Vascular Progenitors Made from Induced Pluripotent Stem Cells Repair Blood Vessels in the Eye Regardless of the Site of Injection

Johns Hopkins University medical researchers have reported the derivation of human induced-pluripotent stem cells (iPSCs) that can repair damaged retinal vascular tissue in mice. These stem cells, which were derived from human umbilical cord-blood cells and reprogrammed into an embryonic-like state, were derived without the conventional use of viruses, which can damage genes and initiate cancers. This safer method of growing the cells has drawn increased support among scientists, they say, and paves the way for a stem cell bank of cord-blood derived iPSCs to advance regenerative medical research.

In a report published Jan. 20 in the journal Circulation, Johns Hopkins University stem cell biologist Elias Zambidis and his colleagues described laboratory experiments with these non-viral, human retinal iPSCs, that were created generated using the virus-free method Zambidis first reported in 2011.

“We began with stem cells taken from cord-blood, which have fewer acquired mutations and little, if any, epigenetic memory, which cells accumulate as time goes on,” says Zambidis, associate professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center. The scientists converted these cells to a status last experienced when they were part of six-day-old embryos.

Instead of using viruses to deliver a gene package to the cells to turn on processes that convert the cells back to stem cell states, Zambidis and his team used plasmids, which are rings of DNA that replicate briefly inside cells and then are degraded and disappear.

Next, the scientists identified and isolated high-quality, multipotent, vascular stem cells that resulted from the differentiation of these iPSC that can differentiate into the types of blood vessel-rich tissues that can repair retinas and other human tissues as well. They identified these cells by looking for cell surface proteins called CD31 and CD146. Zambidis says that they were able to create twice as many well-functioning vascular stem cells as compared with iPSCs made with other methods, and, “more importantly these cells engrafted and integrated into functioning blood vessels in damaged mouse retina.”

Working with Gerard Lutty, Ph.D., and his team at Johns Hopkins’ Wilmer Eye Institute, Zambidis’ team injected these newly iPSC-derived vascular progenitors into mice with damaged retinas (the light-sensitive part of the eyeball). The cells were injected into the eye, the sinus cavity near the eye or into a tail vein. When Zamdibis and his colleagues took images of the mouse retinas, they found that the iPSC-derived vascular progenitors, regardless of injection location, engrafted and repaired blood vessel structures in the retina.

“The blood vessels enlarged like a balloon in each of the locations where the iPSCs engrafted,” says Zambidis. Their vascular progenitors made from cord blood-derived iPSCs compared very well with the ability of vascular progenitors derived from fibroblast-derived iPSCs to repair retinal damage.

Zambidis says that he has plans to conduct additional experiments in diabetic rats, whose conditions more closely resemble human vascular damage to the retina than the mouse model used for the current study, he says.

With mounting requests from other laboratories, Zambidis says he frequently shares his cord blood-derived iPSC with other scientists. “The popular belief that iPSCs therapies need to be specific to individual patients may not be the case,” says Zambidis. He points to recent success of partially matched bone marrow transplants in humans, shown to be as effective as fully matched transplants.

“Support is growing for building a large bank of iPSCs that scientists around the world can access,” says Zambidis, although large resources and intense quality-control would be needed for such a feat. However, Japanese scientists led by stem-cell pioneer Shinya Yamanaka are doing exactly that, he says, creating a bank of stem cells derived from cord-blood samples from Japanese blood banks.

Controlling Transplanted Stem Cells from the Inside Out

Scientists have worked very hard to understand how to control stem cell differentiation.  However, despite how well you direct stem cell behavior in culture, once those stem cells have been transplanted, they will often do as they wish.  Sometimes, transplanted stem cells surprise people.

Several publications describe stem cells that, once transplanted undergo “heterotropic differentiation.” Heterotropic differentiation refers to tissues that form in the wrong place. For example, one lab found that transplantation of mesenchymal stem cells into mouse hearts after a heart attack produced bone (don’t believe me – see Martin Breitbach and others, “Potential risks of bone marrow cell transplantation into infarcted hearts.” Blood 2007 110:1362-1369).  Bone in the heart – that can’t be good. Therefore, new ways to control the differentiation of cells once they have been transplanted are a desirable goal for stem cell research.

From this motivation comes a weird but wonderful paper from Jeffrey Karp and James Ankrum of Brigham and Women’s Hospital and MIT, respectively, that loads stem cells with microparticles that give the transplanted stem cell continuous cues that tell them how to behave over the course of days or weeks as the particles degrade.

“Regardless of where the cell in the body, it’s going to be receiving its cues from the inside,” said Karp. “This is a completely different strategy than the current method of placing cells onto drug-doped microcarriers or scaffolds, which is limiting because the cells need to remain in close proximity to those materials in order to function. Also these types of materials are too large to be infused into the bloodstream.”

