Showing posts with label regenerative Medicine. Show all posts
Showing posts with label regenerative Medicine. Show all posts

Tuesday, April 8, 2014

British Scientists regenerate immune organ in mice

Scientists regenerate immune organ in mice
By Kate Kelland

LONDON Tue Apr 8, 2014 7:07am EDT

(Reuters) - British scientists have for the first time used regenerative medicine to fully restore an organ in a living animal, a discovery they say may pave the way for similar techniques to be used in humans in future.

The University of Edinburgh team rebuilt the thymus - an organ central to the immune system and found in front of the heart - of very old mice by reactivating a natural mechanism that gets shut down with age.

The regenerated thymus was not only similar in structure and genetic detail to one in a young mouse, the scientists said, but was also able to function again, with the treated mice beginning to make more T-cells - a type of white blood cell key to fighting infections.

The regenerated thymus was also more than twice the size of the aged organs in the untreated mice.

"By targeting a single protein, we have been able to almost completely reverse age-related shrinking of the thymus," said Clare Blackburn from Edinburgh's Medical Research Council (MRC) Centre for Regenerative Medicine, who led the research.

"Our results suggest that targeting the same pathway in humans may improve thymus function and therefore boost immunity in elderly patients, or those with a suppressed immune system."

She added however, that while the treated mice were making T-cells, her research could not yet establish whether the immune systems of the older mice were strengthened.

And before the technique can be tested in humans, she said, researchers will need to conduct more animal experiments to make sure the regeneration process can be tightly controlled.

The thymus is the first organ to deteriorate as people age. This shrinking is one of the main reasons the immune system becomes less effective and we lose the ability to fight off new infections, such as flu, as we get older.

Regenerative medicine is a fast-growing area of research, mainly focused on stem cells - the master cells that act as a source for all types of cells and tissues in the body. One of the central aims is to harness the body's own repair mechanisms and manipulate them in a controlled way to treat disease.

Blackburn's team, whose work was published on Tuesday in the journal Development, said they targeted a part of the process by which the thymus degenerates - a protein called FOXN1 that helps control how key genes in the thymus are switched on.

They used genetically modified mice to enable them to increase levels of this protein using chemical signals. By doing so, they managed to instruct immature cells in the thymus - similar to stem cells - to rebuild the organ in the older mice.

Rob Buckle, the MRC's head of regenerative medicine, said this success with the mouse thymus suggests organ regeneration in mammals can be directed by manipulating a single protein - something he said could have broad implications for other areas of regenerative biology.

(Editing by Pravin Char)

Saturday, October 19, 2013

Milk-Maker Hormone May Help Liver Regenerate

Milk-Maker Hormone May Help Liver Regenerate

Oct. 15, 2013 — The hormone prolactin is probably best known for its role in stimulating milk production in mothers after giving birth.

But prolactin also has an important function in the liver. This organ has the highest number of prolactin receptors in the body, ports that allow this hormone to enter liver cells. There, prolactin signals these cells to multiply and new blood vessels to grow to fuel this organ's expansion.

Wondering if these properties might be useful to encourage the liver to regrow after surgery to remove part of it -- sometimes necessary to treat cancer or other liver diseases, or to donate liver tissue for transplants -- Carmen Clapp of the Universidad Nacional Automoma de Mexico and her colleagues worked with animal models on both ends of a prolactin spectrum: rats that overproduced the hormone, and mice specially bred to have no prolactin receptors, the equivalent of a dearth of the hormone since prolactin can't enter these animals' cells.

The researchers found that the animals with extra prolactin had larger livers, regenerated their livers faster after partial removal, and were significantly more likely to survive that liver surgery compared to the animals that couldn't process prolactin.

The article is entitled "Prolactin Promotes Normal Liver Growth, Survival, and Regeneration in Rodents." It appears in the American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, published by the American Physiological Society.

Methodology
 The researchers made rats overproduce prolactin by implanting two extra anterior pituitary glands -- the gland that produces prolactin -- in the animals' backs. To make sure the surgery itself wasn't responsible for any effects they saw, they compared these rats to others that had a sham surgery, in which they made incisions but didn't implant extra anterior pituitary glands. To confirm that prolactin itself was responsible for the effects they saw in the overproducers, the researchers injected some of the rats that had the real surgery with a drug that deactivated extra prolactin, bringing the overproducers' prolactin down to baseline levels.

As a contrast to these prolactin overproducers, the researchers also studied mice that were genetically engineered to not have prolactin receptors. Thus, even though these mice made prolactin, their bodies behaved as if they had none of the hormone because their cells couldn't process it.

The researchers measured the ratio of liver to body weight in each of the rats and mice. They tested how readily liver and liver blood vessel cells were dividing in some of the animals from each group. They also removed portions of the animals' livers, comparing how quickly animals from each group regenerated liver tissue. Additionally, they tested the animals' levels of interleukin-6 (IL-6), a chemical produced by cells and is kept in check by prolactin. IL-6 can stimulate the liver to repair itself at low levels but can hinder this self-repair at higher levels.

Results
The researchers found that rats that overproduced prolactin had larger livers in proportion to their body weight compared to rats that had normal prolactin levels and those that overproduced prolactin but received the nullifying drug. These overproducers also had significantly larger livers in proportion to their body weight compared to the mice that couldn't process prolactin. Liver cells and liver blood vessel cells were multiplying more readily in the prolactin overproducers than in animals in the other groups.

After the researchers removed portions of the animals' livers, the prolactin overproducers regenerated their livers more quickly than animals from the other groups. Mice that didn't process prolactin not only had smaller livers than the normal mice but were also significantly more likely to die in the days after surgery. Tests showed that these mice had elevated levels of IL-6, a factor that could be partially responsible for their slower healing and increased mortality.

Importance of the Findings These findings suggest that prolactin is important both for normal liver growth and for regenerating the liver after part of it is removed, with extra prolactin providing a boost for repair mechanisms. Consequently, enhancing prolactin levels could provide a way to improve regeneration when the liver becomes damaged or diseased, or after surgery.

"The use of current medications known to increase prolactinemia (prolactin production) constitute potential therapeutic options in liver diseases, liver injuries, or after liver surgery and warrants further investigation," the study authors write.

http://www.the-aps.org/mm/hp/Audiences/Public-Press/For-the-Press/releases/13/33.html

Wednesday, September 11, 2013

Scientists grow new stem cells in a living mouse

LONDON | Wed Sep 11, 2013 1:03pm EDT

(Reuters) - Scientists have succeeded in generating new stem cells in living mice and say their success opens up possibilities for the regeneration of damaged tissue in people with conditions ranging from heart failure to spinal cord injury. 
The researchers used the same "recipe" of growth-boosting ingredients normally used for making stem cells in a petri dish, but introduced them instead into living laboratory mice and found they were able to create so-called reprogrammed induced pluripotent stem cells (iPS cells). 
"This opens up new possibilities in regenerative medicine," said Manuel Serrano, who led the study at the Spanish National Cancer Research Centre in Madrid.
Continue reading @ Reuters

 

Thursday, June 14, 2012

Stem Cell Therapies Could Change Medicine-If They Get the Chance

Cell Stem Cell Forum

Stem Cell Therapies Could Change Medicine.
If They Get the Chance

Irving Weissman1,*
Download PDF
1Institute of Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305 USA

*Correspondence: irv@stanford.edu
DOI 10.1016/j.stem.2012.05.014

Stem cell therapies have the potential to revolutionize the way we practice medicine. However, in the current climate several barriers and false assumptions stand in the way of achieving that goal.

The first two precepts of the modified Hippocratic Oath, which all M.D. graduates pledge are, in paraphrase: first, do no harm; and second, the primary obligation of a physician is to the health of the patient (to which I add ‘‘and future patients’’), and a physician will not let issues of race, creed, religion, politics, or personal ethics to stand between the patient’s health and his/her actions.

The stem cell field, probably more than any I know of in medical science, is plagued by failures to act responsibly on both precepts. While I am usually an optimist, I must admit that there is a possibility that we will continue to be in the Dark Ages of medicine for quite some time. I fear that therapies using purified tissue and organ-specific stem cells—the only selfrenewing cells in a tissue or that can regenerate that tissue or organ for life—will remain elusive. Before I go further, just think about that statement: regenerate that tissue or organ for life. No pharmaceutical, no biotech-developed protein, and no other transplanted cells can do that. If we can deliver purified stem cells safely and effectively as a one-time therapy, we can change medicine,especially for diseases that drugs and proteins can’t touch. Moreover, if we manage the costs and charges carefully,this form of therapy could lower overall health care costs dramatically.

This vision is based on solid scientific evidence that stem cells regularly maintain, and, if necessary, regenerate tissues in a homeostatically controlled process. So it’s worth the extra effort to find a way to make it happen.

Doing Harm
One of the barriers to practicing stemcell-based regenerative medicine is the existence of fraudulent clinics and individuals who claim unproven therapies without underlying scientific backing. In many cases, they use cells that have never been tested experimentally for their‘‘stemness,’’ have not been through IRBapproved protocols that demand experimental evidence to justify the human experiment, and lack both independent medical monitoring of patient safety and oversight by a state or country regulatory system such as the FDA. It is critical that,as the community that speaks for stem cell biology and stem cell medicine, we find ways to warn patients and caregivers effectively about these concerns (Taylor et al., 2010).

