the cells that display this protein on their surfaces. The second protein, PD-L1, also targets T cells and when T cells bind to cells that have this protein on their surfaces, they are completely prevented from acting.

CTLA-4 mechanism

Think of it this way: T cells are the “detectives” of the immune system. When they find something fishy in the body (immunologically speaking), they get on their “cell phones” and call in the cavalry. However, when these detectives come upon these cells, their cell phones are inactivated, and their memories are wiped. The detectives wander away and then do not remember that they ever came across these cells.

Further experiments showed that any derivatives of these engineered ESCs, (teratomas, fibroblasts, and heart muscle cells) were completely tolerated by the immune system of these humanized mice.

This is a remarkable paper. However, I have a few questions. Genetic engineering of these cells might be potentially dangerous, depending upon how it was done, where in the genome the introduced genes insert, and how they are expressed. Secondly, if cells experience any mutations during the expansion of these cells, these mutations might cause the cells to be detected by the immune system. Third, do these types of immune repression last long-term? Clearly more work will need to be done, but these questions are potentially addressable.

My final concern is that if this procedure is used widespread, it might lead to the wholesale destruction of human embryos. Human embryos, however, are the youngest, weakest, and most vulnerable among us. What does that say about us if we do not value the weakest among us and dismember them for their cells? Would we allow this with toddlers?

Thus my interest and admiration for this paper is tempered by my concerns for human embryos.

Induced Pluripotent Stem Cells Do Not Cause Immune Rejection


A paper appeared in the journal PLoS One by Liu and others that showed that heart muscle cells made from induced pluripotent stem cells were rejected by the immune system of mice. The way induced pluripotent stem cells (iPSCs) are made introduces mutations, many of which are harmless. However, mutations that alter the cell surface proteins of iPSC derivatives can cause the immune system of the host to attack and destroy any transplanted cells.

Are adult cells made from iPSC recognized by the immune system? Are the mouse experiments merely an anomaly of the mouse system?

Dr. Jun Takahashi of Kyoto University’s Center for iPS Cell Research and Application and his research group have examined how monkeys respond to implanted derivatives of iPSCs. They made iPSCs from monkey cells taken from the inside of the mouth. Then Takahashi and his group made midbrain-specific neurons from them and transplanted them back into the monkeys. Only a minimal immune response against these cells was observed. However if a monkey received midbrain neurons made from another animal’s cells, then a robust immune response followed.

Therefore, in non-human primates, iPSC derivatives are not rejected by the immune system of the host.

Takahashi said of this experiment, “These findings give a rationale to start autologous transplantation – at least of neural cells – in clinical situations.”  Takahashi’s last statement is critically important – “At least of neural cells.” The brain is an immunologically privileged organ that normally does not have immune cells lurking in its midst. The heart, however, is constantly under immunological surveillance. Therefore, even though this experiment shows that IPSC derivatives are not rejected in non-human primates under these circumstances, there might be circumstances under which they are rejected.

Since there are ways to screen iPSCs and their derivatives for mutations that might sensitize the immune system to the host, such screenings could almost certainly decrease the rate of immunological rejection. Such screening were not done in either this experiment or in the experiments of Liu and others.

Embryonic Stem Cells Used to Make Laboratory-Created Thymus


Medical researchesr from UC San Francisco have used embryonic stem cells to construct a functioning mouse thymus in the laboratory. When implanted into a living mouse, this laboratory-made thymus can successfully foster the development of T cells, which the body needs to fight infections and prevent autoimmune reactions.

This achievement marks a significant step toward developing new treatments for autoimmune disorders such as type 1 diabetes and other autoimmune diseases, such as systemic lupus erythematosis and ulcerative colitis.

This research team was led by immunologist Mark Anderson and stem cell researcher Matthias Hebrok. They used a unique combination of growth factors to push the embryonic stem cells into a particular developmental trajectory. After a period of trial and error, they eventually found a formula that produced functional thymus tissue.

In our bodies, the thymus lies just over the top of our heart, and it serves to instruct T lymphocytes (a type of white blood cell) what to attack and what to leave alone. Because T cells serve a vital role in the immune response, the thymus serves a vital function.

thymus

Typically, each T cell attacks a foreign substance that it identifies by binding the foreign substance to its cell surface receptor. This T cell-specific receptor is made in each T cell by a set of genes that are randomly shuffled, and therefore, each T cell has a unique cell receptor that can bind particular foreign molecules. Thus each T cell recognizes and attacks a different foreign substance.

With in the thymus, T cells that attack the body’s own proteins are eliminated. Thymic cells express major proteins from elsewhere in the body. The T cells that enter the thymus first undergo “Positive Selection” in which the T cell comes in contact with self-expressed proteins that are found in almost every cell of the body and are used to tell “you” from something that is not from “you.” In order to destroy cells that do not bear these self-expressed proteins, they must be able to properly identify them. If T cells that enter the thymus cannot properly recognize those self-expressed proteins (known as MHC or major histocompatibility complex proteins for those who are interested), the thymus destroys them. Second, T cells undergo “Negative Selection” in which if the T cell receptor binds to self MHC proteins, that T cell is destroyed to avoid autoimmunity.