Controlling cells in culture is relatively easy. If cells take up the right molecules, they will change their behavior. This level of control, however, is lost after the cell is transplanted. Sometimes implanted cells readily respond to the environment within the body,. but other times, their behavior is erratic and unpredictable. Karp’s strategy, which her called “particle engineering,” corrects this problem by turning cells into pre-programmable units. The internalized particles stably remain inside the transplanted cell and instruct it precisely how to act. It can direct cells to release anti-inflammatory factors, or regenerate lost tissue and heal lesions or wounds.

“Once those particles are internalized into the cells, which can take on the order of 6-24 hours, we can deliver the transplant immediately or even cryopreserve the cells,” said Karp. “When the cells are thawed at the patient’s bedside, they can be administrated and the agents will start to be released inside the cells to control differentiation, immune modulation or matrix production, for example.”

It could take more than a decade for this type of cell therapy to be a common medical practice, but to speed up the pace of this research, Karp published the study to encourage others in the scientific community to apply the technique to their various fields. Karp’s paper also illustrates the range of different cell types that can be controlled by particle engineering, including stem cells, cells of the immune system, and pancreatic cells.

“With this versatile platform, which leveraged Harvard and MIT experts in drug delivery, cell engineering, and biology, we’ve demonstrated the ability to track cells in the body, control stem cell differentiation, and even change the way cells interact with immune cells, said Ankrum, who is a former graduate student in Karp’s laboratory. “We’re excited to see what applications other researchers will imagine using this platform.”

Stem Cell-Based Gene Therapy Restores Normal Skin Function

Michele De Luca from the University of Modena, Italy and his collaborator Reggio Emilia have used a stem cell-based gene therapy to treat an inherited skin disorder.

Epidermolysis bullosa is a painful skin disorder that causes the skin to be very fragile and blister easily. These blisters can lead to life-threatening infections. Unfortunately, no cure exists for this condition and most treatments try to alleviate the symptoms and infections.

Stem cell-based therapy seems to be one of the best ways to treat this disease, there are no clinical studies that have examined the long-term outcomes of such a treatment.

However, De Luca and his colleagues have examined a particular patients with epidermolysis bullosa who was treated with a stem cell-based gene therapy nearly seven years ago as part of a clinical trial.

The treatment of this patient has established that transplantation of a small quantity of stem cells into the skin on this patient’s legs restored normal skin function without causing any adverse side effects.

“These findings pave the way for the future safe use of epidermal stem cells for combined cell and gene therapy of epidermolysis bullosa and other genetic skin diseases,” said Michele De Luca.

De Luca and his research team found that their treatment of their patient, named Claudio, caused the skin covering his upper legs to looker normal and show no signs of blisters. To treat Claudio, De Luca and his colleague extracted skin cells from Claudio’s palm, used genetic engineering techniques to correct the genetic defect in the cells, and then transplanted these cells back into the skin of his upper legs. This was part of a clinical trial conducted at the University of Modena.

Claudio’s legs also showed no signs of tumors and the small number of transplanted cells sufficiently repaired Claudio’s skin long-term. Keep in mind that Claudio’s skin cells had undergone approximately 80 cycles of cell division and still had many of the features of palm skin cells, they show proper elasticity and strength and did not blister.

“This finding suggests that adult stem cell primarily regenerate the tissue in which they normally reside, with little plasticity to regenerate other tissues,” De Luca said. “This calls into question the supposed plasticity of adult stem cells and highlights the need to carefully chose the right type of stem cell for therapeutic tissue regeneration.”

I think De Luca slightly overstates his case here. Certainly choosing the right stem cells is crucial for successful stem cell treatments, but to take stem cells from skin, which are dedicated to making skin and expect them to form other tissues is unreasonable. However, several experiments have shown that stem cells from hair follicles and form neural tissues and several other cell types as well (see Jaks V, Kasper M, Toftgård R. The hair follicle-a stem cell zoo. Exp Cell Res. 2010 May 1;316(8):1422-8).

Adult stem cells have limited plasticity to be sure, but their plasticity is far greater than originally thought and a wealth of experiments have established that.

Despite these quibbles, this is a remarkable experiment that illustrates the feasibility and safety of such a treatment.  A larger problem is that large quantities of cells will be required to treat a person.  It is doubtful that small skin biopsies around the body can provide enough cells to treat the whole person.  Therefore, this might a case for induced pluripotent skin cells, which seriously complicates this treatment strategy.

New Tool for Stem Cell Transplantation into the Heart

Researchers from the famed Mayo Clinic, in collaboration with scientists at a biopharmaceutical biotechnology company in Belgium have invented a specialized catheter for transplanting stem cells into a beating heart.

This new device contains a curved needle with graded openings along the shaft of the needle. The cells are released into the needle and out through the openings in the side of the needle shaft. This results in maximum retention of implanted stem cells to repair the heart.

“Although biotherapies are increasingly more sophisticated, the tools for delivering regenerative therapies demonstrate a limited capacity in achieving high cell retention in the heart,” said Atta Behfar, the lead author of this study and a cardiologist. “Retention of cells is, of course, crucial to an effective, practical therapy.”