There is also a fine line between these clearly fraudulent practices and questionable ones that use the stem cell label, but are not in fact stem cell therapies. For example, cultures of adherent cells from bone marrow, cord blood, or adipose tissue are regularly claimed to be mesenchymal stem cells (MSCs), but in such cultures true stem cells that both selfrenew and differentiate to mesenchymal fates such as bone, cartilage, fibroblasts, and adipocytes are rare. Mesenchymal stromal cells, as a population, may contain cells that produce immunomodulatory and/or angiogenic factors, but are not sufficiently purified or defined to be a characterized entity for research or clinical transplantation.

Finding markers that help define these populations was an important step (Dominici et al., 2006),but until there is a better understanding of how many of these cells can self-renew and give robust regeneration, I do not think they should be called stem cells.

There are also many claims that mesenchymal and/or hematopoietic cells can transdifferentiate without gene modification to make brain, liver, heart, skeletal muscle, or other tissues. However, these claims lack rigorous scientific support (Wagers and Weissman, 2004). Highly visible athletes and politicians are among the many patients who have received such ‘‘treatments.’’

Recently, the Texas Medical Board approved a policy that allows licensed physicians to transplant investigational agents, including MSCs, with IRB approval but without a requirement for FDA approval of safety and efficacy. In my view, this lack of a requirement for FDA oversight and approval for both safety and efficacy is a giant step backward.

Another example of questionable stem cell practices comes from some commercial private cord blood banks. Cord blood does contain both HSCs and mesenchymal progenitors. The number of HSCs in each cord is sufficient to give rapid generation of blood only in infants and very small children, and above the age of 7, several HLA-matched cords are needed. The development of public cord blood banks is an important, lifesaving advance for patients needing hematopoietic cell transplants but lacking matched donors. However, this activity is very different from the private cord blood banks that charge significant amounts to initiate freezing of cord blood cells and then maintain them in case the child from whom the cord is obtained needs therapy. These companies often list a broad range of diseases that now or someday will be treated with stem cells without warning the patients or caregivers that the evidence that cord blood cells will be useful for treating such diseases is still very limited, and in any case the stored cord blood has the same genetic background as the child from whom the cord was obtained. The overall cause of legitimate stem cell therapy would be greatly advanced by greater control and oversight of these and other organizations making unsupported claims about the potential of stem-cell-based treatments.

The Therapeutic Entity Is the Stem Cell Itself
Very few ‘‘adult’’ stem cells have been prospectively isolated, and only prospectively isolated blood-forming stem cells (HSCs) and brain-forming stem cells (NSCs) have been transplanted in clinical trials (Baum et al., 1992; Uchida et al., 2000). Grafts of other tissues, such as skin and bone marrow, depend on the stem cells in that tissue, but prospectively isolated stem cells are usually not used.

Instead of using cells as the therapy, as a general rule, large drug companies are approaching the use of disease specific iPSCs or adult stem cells as tools for chemical or protein screens to find compounds that can be taken as conventional drugs to treat diseases. Some of these efforts are focused on differentiated cells derived from stem cells, but others aim to address diseases where altered or insufficient numbers of stem cells are central to the disease. The principal property of stem cells that makes them special is their ability to self-renew and reconstitute cell populations. Inducing self-renewal in vivo could be difficult to achieve because many factors affect stem cell regulation. It seems unlikely that single molecules will be able to activate all of the necessary pathway genes appropriately to expand a stem cell pool and allow robust and physiologically significant regeneration.

Thus, I think this approach is likely to fall short as a method to replace tissue stem cells in vivo, and efforts will need to focus more on transplanting the cells themselves. However, stem-cell-regulating agents derived from screening could still be used as adjuvants for transplanted stem cells.

At a broader level, HSCs themselves form a foundation on which the rest of the regenerative medicine field could be built. When engrafted, purified HSCs can replace the hematopoietic system. By doing so, they also render the host permanently tolerant to other organs,tissues, or tissue stem cells from the same donor without further immune suppression (Weissman and Shizuru, 2008). In the future, the isolation of HSCs and other tissue stem cells (e.g.,NSCs) from the same donor could come from pluripotent stem cell lines, and not living or recently deceased donors.

Pluripotent ESC or iPSC line production of HSCs is still not practical, and working out the pathways to achieve that objective remains a critical roadblock to expanding the field of regenerative medicine.

In Vivo Veritas
The experiments that validated human,purified HSCs for hematopoietic transplants and human brain-stem-cell derived neurospheres for neural disease transplants used immune-deficient mice that were crucial in testing the potential therapeutic effectiveness of these cells in vivo (Weissman, 2002). Although the derivation of patient- and disease specific iPSCs can allow experiments in a petri dish, the disease pathogenesis caused by inherited mutations would be more completely understood if the cells could mature in a more physiological setting. One way to study them would be to develop blastocyst chimeras that are implanted and allowed to develop. Mouse ESCs and iPSCs can already be studied using this type of approach.

Currently, human ESCs/iPSCs do not form chimeras if placed in mouse blastocysts and implanted. However, human pluripotent stem cell lines are mainly at the epiblast stage, and not the preimplantation blastocyst, and even mouse epiblast cells cannot form long-term blastocyst chimeras. If the substantial practical and ethical issues could be overcome,blastocyst chimeras with human iPSCs might provide insights into the cellular and molecular mechanisms of human disease pathogenesis, and the gene expression programs that allow embryonic tissue stem cells to mature.

An Unexpected but Potent Barrier:
Business Development
Growing up in America, it is obvious to all of us that the transition from discovery to therapy almost always involves for-profit entities. Ingenuity and innovation are hallmarks of our society, and so it is natural that the prospective identification and isolation of adult or tissue stem cells leads to business enterprises. I myself have cofounded several companies that have done discovery, preclinical proof of principle,and even phase I/II clinical trials in the stem cell field. Each has succeeded in the discovery and preclinical phases, but found that the results of the clinical trials can take a back seat to business decisions. For example, SyStemix Inc. was a 1988 Palo Alto startup that identified a method to prospectively isolate and transplant clinically relevant numbers of human HSCs. The company entered a relationship with Sandoz, Inc. to explore autologous and allogeneic HSC therapies. Purification of mobilized peripheral blood HSCs resulted in depletion of various metastatic cancer cells by 115,000- to 245,000-fold (Prohaska and Weissman, 2009), and thus could be used to reconstitute the hematopoietic system after therapy with a reduced risk of reintroducing tumor cells. This finding led to clinical trials.

Twenty-two patients with metastatic breast cancer underwent transplantation of previously mobilized HSCs after veryhigh-dose chemotherapy. Although the trials were small, two hypotheses were tested: (1) can one improve the outcome of patients with chemoresistant metastases? And (2) can one improve the outcomes of relapse patients with both metastases and chemoresponsive cancers? The therapy did not help the patients with chemoresistant breast cancers. However, at 3 years the chemoresponsive cohort who received cancer-depleted HSCs appeared to be doing better than patients with standard mobilized peripheral blood transplants. At that point, Sandoz merged with CIBA to form Novartis, and within a few years the stem cell program was cancelled.

Last year Antonia Mu¨ ller and Judy Shizuru published the follow-up of the patients 13–15 years later (Mu¨ ller et al., 2012). One-third of the patients who received purified HSCs were still alive, contrasting with the 7% overall survival of 78 contemporaneous Stanford patients with stage IV breast cancer who received standard, unpurified, mobilized peripheral blood transplant therapy. Of the five long-term surviving patients who had received purified HSCs, four had no recurrence of their breast cancers. Attempts to reinitiate the program in another startup, helped by Novartis management, were halted then consultant oncologists advised investors that stem cell therapies in breast cancer had failed, citing a study indicating that ‘‘stem cell’’ rescue of high-dose chemotherapy patients with metastatic breast cancer was no better than chemotherapy alone, that is, only 6% disease free survival at 2 years (Stadtmauer et al.,2000). However, Stadtmauer et al. transplanted unfractionated mobilized blood, not purified HSCs, and no amount of evidence about the difference could counter the words ‘‘stem cell’’ in the title of the NEJM article.

This particular problem could have been avoided by more rigorous editorial standards regarding the use of the term ‘‘stem cell,’’ and I would argue that improved accuracy in this respect would benefit many areas of the field.

How can we resolve this conflict of goals, that of a company to make a profit,and that of the biomedical researcher to advance medical science for the benefit of patients?

The largest and best funding experiment I have seen so far comes from the California Institute of Regenerative Medicine. CIRM’s charter allows it to fund promising stem-cell-based discoveries to and through phase I trials, taking out the risk that leaves our field bereft of suitable funds and in the ‘‘valley of death.’’

However, to overcome the types of problems that the SyStemix trial encountered,this funding would need to be taken beyond initial trials to a point at which the evidence for clinical efficacy was irrefutable.

In Closing....
So, whom have I failed to annoy here?

In one way or another, I have called out almost all of the different stakeholder groups involved in developing stem cell therapies. I wish I had a better story to tell, but I am convinced that we need to identify and reveal those who directly or indirectly do harm with phony medicines, and those who generate barriers to finding and transplanting adult tissue/organ stem cells for financial, religious, political, or other reasons. Unless we do, it will be difficult to usher in the era of stem cell regenerative medicine.

Remember, right now our patients,friends, and families are contracting diseases that have a very short window of opportunity in which regenerative therapies can save them, and each delay removes a cohort of them from possible cures. We should not fail them.