The thymus tissue grown in the laboratory in this experiment was able to nurture the growth and development of T cells. It could act as a model system to study patients with fatal diseases from which there are no effective treatments, according the Mark Anderson.

As an example, DiGeorge Syndrome is caused by a small deletion of a small portion of chromosome 22 and infants born with DiGeorge Syndrome are born without a thymus and they usually die during infancy.

Other applications include manipulating the immune system to accept transplanted tissues such as implanted stem cells or organs from donors that are not a match to the recipient.

Anderson said, “The thymus is an environment in which T cells mature and where they also are instructed on the difference between self and nonself.” Some T cells are prepared by the thymus to attack foreign invaders and that includes transplanted tissue. Other T cells that would potentially attack our own tissues are eliminated by the thymus.

Laboratory-induced thymus tissue could be used to retrain the immune system in autoimmune diseases so that the T cells responsible for the autoimmune response eventually ignore the native tissues they are attacking.

Hebrok warns that he and his team have not perfectly replicated a thymus. Only about 15% of the cells are successfully directed to become thymus tissue with the protocols used in this study. Nevertheless, Anderson asserted, “We now have developed a tool that allows us to modulate the immune system in a manner that we never had before.”

The Surprising Ability of Blood Stem Cells to Respond to Emergencies


A research team from Marseille, France has revealed an unexpected role for hematopoietic stem cells (the cells that make blood cells): not only do these cells continuously renew our blood cells, but in emergencies these cells can make white blood cells on demand. that help the body deal with inflammation and infection. This stem cell-based activity could be utilized to protect against infection in patients who are undergoing a bone marrow transplant.

The research team that discovered this previously unknown property of hematopoietic stem cells were from INSERM, CNRS and MDC led by Michael Sieweke of the Centre d’Immunologie de Marseille Luminy and the Max Delbruck Centre for Molecular Medicine, Berlin-Buch.

Cells in our blood feed, clean, and defend our tissues, but their lifespan is limited. The life expectancy of a red blood cell rarely exceeds three months, our platelets die after ten days and the vast majority of our white blood cells survive only a few days.

Therefore, our bodies must produce replacements for these dying cells in a timely manner and in the right quantities and proportions. Blood cells replacement is the domain of the hematopoietic stem cells, which are nested in the bone marrow; that soft tissue inside long bones of the chest, spine, pelvis, upper leg and shoulder. Bone marrow produces and releases billions of new cells into out blood every day. To do this, hematopoietic stem cells must not only divide but their progeny must also differentiate into specialized cells, such as white blood cells, red blood cells, platelets, and so on.

For several years, researchers have been interested in how the process of differentiation and specialization is triggered in stem cell progeny. Sieweke and his colleagues discovered in previous work that hematopoietic stem cell progeny are not preprogrammed to assume a particular cell fate, but respond to environmental cues that direct them to become one cell type or another.

Nevertheless, it is still unclear how stem cells respond during emergencies? How are hematopoietic stem cells able to meet the demand for white blood cells during an infection? Recently, the answer was considered clear: the stem cells neither sensed nor responded to the signals sent to induce their progeny to differentiate into particular cell types. They merely proliferated and their progeny responded to the available signals and differentiated into the necessary cell fates. However, Sieweke’s research team has found that rather than being insensitive to these inductive signals meant for their progeny, hematopoietic stem cells perceive these environmental signals and, in response to them, manufacture the cells that are most appropriate for the danger faced by the individual.

Dr. Sandrine Sarrazin, INSERM researcher and co-author of the publication, said, “We have discovered that a biological molecule produced in large quantities by the body during infection or inflammation directly shows stem cells the path to take.”

Sieweke added, “Now that we have identified this signal, it may be possible in the future to accelerate the production of these cells in patients facing the risk of acute infection.” He continued: “This is the case for 50,000 patients worldwide each year who are totally defenseless against infections just after bone marrow transplantation. Thanks to M-CSF [monocyte-colony stimulating factor], it may be possible to stimulate the production of useful cells while avoiding to produce those that can inadvertently attack the body of these patients. They could therefore protect against infections while their immune system is being reconstituted.”

To reach their conclusions the team had to measure the change of state in each cell. This was a terrifically difficult challenge since the stem cells in question are very rare in the bone marrow: only one cell in 10,000 in the bone marrow of a mouse. Furthermore, the hematopoietic stem cells are, by appearance, indistinguishable from their progeny, the hematopoietic progenitor cells. Therefore, this experiment was tedious and difficult, but it proved that M-CSF could instruct single hematopoietic stem cells to differentiate into the monocyte lineage.

The clinical use of M-CSF will hopefully follow in the near future, but for now, this is certainly an exciting finding that may lead to clinical trials and applications in the future.