Researchers from the Mayo Clinic Center for Regenerative Medicine in Rochester, MN and Cardio3 Biosciences in Mont-Saint-Guibert, Belgium, collaborated to develop the device. Development of this technology began by modeling the dynamic motions of the heart in a computer model. Once the Belgium group had refined this computer model, the model was tested in North America for safety and retention efficiency.

These experiments showed that the new, curved design of the catheter eliminates backflow and minimizes cell loss. The graded holes that go from small to large diameters decrease the pressures in the heart and this helps properly target the cells. This new design works well in healthy and damaged hearts.

Clinical trials are already testing this new catheter. In Europe, the CHART-1 clinical trial is presently underway, and this is the first phase 3 trial to examine the regeneration of heart muscle in heart attack patients.

These particular studies are the culmination of years of basic science research at Mayo Clinic and earlier clinical studies with Cardio3 BioSciences and Cardiovascular Centre in Aalst, Belgium, which were conducted between 2009 and 2010.  This study, the C-CURE or Cardiopoietic stem Cell therapy in heart failURE study examined 47 patients, (15 control and 32 experimental) who received injections of bone marrow-derived mesenchymal stem cells from their own bone marrow into their heart muscle.  Control patients only received standard care.  After six months, those patients who received the stem cell treatment showed an increase in heart function and the distance they could walk in six minutes.   No adverse effects were observed in the stem cell recipients.

This study established the efficacy of mesenchymal stem cell treatments in heart attack patients.  However, other animal and computer studies established the efficacy of this new catheter for injecting heart muscle with stem cells.  Hopefully, the results of the CHART-1 study will be available soon.

Postscript:  The CHART-2 clinical trial is also starting.  See this video about it.

Stem Cells Treat Babies With Brittle Bone Disease While Still in the Womb

A new study published by the journal STEM CELLS Translational Medicine shows that stem cells can be effective in treating brittle bone disease, a debilitating and sometimes lethal genetic disorder.

Also known as osteogenesis imperfecta (OI), this genetic disorder was popularized by actor Samuel T. Jackson in the Bruce Willis movie “Unbreakable.” OI is characterized by fragile bones that cause patients to suffer hundreds of fractures over the course of a lifetime. According to the OI Foundation, other symptoms include muscle weakness, hearing loss, fatigue, joint laxity, curved bones, scoliosis, brittle teeth and short stature. In the more severe cases of OI, restrictive pulmonary disease also occurs. Unfortunately, to date no cure exists for OI.

Physicians use ultrasound to detect OI in babies before they are born. In this study, an international research team treated two patients for the disease with mesenchymal stem cells (MSCs) while the infants were still in the womb. After they were born, the babies were given additional mesenchymal stem cell treatments.

“We had previously reported on the prenatal transplantation for the patient with OI type III, which is the most severe form in children who survive the neonatal period,”said Cecilia Götherström, Ph.D., of the Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden. She and Jerry Chan, M.D., Ph.D., of the Yong Loo Lin School of Medicine and National University of Singapore, and KK Women’s and Children’s Hospital, led the study that also included colleagues from the United States, Canada, Taiwan and Australia.

“The first eight years after the prenatal transplant, our patient did well and grew at an acceptable rate. However, she then began to experience multiple complications, including fractures, scoliosis and reduction in growth, so the decision was made to give her another MSC infusion. In the two years since, she has not suffered any more fractures and improved her growth. She was even able to start dance classes, increase her participation in gymnastics at school and play modified indoor hockey,”Dr. Götherström added.

The second child suffered from a milder form of OI and received a stem cell transfusion 31 weeks into gestation and did not suffer any new fractures for the remainder of the pregnancy or during infancy. She followed her normal growth pattern — just under the third percentile in height, but when she was 13 months old, she stopped growing. Six months later, the doctors gave her another infusion of stem cells and she resumed growing at her previous rate.

“Our findings suggest that prenatal transplantation of autologous stem cells in OI appears safe and is of likely clinical benefit and that re-transplantation with same-donor cells is feasible. However, the limited experience to date means that it is not possible to be conclusive, for which further studies are required,”Dr. Chan said.

“Although the findings are preliminary, this report is encouraging in suggesting that prenatal transplantation may be a safe and effective treatment for this condition,”said Anthony Atala, M.D., editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine.

Umbilical Cord Blood Stem Cells in a Biodegradable Scaffold Regenerate Full-Thickness Skin Defects

In a new study published in the ASAIO Journal by Reza Zeinali and others in the laboratory of Kamal Asadipour, specific stem cell from umbilical cord blood called unrestricted somatic stem cells (USSCs) have been grown on a biodegradable scaffold to promote skin regeneration and wound healing.

USSCs are considered by many stem cell scientists to be a type of mesenchymal stem cell, but USSCs can be grown in the laboratory and have the ability to differentiate into a wide variety of adult cell types.

Asadipour and others used a material called PHBV or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) to make a skin-like scaffold upon which the USSCs were grown. They discovered that attaching a molecule called “chitosan” to the PHBV made it quite resilient and a very good substrate for growing cells. When grown on these scaffolds, the USSCs adhered nicely to them and grew robustly.