REFERENCES
Baum, C.M., Weissman, I.L., Tsukamoto, A.S.,
Buckle, A.M., and Peault, B. (1992). Proc. Natl.
Acad. Sci. USA 89, 2804–2808.
Dominici, M., Le Blanc, K., Mueller, I., Slaper-
Cortenbach, I., Marini, F.C., Krause, D.S., Deans,
R.J., Keating, A., Prockop, D.J., and Horwitz,
E.M. (2006). Cytotherapy 8, 315–317.
Mu¨ ller, A.M., Kohrt, H.E., Cha, S., Laport, G., Klein,
J., Guardino, A.E., Johnston, L.J., Stockerl-
Goldstein, K.E., Hanania, E., Juttner, C., et al.
(2012). Biol. Blood Marrow Transplant. 18,
125–133.
Prohaska, S.S., and Weissman, I.L. (2009). Biology
of Hematopoietic Stem and Progenitor Cells:
Thomas’ Hematopoietic Cell Transplantation, F.
Applebaum, S. Forman, R.S. Negrin, and K. Blume,
eds. (Oxford: Wiley-Blackwell), pp. 36–63.
Stadtmauer, E.A., O’Neill, A., Goldstein, L.J.,
Crilley, P.A., Mangan, K.F., Ingle, J.N., Brodsky,
I., Martino, S., Lazarus, H.M., Erban, J.K., et al;
Philadelphia Bone Marrow Transplant Group.
(2000). N. Engl. J. Med. 342, 1069–1076.
Taylor, P.L., Barker, R.A., Blume, K.G., Cattaneo,
E., Colman, A., Deng, H., Edgar, H., Fox, I.J.,
Gerstle, C., Goldstein, L.S., et al. (2010). Cell
Stem Cell 7, 43–49.
Uchida, N., Buck, D.W., He, D., Reitsma, M.J.,
Masek, M., Phan, T.V., Tsukamoto, A.S., Gage,
F.H., and Weissman, I.L. (2000). Proc. Natl. Acad.
Sci. USA 97, 14720–14725.
Wagers, A.J., and Weissman, I.L. (2004). Cell 116,
639–648.
Weissman, I.L. (2002). N. Engl. J. Med. 346, 1576–
1579.
Weissman, I.L., and Shizuru, J.A. (2008). Blood
112, 3543–3553.

Friday, April 27, 2012

Bioengineered organs may hold the future for transplants


Bioengineered organs may hold the future for transplants

by Erin M. Massey
April 26, 2012 

Erin Massey/MEDILL
A mouse recovering from
a new model of transplant surgery.
Northwestern University researchers are in the beginning stages of bioengineering tissues and entire organs from stem cells of adult rats and mice, said Dr. Jenny Zhang. Zhang directs the Microsurgical Core within the Comprehensive Transplant Center at Feinberg.

Once engineered, Zhang said her team will be able to test the functionality of such organs as transplants in the rodents. For now, Zhang and fellow researchers are using a biodegradable scaffold, a kind-of-skeleton of an organ with all living cells removed, to test the model.

By developing a successful animal model, the stage is set for future testing on primates and eventually humans. Zhang said bioengineered organs would significantly reduce waiting times for people needing transplants and prevent rejection of healthy organs.

Researchers also want to gain a better understanding of the cytomegalovirus (CMV), a particular threat to transplant recipients.



Erin Massey/MEDILL
A researcher performing micro-surgery in rodents to test a new model for transplants.
Bioengineered organs may redefine transplants for humans someday, and even allow damaged organs to regenerate.

CMV is a common virus found in 50-90% of all people, but has a greater chance of being activated after transplantation due to immunosuppression (reduction or complete absence of a healthy immune response or immune system).

The immune systems of healthy individuals are able to fight off the virus or put it in a latent state. Compromised immune systems have a much harder time fighting the virus that can cause respiratory and other health problems.

Zhang talks about her research of ongoing projects and current successful models already established.

Q. You mentioned that the core is working on 10 ongoing projects. What are these?
A. One of the projects is tissue engineering. For example, an idea in the transplant field is regenerative medicine. If you have a damaged organ, you would want it to regenerate, such as in the case of acute kidney failure. Also, a lot of people are on the waiting list for transplants. There is an organ shortage. We are trying to grow tissues and organs in the lab using stem cells from bone marrow or a particular organ of adult mice. In the future, we hope to take cells from something living and grow them. Normally, you can only grow embryonic stem cells. Now, we can use any cells to become pluripotent, meaning any cell.

Another project deals with the CMV virus. We want to study why this virus is activated after transplant surgery so that we can prevent it. It is one of a couple of viruses that suddenly wake up with immunosuppression. CMV can be transferred via saliva, bodily fluids or through the placenta. Only an active CMV agent can be transferred and is a threat only when activated. A great percentage of the population carry the virus in their bodies but it is latent until activated. Immunosuppression in a transplant recipient can greatly affect a person’s ability to fight off the virus.

Q. What organs have you successfully worked with so far?
A. So far, we have had success with the kidney, liver, and heart in these animals. Normally, the mouse or rat can survive two surgeries.

Q. Have you been successful in taking the cells out of an organ, infusing new cells, and transplanting a completely functional organ into a mouse or rat yet?
A. We are in the early stages. What we have been successful doing is removing all the cells from an organ, such as a kidney or liver and injecting new cells, called endothelial cells, on the surface of the organ. We are in the process of testing these organs to see if they can hold blood and function normally.

Q. What have you been able to accomplish thus far with the stem cells?
A. Our research entails using real organs in whole or part from mice and rats. We use a machine to diffuse the organ and take out all of the cells, leaving a scaffold, skeleton-like structure that looks like a honeycomb. Then we infuse new cells that are grown in a bioreactor. The intention of infusing cells is for them to grow into the desired organ or tissue.

The bioreactor is able to simulate a natural environment where these cells would normally be grown. We are able to monitor the environment and determine the best condition for use. Usually the time the organ spends in the machine can range from 10 days or even longer depending on the type and size of the organ.

Q. What is the significance behind choosing to conduct this research on rodents?
A. If we can equate an animal model, we can use it for testing a bioengineered organ and see if it is functional, how long the cells with survive, and if it will form a normal structure. In a mouse we can do all sorts of things we can’t do in humans.

Q. Why do you test both mice and rats?
A. We will test this model on rodents, then primates, and eventually humans. The difference between a rat and a mouse is the size. Rats are 10 times bigger than mice and are easier to work on. We use only inbred rodents. These animals have been studied for years so we know the genetic make-up and can precisely test the type of rejection they will experience.

Q. In how many of the cases will an animal or human experience rejection of an organ?
A. In all cases, animals and humans experience some type of rejection. In the case of humans, regardless if two people are a complete match, some molecules are bound to be different. Each cell has a molecule on the surface and what is called a major histocompatibility complex, or antigens. Humans are more complicated than animals and with the mice it is much easier to control the immunosuppression response.

Q. How are humans treated when they experience rejection of an organ?
A. All people are treated and will take medicine for the rest of their lives. The rejection is variable as is the dosage of medicine a person will take. Examples of medicine prescribed include Tacrolimus or Thymoglobulin. For kidney and liver transplants, there is a current success rate of about 90 percent.

Q. How do you infect the rodents with the CMV virus?
A. We inject the mouse with the virus and let it survive for a couple of months. Initially, it will suffer respiratory problems. Sometimes, it will not happen, because the mouse has cleared the virus from its system. A healthy individual can sometimes clear out the virus, but someone whose immune system is compromised won’t be able to clear it out.

Q. What is the goal of studying rejection?
A. We are in the beginning stages to test tolerance, or the ability to trick the body into not recognizing the organ as foreign. If we can do that, we can prevent rejection partially or wholly, which can possibly translate to a human.


Thursday, April 26, 2012

Teaching old cells new tricks



"The new technology consists of taking cells from skin and reprogramming them so that they become stem cells – cells that are capable of proliferating and differentiating into almost all tissue types."
—Dr Ludovic Vallier
                        

Much hyped by the media, stem cells have tremendous power to improve human health. As part of the Cambridge Stem Cell Initiative, Dr Ludovic Vallier’s research in the Anne McLaren Laboratory for Regenerative Medicine shows how stem cells can further our understanding of disease and help deliver much-needed new treatments.

How do you study a human disease that has no equivalent in animals and where the human cells in question are so hard to grow outside the body they cannot be tested in the laboratory? The answer, until now, was with great difficulty. But by using a new stem cell technique, that is set to change.
Dr. Ludovic Vallier, who holds an MRC Senior Fellowship in the Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery at Cambridge in collaboration with Professor David Lomas (Cambridge Institute for Medical Research and Department of Medicine), works on a group of devastating genetic diseases affecting the liver.

“We target metabolic diseases of the liver, diseases such as alpha 1 antitrypsin deficiency. It’s one of the most common single genetic disorders and the protein it affects – which is only produced by the liver – is really important because it controls activity of elastase in the lung. Without this control, people develop serious lung problems and the disease also affects the liver, so these patients develop liver failure,” he explained.

The problem is that these diseases cannot be studied in vitro – in a dish – in the laboratory, he said: “You can’t take cells from the liver of these very sick patients, and if you could they wouldn’t grow, which means you don’t have any way of screening drugs that could help treat these diseases.”
Without effective drugs, the only current treatment is a liver transplant. “There is a huge shortage of organs and transplantation involves taking immunosuppressive drugs, which is heavy treatment especially in already fragile patients,” Dr. Vallier said. “And the disease is progressive so it’s very complicated to manage.” Understandably, Dr. Vallier is excited that a new method of producing stem cells developed in Japan has given him and other researchers a way of studying these diseases and screening potential drugs to treat them.