Then Zeinali and his colleagues used these cell-impregnated scaffolds to treat open surgical wounds in laboratory rodents. After three weeks, the group treated with the cell grown on the scaffolds healed significantly better than those animals treated with just cells, just scaffolds, or neither.

Thus it seems likely that tissue-engineered skin made from modified PHBV scaffolds and embedded umbilical cord blood-based stem cells might be a potent treatment for wound patients with large injuries that do heal slowly.  In the words of the abstract of this paper, “These data suggest that chitosan-modified PHBV scaffold loaded with CB-derived USSCs could significantly contribute to wound repair and be potentially used in the tissue engineering.”

Some larger animal studies should further test this protocol and if it can augment the healing of large animal wounds, then human clinical trials should be considered.

A Patient’s Own Bone Marrow Stem Cells Defeat Drug-Resistant Tuberculosis

People infected with multidrug-resistant forms of tuberculosis could, potentially, be treated with stem cells from their own bone marrow. Even though this treatment is in the early stage of its development, the results of an early-stage trial of the technique show immense promise.

British and Swedish scientists have tested this procedure, which could introduce a new treatment strategy for the estimated 450,000 people worldwide who have multi drug-resistant (MDR) or extensively drug-resistant (XDR) TB.

This study, which was published in the medical journal, The Lancet, showed that over half of 30 drug-resistant TB patients treated with a transfusion of their own bone marrow stem cells were cured of the disease after six months.

“The results … show that the current challenges and difficulties of treating MDR-TB are not insurmountable, and they bring a unique opportunity with a fresh solution to treat hundreds of thousands of people who die unnecessarily,” said TB expert Alimuddin Zumla at University College London, who co-led the study.

TB initially infects the lungs but can rapidly spread from one person to another through coughing and sneezing. Despite its modern-day resurgence, TB is often regarded as a disease of the past. However, recently, drug-resistant strains of Mycobacterium tuberculosis, the microorganism that causes TB, have spread globally, rendering standard anti-TB drug treatments obsolete.

The World Health Organisation (WHO) estimates that in Eastern Europe, Asia and South Africa 450,000 people have MDR-TB, and close to half of these cases will fail to respond to existing treatments.

Mycobacterium tuberculosis, otherwise known as the “tubercle bacillus, trigger a characteristic inflammatory response (granulomatous response) in the surrounding lung tissue that elicits tissue damage (caseation necrosis).

Bone-marrow stem cells are known to migrate to areas of lung injury and inflammation. Upon arrival, they initiate the repair of damaged tissues. Since bone marrow stem cells also they also modify the body’s immune response, they can augment the clearance of tubercle bacilli from the body. Therefore, Zumla and his colleague, Markus Maeurer from Stockholm’s Karolinska University Hospital, wanted to test bone marrow stem cell infusions in patients with MDR-TB.

In a phase 1 trial, 30 patients with either MDR or XDR TB aged between 21 and 65 who were receiving standard TB antibiotic treatment were also given an infusion of around 10 million of their own bone marrow-derived stem cells.

The cells were obtained from the patient’s own bone marrow by means of a bone marrow aspiration, and then grown into large numbers in the laboratory before being re-transfused into the same patient.

During six months of follow-up, Zumla and his team found that the infusion treatment was generally safe and well tolerated, and no serious side effects were observed. The most common non-serious side effects were high cholesterol levels, nausea, low white blood cell counts and diarrhea.

Although a phase 1 trial is primarily designed only to test a treatment’s safety, the scientists said further analyses of the results showed that 16 patients treated with stem cells were deemed cured at 18 months compared with only five of 30 TB patients not treated with their own stem cells.

Maeurer stressed that further trials with more patients and longer follow-up were needed to better establish how safe and effective the stem cell treatment was.

But if future tests were successful, he said, this could become a viable extra new treatment for patients with MDR-TB who do not respond to conventional drug treatment or for those patients with severe lung damage.

Stem Cell Treatments for Aortic Aneurysms

The aorta is the largest blood vessel in our bodies and it emerges from the left ventricle of the heart, takes a U-turn, and swings down toward the legs (descending or dorsal aorta). There are several branches of the aorta as it sharply turns that extend towards the head and upper extremities.

Aorta structure

Sometimes, as a result of inflammation of the aorta or other types of problems, the elastic matrix that surrounds and reinforces the aorta breaks down.  This weakens the wall of the aorta and it bulges out.  This bulge is called an aortic aneurysm and it is a dangerous condition because the aneurysm can burst, which will cause the patient to bleed to death.

Aortic Aneurysm

If an aneurysm is discovered through medical imaging techniques, drugs are given to lower blood pressure and take some of the pressure off the aorta.  Also, drugs that prevent further degradation of the elastic matrix are also used.  Ultimately, for large or fast-growing aneurysms, surgical repair of the aorta is necessary.  For aneurysms of the abdominal aorta, a surgical procedure called abdominal aortic aneurysm open repair is the “industry standard.”  For this surgery, the abdomen is cut open, and the aneurysm is repaired by the use of a long cylinder-like tube called a graft.  Such grafts are made of different materials that include Dacron (textile polyester synthetic graft) or polytetrafluoroethylene (PTFE, a nontextile synthetic graft).  The surgeon sutures the graft to the aorta, and connects one end of the aorta at the site of the aneurysm to the other end.