“The new technology consists of taking cells from skin and reprogramming them so that they become stem cells – cells that are capable of proliferating and differentiating into almost all tissue types,” he said.

This reprogramming means a cell with a previously fixed identity can be taught a new one – in this case taking skin cells and reprogramming them to become liver cells. When the skin cells come from a patient with liver disease, these skin-turned-liver cells also have the disease, making them ideal for studying the disease and screening potential drugs to treat it.

According to Dr. Vallier: “Because we can generate liver cells that mimic the disease of the original patient in vitro, that allows us to do basic studies that were impossible by biopsy or primary culture and also to do drug screening.” And because the skin cells can come from a whole range of people, it gives researchers access to a broad diversity of patients as well as overcoming some of the ethical concerns associated with embryonic stem cells.

“That’s a very important step because it solves the problems associated with a limited stock of stem cells,” he said, “and because it’s a simple method, it’s easily accessible to a wide number of laboratories.”

Showing this can be done in a small number of liver patients in Cambridge is an important proof of concept, and supports the possibility that a similar approach might be applicable to a wide range of other serious diseases that still lack effective treatments, including neurodegenerative diseases such as Parkinson’s and Alzheimer’s Disease as well as heart diseases.

And Cambridge – which now has almost 30 groups doing stem cell research and strong links between academic researchers and clinicians – is perfectly positioned to make the most of this new technique.
“The Laboratory for Regenerative Medicine is starting to become an expert in this disease modelling and we are all part of a larger consortium, the Cambridge Stem Cell Initiative (SCI),” said Dr. Vallier. “Together, we are putting together resources and scientific interest to really develop stem cells and their clinical application. The SCI is a unique consortium because it brings together a wealth of complementary expertise.”

While this first revolution involves in vitro disease modelling and drug screening, Dr. Vallier hopes this work will ultimately lead to personalized cell-based therapies where liver cells reprogrammed from a patient’s own skin cells could be used in place of a liver transplant. “It will take time for us to assess this clinical use and show that it is safe as well as effective,” he explained, “but if you ask me again in five years I should be able to tell you whether we are going to do it.”

Provided by University of Cambridge
http://www.cam.ac.uk/research/features/teaching-old-cells-new-tricks/

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Friday, April 20, 2012

EASL: Gallbladder shown as potential stem cell source for regenerative liver and metabolic disease

Posted On: April 19, 2012 - 9:31am

A new study presented today at the International Liver Congress™ 2012 indicates the potential for gallbladder tissue (which is routinely discarded from organ donors and surgical interventions) to be a highly available candidate source for multipotential stem cells.(1)

Biliary tree stem/progenitor cells (BTSCs) have previously been identified in the glands of normal adult human extrahepatic bile ducts and been shown to generate in vitro and in vivo mature cells of the hepato-biliary and pancreatic endocrine lineages.

The study found both normal and pathological gallbladders contained easily isolable cells with the phenotype and biological properties of BTSCs. Interestingly, in an animal model, these cells were able to repopulate the injured liver and to improve synthetic functions.

These data open novel perspectives for the collection and use of multipotent stem cells in regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes

Source: European Association for the Study of the Liver

Sunday, March 4, 2012

Scientists have shed light on how the liver repairs itself

Boosting cell production could help treat liver disease

Source

Scientists have shed light on how the liver repairs itself with research that could help develop drugs to treat liver disease

Researchers at the Medical Research Council (MRC) Centre for Regenerative Medicine at the University of Edinburgh have discovered how to enhance the production of key cells needed to repair damaged liver tissue.

The study, published in the journal Nature Medicine, could help heal livers affected by diseases such as cirrhosis or chronic hepatitis.

Scientists were able to unpick the process of how different cells in the liver are formed.
When the liver is damaged it produces too many bile duct cells and not enough cells called hepatocytes, which the liver needs to repair damaged tissue.

They found they could increase the number of hepatocyte cells – which detoxify the liver – by encouraging these cells to be produced instead of bile duct cells.

Understanding how liver cells are formed could help to develop drugs to encourage the production of hepatocytes to repair liver tissue. This could eventually ease the pressure on waiting lists for liver transplants.
Professor Stuart Forbes, Associate Director at the MRC Centre for Regenerative Medicine at the University of Edinburgh, who is a consultant hepatologist and was the academic leader of the study, said: "Liver disease is on the increase in the UK and is one of the top five killers. Increasing numbers of patients are in need of liver transplants, but the supply of donated organs is not keeping pace with the demand. If we can find ways to encourage the liver to heal itself then we could ease the pressure on waiting lists for liver transplants."
Liver disease is the fifth biggest killer in the UK. There are almost 500 people waiting for a liver transplant, compared to just over 300 five years ago.

The production of hepatocyte cells was increased by altering the expression of certain genes in early stage liver cells.

Dr Luke Boulter, of the University of Edinburgh's MRC Centre for Regenerative Medicine and first author on the paper, said: "This research helps us know how to increase numbers of cells that are needed for healthy liver function and could pave the way for finding drugs that help liver repair. Understanding the process in which cells in the liver are formed is key in looking at ways to repair damaged liver tissue."

Dr Rob Buckle, Head of Regenerative Medicine at the MRC, said: "Liver transplants have saved countless lives over the years, but demand will inevitably outstrip supply and in the long term we need to look beyond replacing damaged tissues to exploiting the regenerative potential of the human body. The MRC continues to invest heavily across the breadth of approaches that might deliver the promise of regenerative medicine, and this study opens up the possibility of applying our increasing knowledge of stem cell biology to stimulate the body's own dormant repair processes as a basis for future therapy."

###

The study was carried out in collaboration with the University's MRC Centre for Inflammation Research, the Beatson Institute for Cancer Research in Glasgow and the K.U. Leuven in Belgium.

Contact: Catriona Kelly
Catriona.Kelly@ed.ac.uk
44-131-651-4401
University of Edinburgh

Thursday, June 30, 2011

Stem-cell scientists grapple with clinics;Educating patients about unproven treatments

Nature today has an article written by Heidi Ledford discussing the worldwide rapid increase of stem-cell clinics and the dangers of these unproven treatments.

Scope has a nice write up on the article by Eva Valenti, she writes;

Regenerative medicine such as stem cell therapy has cast a ray of hope into many patients’ lives. Stem cell clinics, however, do not always offer patients the most effective treatments. According to a recent Nature article:
Many of the treatments such clinics offer — injecting a patient’s own stem cells back into his or her body in a bid to treat conditions ranging from Parkinson’s disease to spinal-cord injuries — are at best a waste of money, and at worst dangerous. “There’s real potential to damage the legitimacy of the field,” says Timothy Caulfield, who studies health law and policy at the University of Alberta in Edmonton, Canada.
The potential danger of these clinics is clear: In May, Europe’s largest stem-cell clinic was shut down after its treatments were linked to a child’s death....Please do go read the more at Scope and the original article from Nature.

Related on this blog; Stem Cells; Searching For A Cure " When It Becomes Dangerous"

Friday, February 4, 2011

Scientists Unlock One Mystery of Tissue Regeneration

ScienceDaily (Feb. 4, 2011) — The human body has a remarkable ability to heal itself. Due to the presence of dedicated stem cells, many organs can undergo continuous renewal. When an organ becomes damaged, stem cells in the organ are typically activated, producing new cells to regenerate the tissue. This activity of stem cells, however, has to be carefully controlled, as too much stem cell activity can cause diseases like cancer. Current research in stem cell biology is starting to unravel the control mechanisms that maintain a balance between efficient regeneration and proper control of stem cell function.

Strikingly, it is becoming evident that oxidative stress is at the heart of this regulation. Researchers at the University of Rochester have now identified a genetic switch that controls oxidative stress in stem cells and thus governs stem cell function.

The work was done by biologists Heinrich Jasper, Christine Hochmuth and Benoit Biteau, and geneticist Dirk Bohmann of the University of Rochester Medical Center, who hoped to gain some insight into human stem cell processes by studying the intestinal stem cells of Drosophila (fruit flies), which have genetic structures that, in many ways, mimic those that are found in humans. The researchers studied the function of two genes, Nrf2 and Keap1, which were already known as regulators of cellular responses to oxidative stress. The research team was surprised to discover that, in contrast to other cell types, Nrf2 was active within the stem cells even in the absence of stress. This finding suggested that Nrf2 might have an unusual role in the control of stem cell function.

Indeed, the researchers found that Nrf2 prevents stem cells from dividing, and that only when Nrf2 is repressed can stem cell division take place. That's where the other gene, Keap1, comes into play.

When the intestine of the fruit fly is damaged, proteins secreted from the damaged cells send signals that activate stem cells. Jasper and colleagues learned that Keap1 inhibits the function of Nrf2 in stem cells experiencing such signals, making it possible for the stem cells to divide and regenerate the intestinal tissue.

Interestingly, Nrf2 controls stem cell activity by influencing the level of ROS (reactive oxygen species) in these cells. ROS are highly reactive molecules that, though occurring naturally in cells, can harm the cell structure if their concentration increases significantly. Nrf2 reduces the ROS levels in cells -- and that's the mechanism by which Nrf2 helps to determine whether stem cells divide in fruit flies: Intestinal stem cell division can only take place when ROS levels go up, and as long as Nrf2 does its job, that won't happen. But when Keap1 represses Nrf2, ROS levels increase, allowing stem cells to divide and initiate regeneration. This switch is thus a critical stress sensor that allows proper control of stem cell activity in the intestine. Accordingly, the researchers found that when Nrf2 function is disrupted, the fly intestine degenerates due to excessive production of new cells by the stem cells.

Their work is being published in the February 4 issue of the scientific journal Cell Stem Cell.

Jasper expects other scientists to start testing whether stem cell regulation works the same way in small vertebrates and humans. "If it does, it would encourage the adaptation of these findings to new therapies. And scientists may eventually learn how to control stem cell function to safely replace damaged tissue in humans."

The University of Rochester researchers are now trying to learn more about the processes behind Keap1 and Nrf2 activity. "How does Keap1 know that there's a signal from the damaged tissue?" asked Jasper. "We're trying to understand what happens upstream and what happens downstream -- before and after Keap1 is activated."

The research project was funded by NYSTEM (New York State Stem Cell Initiative), National Institute on Aging (part of the National Institutes of Health), and the Ellison Medical Foundation.

Thursday, February 3, 2011

UC Davis scientists successfully bioengineer functioning liver tissue

UC Davis scientists successfully bioengineer functioning liver tissue
03/02/2011 04:17:00

Study holds promise for creating organs to treat liver disease

(SACRAMENTO, Calif.) — UC Davis researchers have announced that they have used a novel technique to transplant human liver cells into an animal model that enabled the cells to function well for a considerably longer period of time than methods used in previous studies.
The technique has the potential to one day create a working liver for transplantation into people with severe liver disease, or be used as an interim measure for a patient who must wait until a conventional donor organ becomes available.

The study, “Decellularized liver matrix as a carrier for transplantation of human fetal and primary hepatocytes in mice,” has been accepted by the journal Liver Transplantation and is now online.

The innovative technique devised by the UC Davis scientists working in Sacramento, Calif., involved “decellularizing” a mouse liver – stripping all cells out of the organ while preserving its protein structure and blood supply framework. Using the decellularized liver as a scaffold, the researchers inserted human liver cells into the structure, prompting the cells to survive longer and function better in this native liver scaffold. The scaffold with liver cells was then implanted into the fatty tissue of a mouse abdomen, where it functioned well for two months after transplantation. Researchers say that having the cells function for at least 60 days is an important milestone in the research work now under way to create new organs and tissues from stem cells.

“We have demonstrated the best results to date for the efficacy of transplanting cells into an animal model using a decellularized liver matrix,” said Jian Wu, an adjunct professor of internal medicine and senior author of the study. “This is an important step in the pathway to providing people suffering from liver failure with more hope and a much better chance of survival.”
New approaches to treating liver failure are critically needed, according to Mark Zern, a senior member of the research team, professor of internal medicine and director of the UC Davis Transplant Research Program. “There are not enough organs available for transplant, and many patients die waiting for one,” said Zern.

About 25,000 people are on waiting lists around the country for liver transplants, but only 6,000 to 7,000 organs become available each year. The liver is essential for life and has many complex functions: It stores glucose, as well as many vitamins and minerals; makes blood-clotting factors and the building blocks of proteins; and detoxifies impurities that enter the bloodstream.

The method of decellularizing an organ and transplanting new working cells into animals using this scaffold has been used experimentally in research to regenerate other organs such as the heart, trachea, lungs and kidneys. Until now, other studies to create a functioning liver have been rudimentary and have only resulted in hours or days of activity rather than months.
Ping Zhou, the first author of the study, along with Wu and their UC Davis colleagues, are continuing their research, with a focus on regenerating livers that will function well over an extended period.

“We are very excited about the clinical applications of this research,” said Jan A. Nolta, another senior member of the research team and a professor of cell biology and human anatomy, as well as the director of the UC Davis stem cell program and its Institute for Regenerative Cures. “Our ultimate goal is to ‘scale it up’ to help humans in the future.”

Other authors of the study are Nataly Lessa, Daniel C. Estrada, Ella B. Severson and Shilpa Lingala, all from the UC Davis stem cell or transplant research programs.
The study was supported by grants from the National Institutes of Health, the California Institute for Regenerative Medicine, as well as UC Davis Stem Cell Program start-up funding and the UC Davis Technology Transfer Fund.

UC Davis is playing a leading role in regenerative medicine, with nearly 150 scientists working on a variety of stem cell-related research projects at campus locations in both Davis and Sacramento. The UC Davis Institute for Regenerative Cures, a facility supported by the California Institute for Regenerative Medicine (CIRM), opened in 2010 on the Sacramento campus. This $62 million facility is the university's hub for stem cell science. It includes Northern California's largest academic Good Manufacturing Practice laboratory, with state-of-the-art equipment and manufacturing rooms for cellular and gene therapies. UC Davis also has a Translational Human Embryonic Stem Cell Shared Research Facility in Davis and a collaborative partnership with the Institute for Pediatric Regenerative Medicine at Shriners Hospital for Children Northern California.

All of the programs and facilities complement the university's Clinical and Translational Science Center, and focus on turning stem cells into cures. For more information, visit www.ucdmc.ucdavis.edu/stemcellresearch.


Saturday, January 22, 2011

Future of transplant medicine : Stem Cells


Regrowing Organs
3 min - Jan 7, 2011


Dr. Manny sits down with physicist, Dr. Michio Kaku, to talk about the future of transplant medicine and how doctors are regrowing organs in labs

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Sunday, January 16, 2011

Cell and gene therapies: moving from research to clinic

Also See: Future research and therapeutic applications of human stem cells: general, regulatory, and bioethical aspects

Editorial

Cell and gene therapies: moving from research to clinic

"Translational studies have been and will continue to be critical to progress in cellular and gene therapy. The converging nature of gene therapy, immune therapy for cancer, HSC transplantation, regenerative medicine and tissue engineering make the rapid and widespread exchange of information essential".

David F Stroncek1 and Raj K Puri2
1 Department of Transfusion Medicine, Clinical Center, NIH, Bethesda, Maryland, USA
2 Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA
author email corresponding author email
Journal of Translational Medicine 2010, 8:31doi:10.1186/1479-5876-8-31

The electronic version of this article is the complete one and can be found online at: http://www.translational-medicine.com/content/8/1/31
Received:8 March 2010Accepted:29 March 2010
Published:29 March 2010

© 2010 Stroncek and Puri; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Editorial
Cell and gene therapy clinical trials began more than 40 years ago. At some institutions cellular therapy laboratories were started to support marrow transplantation. These early laboratories removed red blood cells and plasma from aspirated bone marrow that was used for allogeneic transplants, cyropreserved autologous marrow, depleted T cells from allogeneic grafts and depleted leukemic or cancerous cells from autologous grafts [1]. At other institutions cellular therapy laboratories were started to isolate and expand tumor infiltrating lymphocytes (TIL) that were used as investigational treatments for patients with melanoma or to prepare transfected autologous lymphocytes to treat severe combined immune deficiencies.
For many years cellular and gene therapies were primarily highly experimental therapies which were developed and used in highly specialized academic health care centers. Now these forms of therapies are used in numerous clinical trials throughout the US and worldwide. While the field has advanced, progress has been slow. Some therapies have not been effective and some have been associated with unacceptable adverse out comes. However, both cell and gene therapies have now become important potential therapies for incurable diseases.

Hematopoietic stem cell transplants have changed dramatically and have become very successful for hematopoietic reconstitution. Hematopoietic stem cell transplants now make use of marrow, mobilized peripheral blood stem cells and umbilical cord blood for transplants involving HLA matched siblings and unrelated subjects as well as autologous transplants. Recently, there have been important advances in immune therapy of cancer. Immune therapy for melanoma protocols that involve TIL make use of lymphodepletion and autologous CD34+ cell rescue have been reported to result in a greater than 50% objective clinical response rates [2].

Gene therapy is being used as investigational treatment for severe combined immune deficiency (SCID), Leber's Congenital Amaurosis (LCA) and chronic granulomatous disease (CGD) [3] and may soon be used in clinical trials to treat sickle cell disease.

The successful clinical results of some cellular and gene therapy clinical trials and the increased understanding of immunology, cancer, and stem cell biology have lead to the development of many potential new therapies. Natural killer (NK) cells and dendritic cells (DCs) are important parts of many cancer immune therapy investigational protocols. Genetically engineered T cells and DCs are being tested for immune therapy for cancer. Vectors containing tumor reactive T cell receptors are being introduced into T cells. Chimeric receptors containing antibodies specific to antigens expressed by leukemic cells along with T cell costimulatory molecules are being transferred into T cells that are being used therapeutically. Artificial antigen presenting cells are being made by introducing costimulatory molecules into cell lines and these cells are being used to expand cytotoxic T cells in vitro.

Regenerative medicine is an emerging new field. Marrow and mobilized PBSCs injected into ischemic myocardium was been reported to increase cardiac function [4]. Meschenchymal stem cells or bone marrow stromal cells (BMSCs) are also being used to as investigational treatments for ischemic heart disease. BMSCs are also being tested for the treatment of acute renal failure, nerve injury, acute GVHD and autoimmune disease [5]. Induced pluripotent stem (IPS) cells harbor great potential for regenerative medicine applications and for a number of hematopoietic and immune disorders. Work with IPS cells is moving quickly, but the routine clinical application of IPS cells is still many years away.