A “kinder, gentler” way to fix an aneurysm is to use a procedure called endovascular aneurysm repair (EVAR).  EVAR uses these devices called “stents” to support the wall of the aorta.  A small insertion is made in the groin and the collapsed stent is inserted through the large artery in the leg.  Then the stent, which is long cylinder-like tube made of a thin metal framework and covered with various materials such as Dacron or polytetrafluoroethylene (PTFE), is inserted into the aneurysm.  Once in place, the stent-graft will be expanded in a spring-like fashion to attach to the wall of the aorta and support it.  The aneurysm will eventually shrink down onto the stent-graft.

In some cases, the patient is too weak for surgery, and is not a candidate for EVAR.  A much better option would be to non-surgically repair the elastic support framework that surrounds the aorta, and stem cells are candidates for such repair.

To repair the elastic mesh work that surrounds the wall of the aorta, smooth muscle cells that secrete the protein “elastin” must be introduced into the wall of the aorta.  Also, using the patient’s own stem cells offers a better strategy at this point, since this circumvents such issues as immune rejection of implanted tissues and so on.  The sources of stem cells for smooth muscle cells include bone marrow stem cells, fat-based stem cells, and stem cells from peripheral blood.  All three of these stem cell sources have problems with finding enough cells in the body and expanding them to high enough numbers in order to properly treat the aneurysm.

Fortunately, the use of induced pluripotent stem cells, which are made from a patient’s mature cells and have many, though not all of the characteristics of embryonic stem cells, can provide large quantities of elastin-secreting smooth muscle cells.  Also, one laboratory in particular has reported differentiating human induced pluripotent stem cells into smooth muscle cells (Lee TH, Song SH, Kim KL, et al. Circ Res 106:120–128).  While there are challenges to making functional elastin, there are possibilities that many of these can be overcome.

Ideal characteristics and expected roles of iPSCs and differentiated SMC-like derivatives for treating AAAs. Shown are several of the necessary properties for expansion/differentiation in culture, delivery to the AAA, and elastogenesis within the tunica media microenvironment. Abbreviations: AAA, abdominal aortic aneurysm; ECM, extracellular matrix; Eln, elastin; iPSC, induced pluripotent stem cell; LOX, lysyl oxidase; MMPs, matrix metalloproteinases; SMC, smooth muscle cell; TNFα, tumor necrosis factor-α.
Ideal characteristics and expected roles of iPSCs and differentiated SMC-like derivatives for treating AAAs. Shown are several of the necessary properties for expansion/differentiation in culture, delivery to the AAA, and elastogenesis within the tunica media microenvironment. Abbreviations: AAA, abdominal aortic aneurysm; ECM, extracellular matrix; Eln, elastin; iPSC, induced pluripotent stem cell; LOX, lysyl oxidase; MMPs, matrix metalloproteinases; SMC, smooth muscle cell; TNFα, tumor necrosis factor-α.

In addition to induced pluripotent stem cells, other laboratories have examined umbilical cord mesenchymal stem cells and their ability to decrease the inflammation within the aorta that leads to aneurysms.  The researchers discovered that all the indicators of inflammation decreased, but the synthesis of new elastin was not examined.  However, a Japanese laboratory used mouse mesenchymal stem cells from bone marrow and found that not only did these cells shut down enzymes that tend to degrade elastin, but also initiated new elastin synthesis in culture.  The same study also showed that MSCs implanted into the vessel walls of an aorta that was experiencing an aneurysm stabilized the aneurysm by inhibiting the elastin-degrading enzymes, and increasing the elastin content of the vessel wall.  This had the net effect of stabilizing the aneurysms and preventing them from growing further (see Hashizume R, Yamawaki-Ogata A, Ueda Y, et al. J Vasc Surg 54:1743–1752).  

These experiments show that stem cell treatments for abdominal aneurysms are feasible and would definitely be a much-needed nonsurgical treatment option for the high-risk elderly demographic, which is rapidly growing in the developed world.

For more information on this interesting topic, see Chris A. BashuraRaj R. Raob and Anand Ramamurthia. Perspectives on Stem Cell-Based Elastic Matrix Regenerative Therapies for Abdominal Aortic Aneurysms.  Stem Cells Trans Med June 2013 vol. 2 no. 6 401-408.

New Approach for Corneal Stem Cell Treatments

More than 8 million people worldwide suffer from corneal blindness; a form of blindness that results from cloudiness of the outermost covering of the eye, the cornea.

Usually, the cornea copes quite well with minor injuries or scrapes and scratches. If the cornea is scratched, healthy cells slide over quickly and patch the injury before infection occurs and vision is not adversely affected. However, if the scratch penetrates the cornea more deeply, then the healing process takes longer and can result in greater pain, blurred vision, tearing, redness, and extreme sensitivity to light. Such scratches may require professional treatment. Even deeper scratches can also cause corneal scarring, which results in a haze on the cornea that can greatly impair vision, and the patient might require a corneal transplant.