Translational studies have been and will continue to be critical to progress in cellular and gene therapy. The converging nature of gene therapy, immune therapy for cancer, HSC transplantation, regenerative medicine and tissue engineering make the rapid and widespread exchange of information essential. The goal the JTM Cell and Gene Therapy Section is to advance this field by reporting the results of translational medicine studies and by being a forum for the exchange and discussion of new information, ideas and hypothesis. We welcome contributions from all those participating in this field; clinicians, scientists, and engineers from academia, industry and the regulatory community.


References
Lasky LC, Warkentin PI, Kersey JH, Ramsay NK, McGlave PB, McCullough J: Hemotherapy in patients undergoing blood group incompatible bone marrow transplantation.
Transfusion 1983 , 23:277-285. PubMed Abstract Publisher Full Text
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Rosenberg SA, Dudley ME: Adoptive cell therapy for the treatment of patients with metastatic melanoma.
Curr Opin Immunol 2009 , 21:233-240. PubMed Abstract Publisher Full Text
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Aiuti A, Roncarolo MG: Ten years of gene therapy for primary immune deficiencies.
Hematology Am Soc Hematol Educ Program 2009 , 682-689. PubMed Abstract Publisher Full Text
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Herrmann JL, Abarbanell AM, Weil BR, Wang Y, Wang M, Tan J, Meldrum DR: Cell-based therapy for ischemic heart disease: a clinical update.
Ann Thorac Surg 2009 , 88:1714-1722. PubMed Abstract Publisher Full Text
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Kode JA, Mukherjee S, Joglekar MV, Hardikar AA: Mesenchymal stem cells: immunobiology and role in immunomodulation and tissue regeneration.
Cytotherapy 2009 , 11:377-391. PubMed Abstract Publisher Full Text
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Thursday, January 13, 2011

Lasers, stem cells, and COPD

Lasers, stem cells, and COPD

eng Lin1* , Steven F Josephs1* , Doru T Alexandrescu2* , Famela Ramos1 , Vladimir Bogin3 , Vincent Gammill4 , Constantin A Dasanu5 , Rosalia De Necochea-Campion6 , Amit N Patel7 , Ewa Carrier6 and David R Koos1
1 Entest BioMedical, San Diego, CA, USA
2 Georgetown Dermatology, Washington DC, USA
3 Cromos Pharma Services, Longview, WA, USA
4 Center for the Study of Natural Oncology, Del Mar, CA, USA
5 Department of Hematology and Medical Oncology, St Francis Hospital and Medical Center, Hartford, CT, USA
6 Moores Cancer Center, University of California San Diego, CA, USA
7 Department of Cardiothoracic Surgery, University of Utah, Salt Lake City, UT, USA
author email corresponding author email* Contributed equally
Journal of Translational Medicine 2010, 8:16doi:10.1186/1479-5876-8-16
The electronic version of this article is the complete one and can be found online at:
http://www.translational-medicine.com/content/8/1/16
Received:
7 January 2010
Accepted:
16 February 2010
Published:
16 February 2010
© 2010 Lin et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract
The medical use of low level laser (LLL) irradiation has been occurring for decades, primarily in the area of tissue healing and inflammatory conditions. Despite little mechanistic knowledge, the concept of a non-invasive, non-thermal intervention that has the potential to modulate regenerative processes is worthy of attention when searching for novel methods of augmenting stem cell-based therapies. Here we discuss the use of LLL irradiation as a "photoceutical" for enhancing production of stem cell growth/chemoattractant factors, stimulation of angiogenesis, and directly augmenting proliferation of stem cells. The combination of LLL together with allogeneic and autologous stem cells, as well as post-mobilization directing of stem cells will be discussed.


Introduction (Personal Perspective)
We came upon the field of low level laser (LLL) therapy by accident. One of our advisors read a press release about a company using this novel technology of specific light wavelengths to treat stroke. Given the possible role of stem cells in post-stroke regeneration, we decided to cautiously investigate. As a background, it should be said that our scientific team has been focusing on the area of cord blood banking and manufacturing of disposables for processing of adipose stem cells for the past 3 years. Our board has been interested in strategically refocusing the company from services-oriented into a more research-focused model. An unbiased exploration into the various degenerative conditions that may be addressed by our existing know-how led us to explore the condition of chronic obstructive pulmonary disease (COPD), an umbrella term covering chronic bronchitis and emphysema, which is the 4th largest cause of death in the United States. As a means of increasing our probability of success in treatment of this condition, the decision was made to develop an adjuvant therapy that would augment stem cell activity. The field of LLL therapy attracted us because it appeared to be relatively unexplored scientific territory for which large amounts of clinical experience exist. Unfortunately, it was difficult to obtain the cohesive "state-of-the-art" description of the molecular/cellular mechanisms of this therapy in reviews that we have searched. Therefore we sought in this mini-review to discuss what we believe to be relevant to investigators attracted by the concept of "regenerative photoceuticals". Before presenting our synthesis of the field, we will begin by describing our rationale for approaching COPD with the autologous stem cell based approaches we are developing.


COPD as an Indication for Stem Cell Therapy
COPD possesses several features making it ideal for stem cell based interventions: a) the quality of life and lack of progress demands the ethical exploration of novel approaches. For example, bone marrow stem cells have been used in over a thousand cardiac patients with some indication of efficacy [1,2]. Adipose-based stem cell therapies have been successfully used in thousands of race-horses and companion animals without adverse effects [3], as well as numerous clinical trials are ongoing and published human data reports no adverse effects (reviewed in ref [4]). Unfortunately, evaluation of stem cell therapy in COPD has lagged behind other areas of regenerative investigation; b) the underlying cause of COPD appears to be inflammatory and/or immunologically mediated. The destruction of alveolar tissue is associated with T cell reactivity [5,6], pathological pulmonary macrophage activation [7], and auto-antibody production [8]. Mesenchymal stem cells have been demonstrated to potently suppress autoreactive T cells [9,10], inhibit macrophage activation [11], and autoantibody responses [12]. Additionally, mesenchymal stem cells can be purified in high concentrations from adipose stromal vascular tissue together with high concentrations of T regulatory cells [4], which in animal models are approximately 100 more potent than peripheral T cells at secreting cytokines therapeutic for COPD such as IL-10 [13,14]. Additionally, use of adipose derived cells has yielded promising clinical results in autoimmune conditions such as multiple sclerosis [4]; and c) Pulmonary stem cells capable of regenerating damaged parenchymal tissue have been reported [15]. Administration of mesenchymal stem cells into neonatal oxygen-damaged lungs, which results in COPD-like alveoli dysplasia, has been demonstrated to yield improvements in two recent publications [16,17].
Based on the above rationale for stem cell-based COPD treatments, we began our exploration into this area by performing several preliminary experiments and filing patents covering combination uses of stem cells with various pharmacologically available antiinflammatories, as well as methods of immune modulation. These have served as the basis for two of our pipeline candidates, ENT-111, and ENT-894. As a commercially-oriented organization, we needed to develop a therapeutic candidate that not only has a great potential for efficacy, but also can be easily implemented as part of the standard of care. Our search led us to the area of low level laser (LLL) therapy. From our initial perception as neophytes to this field, the area of LLL therapy has been somewhat of a medical mystery. A pubmed search for "low level laser therapy" yields more than 1700 results, yet before stumbling across this concept, none of us, or our advisors, have ever heard of this area of medicine.
On face value, this field appeared to be somewhat of a panacea: clinical trials claiming efficacy for conditions ranging from alcoholism [18], to sinusitis [19], to ischemic heart disease [20]. Further confusing was that many of the studies used different types of LLL-generating devices, with different parameters, in different model systems, making comparison of data almost impossible. Despite this initial impression, the possibility that a simple, non-invasive methodology could exist that augments regenerative potential in a tissue-focused manner became very enticing to us. Specific uses envisioned, for which intellectual property was filed included using light to concentrate stem cells to an area of need, to modulate effects of stem cells once they are in that specific area, or even to use light together with other agents to modulate endogenous stem cells.
The purpose of the current manuscript is to overview some of the previous work performed in this area that was of great interest to our ongoing work in regenerative medicine. We believe that greater integration of the area of LLL with current advancements in molecular and cellular biology will accelerate medical progress. Unfortunately, in our impression to date, this has been a very slow process.