Alternatively, corneal stem cells can help heal a damaged cornea; especially in those cases where the cornea has been damaged to the point where the native stem cell population has suffered irreparable damage (e.g., chemical burns, eye infections, or cases where the patient was born with a corneal stem cell deficiency).

A feasible treatment for such cases is a corneal stem cell transplant from another eye or from cultured corneal stem cells. Unfortunately, this procedure has not yet been standardized to date.

Fortunately, researchers at the Eye Program at the Cedar-Sinai Regenerative Medicine Institute have designed a fast, new procedure for preparing human amniotic membrane to use as a scaffold for corneal stem cells. The membrane provides a foundation that supports the growth of stem cells that can be grafted onto the cornea.

To date, a standardized method does not exist for the preparation of amniotic membranes for culturing corneal stem cells. Many methods use chemicals and may leave behind amniotic cells and membrane components.

This new procedure, however, takes less than one minute and ensures complete amniotic cell removal and preservation of amniotic membrane components, and, as an added bonus, supports the overall growth of various stem and tissue cells.

“We believe that this straightforward and relatively fast procedure would allow easier standardization of amniotic membrane as a valuable stem cell support and improve the current standard of care in corneal stem cell transplantation,” said the lead author of this work Alexander Ljubimov, the director of the Eye Program at the Cedar-Sinai Regenerative Medicine Institute. “This new method may provide a better method for researchers, transplant corneal surgeons, and manufacturing companies alike.”

The amniotic membrane has several beneficial properties for corneal stem cells culturing and use in corneal transplantations. For this reason it is an attractive framework for the growth and culture of corneal stem cells and for corneal transplantations.

The new method for amniotic membrane preparation will provide a fast way to create scaffolds for cell expansion and might potentially streamline clinical applications of cell therapies.

Priming Cocktail for Cardiac Stem Cell Grafts

Approximately 700,000 Americans suffer a heart attack every year and stem cells have the potential to heal the damage wrought by a heart attack. Stem cells therapy has tried to take stem cells cultured in the laboratory and apply them to damaged tissues.

In the case of the heart, transplanted stem cells do not always integrate into the heart tissue. In the words of Jeffrey Spees, Associate Professor of Medicine at the University of Vermont, “many grafts simply didn’t take. The cells would stick or would die.”

To solve this problem, Spees and his colleagues examined ways to increase the efficiency of stem cell engraftment. In his experiments, Spees and others used mesenchymal stem cells from bone marrow. Mesenchymal stem cells are also called stromal cells because they help compose the spider web-like filigree within the bone marrow known as “stroma.” Even though the stroma does not make blood cells, it supports the hematopoietic stem cells that do make all blood cells.  Here is a picture of bone marrow stroma to give you an idea of what it looks like:

Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Stromal cells are known to secrete a host of molecules that protect injured tissue, promote tissue repair, and support the growth and proliferation of stem cells.

Spees suspected that some of the molecules made by bone marrow stromal cells could enhance the engraftment of stem cells patches in the heart. To test this idea, Spees and others isolated proteins from the culture medium of bone marrow stem cells grown in the laboratory and tested their ability to improve the survival and tissue integration of stem cell patches in the heart.

Spees tenacity paid off when he and his team discovered that a protein called “Connective tissue growth factor” or CTGF plus the hormone insulin were in the culture medium of these stem cells. Furthermore, when this culture medium was injected into the heart prior to treating them with stem cells, the stem cell patches engrafted at a higher rate.

“We broke the record for engraftment,” said Spees. Spees and his co-workers called their culture medium from the bone marrow stem cells “Cell-Kro.” Cell-Kro significantly increases cell adhesion, proliferation, survival, and migration.

Spees is convinced that the presence of CTGF and insulin in Cell-Kro have something to do with its ability to enhance stem cell engraftment. “Both CTGF and insulin are protective,” said Spees. “Together they have a synergistic effect.”

Spees is continuing to examine Cell-Kro in rats, but he wants to take his work into human trials next. His goal is to use cardiac stem cells (CSCs) from humans, which already have a documented ability to heal the heart after a heart attack. See here, here, and here.

“There are about 650,000 bypass surgeries annually,” said Spees. “These patients could have cells harvested at their first surgery and banked for future application. If they return for another procedure, they could then receive a graft of their own cardiac progenitor cells, primed in Cell-Kro, and potentially re-build part of their injured heart.”

New Model for Kidney Regeneration

Harvard Stem Cell Institute Kidney Diseases Program Leader Benjamin Humphreys has examined tissue regeneration in the kidney. His interest in kidney regeneration has occupied a major part of his career, but some of his more recent work resulted from his skepticism of a particular theory of kidney regeneration.

The kidney stem cell repair model postulates that scattered throughout the kidney are small stem cell populations and are activated after the kidney is injured to repair it. This theory, however, conflicts with another view of kidney regeneration. Namely that after injury, the cells of the kidney dedifferentiate into more primordial versions of themselves and proliferate, after which they differentiate into the various tissues of the kidney.