What is Low Level Laser Irradiation?
Lasers (Light amplification by stimulated emission of radiation) are devices that typically generate electromagnetic radiation which is relatively uniform in wavelength, phase, and polarization, originally described by Theodore Maiman in 1960 in the form of a ruby laser [21]. These properties have allowed for numerous medical applications including uses in surgery, activation of photodynamic agents, and various ablative therapies in cosmetics that are based on heat/tissue destruction generated by the laser beam [22-24]. These applications of lasers are considered "high energy" because of their intensity, which ranges from about 10-100 Watts. The subject of the current paper will be another type of laser approach called low level lasers (LLL) that elicits effects through non-thermal means. This area of investigation started with the work of Mester et al who in 1967 reported non-thermal effects of lasers on mouse hair growth [25]. In a subsequent study [26], the same group reported acceleration of wound healing and improvement in regenerative ability of muscle fibers post wounding using a 1 J/cm2 ruby laser. Since those early days, numerous in vitro and in vivo studies have been reported demonstrating a wide variety of therapeutic effects involving LLL, a selected sample of which will be discussed below. In order to narrow our focus of discussion, it is important to first begin by establishing the current definition of LLL therapy. According to Posten et al [27], there are several parameters of importance: a) Power output of laser being 10-3 to 10-1 Watts; b) Wavelength in the range of 300-10,600 nm; c) Pulse rate from 0, meaning continuous to 5000 Hertz (cycles per second); d) intensity of 10-2-10 W/cm(2) and dose of 0.01 to 100 J/cm2. Most common methods of administering LLL radiation include lasers such as ruby (694 nm), Ar (488 and 514 nm), He-Ne (632.8 nm), Krypton (521, 530, 568, and 647 nm), Ga-Al-As (805 or 650 nm), and Ga-As (904 nm). Perhaps one of the most distinguishing features of LLL therapy as compared to other photoceutical modalities is that effects are mediated not through induction of thermal effects but rather through a process that is still not clearly defined called "photobiostimulation". It appears that this effect of LLL is not depend on coherence, and therefore allows for use of non-laser light generating devices such as inexpensive Light Emitting Diode (LED) technology [28].
To date several mechanisms of biological action have been proposed, although none are clearly established. These include augmentation of cellular ATP levels [29], manipulation of inducible nitric oxide synthase (iNOS) activity [30,31], suppression of inflammatory cytokines such as TNF-alpha, IL-1beta, IL-6 and IL-8 [32-36], upregulation of growth factor production such as PDGF, IGF-1, NGF and FGF-2 [36-39], alteration of mitochondrial membrane potential [29,40-42] due to chromophores found in the mitochondrial respiratory chain [43,44] as reviewed in [45], stimulation of protein kinase C (PKC) activation [46], manipulation of NF-κB activation [47], direct bacteriotoxic effect mediated by induction of reactive oxygen species (ROS) [48], modification of extracellular matrix components [49], inhibition of apoptosis [29], stimulation of mast cell degranulation [50], and upregulation of heat shock proteins [51]. Unfortunately these effects have been demonstrated using a variety of LLL devices in non-comparable models. To add to confusion, dose-dependency seems to be confined to such a narrow range or does not seem to exist in that numerous systems therapeutic effects disappear with increased dose.


In vitro studies of LLL
In areas of potential phenomenology, it is important to begin by assessing in vitro studies reported in the literature in which reproducibility can be attained with some degree of confidence, and mechanistic dissection is simpler as compared with in vivo systems. In 1983, one of the first studies to demonstrate in vitro effects of LLL was published. The investigators used a helium neon (He-Ne) laser to generate a visible red light at 632.8 nm for treatment of porcine granulosa cells. The paper described upregulation of metabolic and hormone-producing activity of the cells when exposed for 60 seconds to pulsating low power (2.8 mW) irradiation [52]. The possibility of modulating biologically-relevant signaling proteins by LLL was further assessed in a study using an energy dose of 1.5 J/cm2 in cultured keratinocytes. Administration of He-Ne laser emitted light resulted in upregulated gene expression of IL-1 and IL-8 [53]. Production of various growth factors in vitro suggests the possibility of enhanced cellular mitogenesis and mobility as a result of LLL treatment. Using a diode-based method to generate a similar wavelength to the He-Ne laser (363 nm), Mvula et al reported in two papers that irradiation at 5 J/cm2 of adipose derived mesenchymal stem cells resulted in enhanced proliferation, viability and expression of the adhesion molecule beta-1 integrin as compared to control [54,55]. In agreement with possible regenerative activity based on activation of stem cells, other studies have used an in vitro injury model to examine possible therapeutic effects. Migration of fibroblasts was demonstrated to be enhanced in a "wound assay" in which cell monolayers are scraped with a pipette tip and amount of time needed to restore the monolayer is used as an indicator of "healing". The cells exposed to 5 J/cm2 generated by an He-Ne laser migrated rapidly across the wound margin indicating a stimulatory or positive influence of phototherapy. Higher doses (10 and 16 J/cm2) caused a decrease in cell viability and proliferation with a significant amount of damage to the cell membrane and DNA [56]. In order to examine whether LLL may positively affect healing under non-optimal conditions that mimic clinical situations treatment of fibroblasts from diabetic animals was performed. It was demonstrated that with the He-Ne laser dosage of 5 J/cm2 fibroblasts exhibited an enhanced migration activity, however at 16 J/cm2 activity was negated and cellular damage observed [57]. Thus from these studies it appears that energy doses from 1.5 J/cm2 to 5 J/cm2 are capable of eliciting "biostimulatory effects" in vitro in the He-Ne-based laser for adherent cells that may be useful in regeneration such as fibroblasts and mesenchymal stem cells.
Studies have also been performed in vitro on immunological cells. High intensity He-Ne irradiation at 28 and 112 J/cm2 of human peripheral blood mononuclear cells, a heterogeneous population of T cells, B cells, NK cells, and monocytes has been described to induce chromatin relaxation and to augment proliferative response to the T cell mitogen phytohemaglutin [58]. In human peripheral blood mononuclear cells (PBMC), another group reported in two papers that interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), and interferon-gamma (IFN-gamma) at a protein and gene level in PBMC was increased after He-Ne irradiation at 18.9 J/cm2 and decreased with 37.8 J/cm2 [59,60]. Stimulation of human PBMC proliferation and murine splenic lymphocytes was also reported with He-Ne LLL [61,62]. In terms of innate immune cells, enhanced phagocytic activity of murine macrophages have been reported with energy densities ranging from 100 to 600 J/cm2, with an optimal dose of 200 J/cm2 [63]. Furthermore, LLL has been demonstrated to augment human monocyte killing mycobacterial cells at similar densities, providing a functional correlation [64].
Thus from the selected in vitro studies discussed, it appears that modulation of proliferation and soluble factor production by LLL can be reliably reproduced. However the data may be to some extent contradictory. For example, the over-arching clinical rationale for use of LLL in conditions such as sinusitis [65], arthritis [66,67], or wound healing [68] is that treatment is associated with anti-inflammatory effects. However the in vitro studies described above suggested LLL stimulates proinflammatory agents such as TNF-alpha or IL-1 [59,60]. This suggests the in vivo effects of LLL may be very complex, which to some extent should not be surprising. Factors affecting LLL in vivo actions would include degree of energy penetration through the tissue, the various absorption ability of cells in the various tissues, and complex chemical changes that maybe occurring in paracrine/autocrine manner. Perhaps an analogy to the possible discrepancy between LLL effects in vitro versus in vivo may be made with the medical practice of extracorporeal ozonation of blood. This practice is similar to LLL therapy given that it is used in treatment of conditions such as atherosclerosis, non-healing ulcers, and various degenerative conditions, despite no clear mechanistic understanding [69-71]. In vitro studies have demonstrated that ozone is a potent oxidant and inducer of cell apoptosis and inflammatory signaling [72-74]. In contrast, in vivo systemic changes subsequent to administration of ozone or ozonized blood in animal models and patients are quite the opposite. Numerous investigators have published enhanced anti-oxidant enzyme activity such as elevations in Mg-SOD and glutathione-peroxidase levels, as well as diminishment of inflammation-associated pathology [75-78]. Regardless of the complexity of in vivo situations, the fact that reproducible, in vitro experiments, demonstrate a biological effect provided support for us that there is some basis for LLL and it is not strictly an area of phenomenology.
Animal Studies with LLL
As early as 1983, Surinchak et al reported in a rat skin incision healing model that wounds exposed He-Ne radiation of fluency 2.2 J/cm2 for 3 min twice daily for 14 days demonstrated a 55% increase in breaking strength over control rats. Interestingly, higher doses yielded poorer healing [79]. This application of laser light was performed directly on shaved skin. In a contradictory experiment, it was reported that rats irradiated for 12 days with four levels of laser light (0.0, 0.47, 0.93, and 1.73 J/cm2) a possible strengthening of wounds tension was observed at the highest levels of irradiation (1.73 J/cm2), however it did not reach significance when analyzed by resampling statistics [80]. In another wound-healing study Ghamsari et al reported accelerated healing in the cranial surface of teats in dairy cows by administration of He-Ne irradiation at 3.64 J/cm2 dose of low-level laser, using a helium-neon system with an output of 8.5 mW, continuous wave [81]. Collagen fibers in LLL groups were denser, thicker, better arranged and more continuous with existing collagen fibers than those in non-LLL groups. The mean tensile strength was significantly greater in LLL groups than in non-LLL groups [82]. In the random skin flap model, the use of He-Ne laser irradiation with 3 J/cm2 energy density immediately after the surgery and for the four subsequent days was evaluated in 4 experimental groups: Group 1 (control) sham irradiation with He-Ne laser; Group 2 irradiation by punctual contact technique on the skin flap surface; Group 3 laser irradiation surrounding the skin flap; and Group 4 laser irradiation both on the skin flap surface and around it. The percentage of necrotic area of the four groups was determined on day 7-post injury. The control group had an average necrotic area of 48.86%; the group irradiated on the skin flap surface alone had 38.67%; the group irradiated around the skin flap had 35.34%; and the group irradiated one the skin flap surface and around it had 22.61%. All experimental groups reached statistically significant values when compared to control [83]. Quite striking results were obtained in an alloxan-induced diabetes wound healing model in which a circular 4 cm2 excisional wound was created on the dorsum of the diabetic rats. Treatment with He-Ne irradiation at 4.8 J/cm2 was performed 5 days a week until the wound healed completely and compared to sham irradiated animals. The laser-treated group healed on average by the 18th day whereas, the control group healed on average by the 59th day [84].
In addition to mechanically-induced wounds, beneficial effects of LLL have been obtained in burn-wounds in which deep second-degree burn wounds were induced in rats and the effects of daily He-Ne irradiation at 1.2 and 2.4 J/cm2 were assessed in comparison to 0.2% nitrofurazone cream. The number of macrophages at day 16, and the depth of new epidermis at day 30, was significantly less in the laser treated groups in comparison with control and nitrofurazone treated groups. Additionally, infections with S. epidermidis and S. aureus were significantly reduced [85].
While numerous studies have examined dermatological applications of LLL, which may conceptually be easier to perform due to ability to topically apply light, extensive investigation has also been made in the area of orthopedic applications. Healing acceleration has been observed in regeneration of the rat mid-cortical diaphysis of the tibiae, which is a model of post-injury bone healing. A small hole was surgically made with a dentistry burr in the tibia and the injured area and LLL was administered over a 7 or 14 day course transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 J/cm2 were applied during the period of the tibia wound healing. Increased angiogenesis was observed after 7 days irradiation at an energy density of 94.5 J/cm2, but significantly decreased the number of vessels in the 14-day irradiated tibiae, independent of the dosage [86]. In an osteoarthritis model treatment with He-Ne resulted in augmentation of heat shock proteins and pathohistological improvement of arthritic cartilage [87]. The possibility that a type of preconditioning response is occurring, which would involve induction of genes such as hemoxygenase-1 [88], remains to be investigated. Effects of LLL therapy on articular cartilage were confirmed by another group. The experiment consisted of 42 young Wistar rats whose hind limbs were operated on in order to immobilize the knee joint. One week after operation they were assigned to three groups; irradiance 3.9 W/cm2, 5.8 W/cm2, and sham treatment. After 6 times of treatment for another 2 weeks significantpreservation of articular cartilage stiffness with 3.9 and 5.8 W/cm2 therapy was observed [89].


Muscle regeneration by LLL was demonstrated in a rat model of disuse atrophy in which eight-week-old rats were subjected to hindlimb suspension for 2 weeks, after which they were released and recovered. During the recovery period, rats underwent daily LLL irradiation (Ga-Al-As laser; 830 nm; 60 mW; total, 180 s) to the right gastrocnemius muscle through the skin. After 2-weeks the number of capillaries and fibroblast growth factor levels exhibited significant elevation relative to those of the LLL-untreated muscles. LLL treatment induced proliferation in satellite cells as detected by BRdU [90].
Other animal studies of LLL have demonstrated effects in areas that appear unrelated such as suppression of snake venom induced muscle death [91], decreasing histamine-induced vasospasms [92], inhibition of post-injury restenosis [93], and immune stimulation by thymic irradiation [94].


Clinical Studies Using LLL
Growth factor secretion by LLL and its apparent regenerative activities have stimulated studies in radiation-induced mucositis. A 30 patient randomized trial of carcinoma patients treated by radiotherapy alone (65 Gy at a rate of 2 Gy/fraction, 5 fractions per week) without prior surgery or concomitant chemotherapy suffering from radiation-induced mucositis was performed using a He-Ne 60 mW laser. Grade 3 mucositis occured with a frequency of 35.2% in controls and at 7.6% of treated patients. Furthermore, a decrease in "severe pain" (grade 3) was observed in that 23.8% in the control group experienced this level of pain, as compared to 1.9% in the treatment group [95]. A subsequent study reported similar effects [96].
Healing ability of lasers was also observed in a study of patients with gingival flap incisions. Fifty-eight extraction patients had one of two gingival flap incisions lased with a 1.4 mW He-Ne (670 nm) at 0.34 J/cm2. Healing rates were evaluated clinically and photographically. Sixty-nine percent of the irradiated incisions healed faster than the control incisions. No significant difference in healing was noted when patients were compared by age, gender, race, and anatomic location of the incision [97]. Another study evaluating healing effects of LLL in dental practice examined 48 patients subjected to surgical removal of their lower third molars. Treated patients were administered Ga-Al-As diode generated 808 nm at a dose of 12 J. The study demonstrated that extraoral LLL is more effective than intraoral LLL, which was more effective than control for the reduction of postoperative trismus and swelling after extraction of the lower third molar [98].
Given the predominance of data supporting fibroblast proliferative ability and animal wound healing effects of LLL therapy, a clinical trial was performed on healing of ulcers. In a double-blinded fashion 23 diabetic leg ulcers from 14 patients were divided into two groups. Phototherapy was applied (<1.0 name="IDAKAOLF">[68].
As previously mentioned, LLL appears to have some angiogenic activity. One of the major problems in coronary artery disease is lack of collateralization. In a 39 patient study advanced CAD, two sessions of irradiation of low-energy laser light on skin in the chest area from helium-neon B1 lasers. The time of irradiation was 15 minutes while operations were performed 6 days a week for one month. Reduction in Canadian Cardiology Society (CCS) score, increased exercise capacity and time, less frequent angina symptoms during the treadmill test, longer distance of 6-minute walk test and a trend towards less frequent 1 mm ST depression lasting 1 min during Holter recordings was noted after therapy [99].
Perhaps one of the largest clinical trials with LLL was the NEST trial performed by Photothera. In this double blind trial 660 stroke patients were recruited and randomized: 331 received LLL and 327 received sham. No prespecified test achieved significance, but a post hoc analysis of patients with a baseline National Institutes of Health Stroke Scale score of <16 name="IDAYAOLF">[100]. Currently Photothera is in the process of repeating this trial with modified parameters.


Relevance of LLL to COPD
A therapeutic intervention in COPD would require addressing the issues of inflammation and regeneration. Although approaches such as administration of bone marrow stem cells, or fat derived cellular components have both regenerative and anti-inflammatory activity in animal models, the need to enhance their potency for clinical applications can be seen in the recent Osiris's COPD trial interim data which reported no significant improvement in pulmonary function [101]. Accordingly, we sought to develop a possible rationale for how LLL may be useful as an adjunct to autologous stem cell therapy.
Table 1 depicts some of the properties of LLL that provide a rationale for the combined use with stem cells. One of the basic properties of LLL seems to be ability to inhibit inflammation at the level of innate immune activation. Representative studies showed that LLL was capable of suppressing inflammatory genes and/or pathology after administration of lipopolysaccharide (LPS) as a stimulator of monocytes [102] and bronchial cells [34], in vitro, and leukocyte infiltration in vivo [103,104]. Inflammation induced by other stimulators such as zymosan, carrageenan, and TNF-alpha was also inhibited by LLL [32,105,106]. Growth factor stimulating activity of LLL was demonstrated in both in vitro and in vivo experiments in which augmentation of FGF-2, PDGF and IGF-1 was observed [36,37,107]. Endogenous production of these growth factors may be useful in regeneration based on activation of endogenous pulmonary stem cells [108,109]. Another aspect of LLL activities of relevance is ability to stimulate angiogenesis. In COPD, the constriction of blood vessels as a result of poor oxygen uptake is results in a feedback loop culminating in pulmonary hypertension. Administration of angiogenic factors has been demonstrated to be beneficial in several animal models of pulmonary pathology [110,111]. The ability of LLL to directly induce proliferation of HUVEC cells [112], as well as to augment production of angiogenic factors such as VEGF [113], supports the possibility of creation of an environment hospitable to neoangiogenesis which is optimal for stem cell growth. In fact, a study demonstrated in vivo induction of neocapillary formation subsequent to LLL administration in a hindlimb ischemia model [114]. The critical importance of angiogenesis in stem cell mediated regeneration has previously been demonstrated in the stroke model, where the major therapeutic activity of exogenous stem cells has been attributed to angiogenic as opposed to transdifferentiation effects [115].
Table 1. Examples of LLL Properties Relevant to COPD

Direct evidence of LLL stimulating stem cells has been obtained using mesenchymal stem cells derived both from the bone marrow and from the adipose tissue [116,117]. Interestingly in vivo administration of LLL stimulated MSC has resulted in 50% decrease in cardiac infarct size [118]. Clinical translation of LLL has been performed in the area of stroke, in which a 660 patient trial
demonstrated statistically significant effects in post trial subset analysis [100].


Conclusions
Despite clinical use of LLL for decades, the field is still in its infancy. As is obvious from the wide variety of LLL sources, frequencies, and intensities used, no standard protocols exist. The ability of LLL to induce growth factor production, inhibition of inflammation, stimulation of angiogenesis, and direct effects on stem cells suggests the urgent need for combining this modality with regenerative medicine, giving birth to the new field of "regenerative photoceuticals". Development of a regenerative treatment for COPD as well as for other degenerative diseases would be of considerable benefit. Regarding COPD, such treatment would be life-saving/life extending for thousands of affected individuals. Ceasing smoking or not starting to smoke would considerably impact this disease.


Competing interests
David R Koos is a shareholder, as well as Chairman and CEO of Entest Bio. Feng Lin is research director of Entest Bio. All other authors declare no competing interest.
Authors' contributions
FL, SFJ, DTA, FR, VB, VG, CAD, RDNC, ANP, EC, DRK contributed to literature review, analysis and discussion, synthesis of concepts, writing of the manuscript and proof-reading of the final draft.
Acknowledgements
The authors thank Victoria Dardov and Matthew Gandjian for critical discussions and input.


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