Humphreys and his colleagues now have evidence that strongly suggests that all the cells of the kidney have the capacity to divide after injury and contribute to kidney regeneration.

Their evidence comes in the form of experiments in mice in which the cells of the kidney were genetically tagged, and then the kidneys were injured to determine what cells contributed to the regeneration of the kidney.

The tagging in this experiment is complicated, but quite technically brilliant. The kidney is composed of myriads of tiny functional units called nephrons. Each nephron is fed by a tiny knot of blood vessels called a glomerulus.  The structure of a nephron is shown below.  


The blood supply to the kidney comes from branches off the descending aorta knows as renal arteries.  After entering the kidneys, the renal arteries branch multiple times until they become tiny vessels that feed into each nephron known as afferent arterioles.  The afferent arterioles forms a dense network of knot-like vessels that form the glomerulus and the portion of the nephron that interacts with the glomerulus is known as the Bowman’s capsule..  The blood vessels of the glomerulus are very special because they are exceptionally porous.  However, the Bowman’s capsule has a series of cells with foot-like extensions that coat the glomerulus called “podocytes.”  An especially beautiful picture of podocytes wrapped around a glomerular vessel is shown below.


The podocytes cover the pores of the glomerulus and only allows water and things dissolved in water through the pores.  Proteins do not make it through – they are too heavily charged.  Cells also do not make it through – they are too big.  But water, sodium ions, potassium ions, hydrogen ions, some drugs, metabolites, waste products, and things like that all make it through the podocyte-guarded pores.  For this reason, if you have excessive protein or some blood cells in your urine, it is usually an indication that something is wrong.  

Now, rest of the tubing attached to the nephron serve to reabsorb all the things you do not want to get rid of and not absorb all the things you do want to get rid of.  The amount of water you eliminate depends on your degree of hydration and is controlled by a hormone called antidiuretic hormone, which is release by the posterior lobe of your pituitary gland when you are dehydrated.  In the presence of ADH, the posterior tubing reabsorbs more water, and in lower concentrations of this hormone, it reabsorbs far less.  

Now that we know something about the kidney, here’s how Humphreys and others genetically marked the kidneys of their mice.  The sodium-dependent inorganic phosphate transporter (SLC34a1) is only expressed in mature proximal tubule cells.  Tetsuro Kusaba, the first author on this paper, and his colleagues inserted a CreERT2 cassette into this gene.  If you are lost at this point all you need to remember is this: the CreERT2 cassette is inserted into a gene that is ONLY expressed in specific kidney cells.  The Cre gene encodes a recombinase that clips out specific bits of DNA from a chromosome.  Kusaba and others crossed these engineered mice with another strain of mice that had the gene for a bright red dye inserted into another gene, but this dye could not be expressed because another piece of DNA was in the way.  When these hybrid mice were fed a drug called tamoxifen, it activated the expression of the Cre protein, but only in the proximal tubule cells of the kidney and this Cre protein clipped out the piece of DNA that was preventing the red dye gene from being expressed.  Therefore, these mice had a particular part of their nephrons, the proximal tubules glowing bright red.  This is a stroke of shear genius and it genetically marks these cells specifically and strongly.  

Next, Kusaba and colleagues used unilateral ischemia reperfusion injury (IRI) to damage the kidneys.  In IRI, the blood supply is stopped to one kidney but not the other for a short period of time (26 minutes).  This causes cell death and kidney damage.  The other kidney is not damaged and serves as a control for the experiment.  

Examination of the damaged kidneys showed that  red-glowing cells were found in areas other than the proximal tubules.  The only way these cells could have ended up in these places was if the differentiated cells divided and helped repair the damaged parts of the nephrons.  

Other research groups have seen similar results, but interpreted them as evidence of stem cell populations in the kidney.  However, Humphreys groups discovered something even more fascinating.  These “stem cell-markers” in the kidney are actually markers of kidney damage and regeneration and all cells in the kidney express them.  In Humphreys words, “What was really interesting is when we looked at the appearance and expression patterns of these differentiated cells, we found that they expressed the exact same ‘stem cell markers’ that these other groups claimed to find in their stem cell populations.  And so, if a differentiated cell is able to express a ‘stem cell marker’ after injury, then what our work shows is that that’s an injury marker – is doesn’t define a stem cell.”  

Indeed, several genes that have been taken to be signs of a kidney stem cell population (CD133, CD24, vimentin, and KIM-1) were expressed in red-glowing cells.  A stem cell population should not be fully differentiated and therefore, should not be able to express the red dye.  However, red-glowing cells clearly expressed these found genes after injury.  This rather definitely shows that it is the fully differentiated cells that are doing the regeneration in the kidney and not a resident stem cell population.  This does not prove that there is no resident stem cell population in the kidney, but only that the lion’s share of the regeneration is done by differentiated cells, and that under these conditions, no stem cell population was detected.  

This new interpretation of kidney repair suggests that cells can reprogram themselves in a way that resembles the way mature cells are chemically manipulated to revert to an induced pluripotent state.  

See Tetsuro Kusaba, Matthew Lalli, Rafael Kramann, Akio Kobayashi, and Benjamin D. Humphreys. Differentiated kidney epithelial cells repair injured proximal tubule. PNAS (October 14, 2013); doi:10.1073/pnas.1310653110.  

The Mechanism Behind Blood Stem Cell Longevity

The blood stem cells that live in bone marrow divide and send their progeny down various pathways that ultimately produce red cells, white cells and platelets. These “daughter” cells must be produced at a rate of about one million cells per second in order to constantly replenish the body’s blood supply.

A nagging question is how these stem cells to persist for decades even though their progeny last for days, weeks or months before they need to be replaced. A study from the University of Pennsylvania has uncovered one of the mechanisms, and these cellular mechanisms allow these stem cells to keep dividing in perpetuity.

Dennis Discher and his colleagues in the Department of Chemical and Biomolecular Engineering in the School of Engineering and Applied Science found that a form of a protein called “myosin,” the motor protein that allow muscles to contract, helps bone marrow stem cells divide asymmetrically. This asymmetric cell division helps one cell remains a stem cell while the other cell becomes a daughter cell. Discher’s findings might provide new insights into blood cancers, such as leukemia, and eventually lead to ways of growing transfusable blood cells in a laboratory.

The participants in this study were members of the Discher laboratory, which include lead author Jae-Won Shin, Amnon Buxboim, Kyle R. Spinler, Joe Swift, Dave P. Dingal, Irena L. Ivanovska and Florian Rehfeldt. Discher collaborated with researchers at the Univ. de Strasbourg, Lawrence Berkeley National Laboratory and Univ. of California, San Francisco. This paper was published in Cell Stem Cell.

“Your blood cells are constantly getting worn out and replaced,” Discher said. “We want to understand how the stem cells responsible for making these cells can last for decades without being exhausted.”

Presently, scientists understand the near immortality of hematopoietic stem cells (HSCs) as a result of their asymmetric cell division, although how this asymmetric cell division enables stem cell longevity was unknown. To ferret out this mechanism, Discher and his coworkers analyzed all of the genes expressed in the stem cells and compared them with the genes expression in their more rapidly dividing progeny. Those proteins that only went to one side of the dividing cell might play a role in partitioning other key factors responsible for keeping one of the cells a stem cell and the other a progeny cell.

One of the proteins that showed a distinct expression pattern was the motor protein myosin II, which has two forms, myosin A and myosin B. Myosin II is the protein that enables the body’s muscles to contract, but in nonmuscle cells also it used during cell division. During the last phase of cell division, known as cytokinesis, myosin II helps cleave and close off the cell membranes as the cell splits apart.

“We found that the stem cell has both types of myosin,” Shin said, “whereas the final red and white blood cells only had the A form. We inferred that the B form was key to splitting the stem cells in an asymmetric way that kept the B form only in the stem cell.”

With these myosins as their top candidate, Discher and others labeled key proteins in dividing stem cells with different colors and put them under the microscope.

“We could see that the myosin IIB goes to one side of the dividing cell, which causes it to cleave differently,” Discher said. ”It’s like a tug of war, and the side with the B pulls harder and stays a stem cell.”

The researchers then performed in vivo tests using mice that had human stem cells injected into their bone marrow. By genetically inhibiting myosin IIB production, Shin and others saw the stem cells and their early progeny proliferating while the amount of downstream blood cells dropped.

“Because the stem cells were not able to divide asymmetrically, they just kept making more of themselves in the marrow at the expense of the differentiated cells,” Discher said.

HSC cell division mechanism

Discher and his team then used a drug that temporarily blocked both myosin A and myosin B. They observed that myosin inhibition increased the prevalence of non-dividing stem cells, blocking the more rapid division of progeny.

Discher believes that these findings could eventually help regrow blood stem cells after chemotherapy treatments for blood cancers or even grow blood products in the lab. Finding a drug that can temporarily shut down only the B form of myosin, while leaving the A form alone, would allow the stem cells to divide symmetrically and make more of themselves without preventing their progeny from dividing themselves.

“Nonetheless, the currently available drug that blocks both the A and B forms of myosin II could be useful in the clinic,” Shin said, “because donor bone marrow cultures can now easily be enriched for blood stem cells, and those are the cells of interest in transplants. Understanding the forces that stem cells use to divide can thus be exploited to better control these important cells.”

Stem Cell Transplant Repairs the Damage that Results from Inflammatory Bowel Disease

A source of stem cells from the digestive tract can repair a type of inflammatory bowel disease when transplanted into mice has been identified by British and Danish scientists.

This work resulted from a collaboration between stem cell scientists at the Wellcome Trust-Medical Research Council/Cambridge Stem Cell Institute at Cambridge University, and the Biotech Research and Innovation Centre (BRIC) at the University of Copenhagen, Denmark. This research paves the way for patient-specific regenerative therapies for inflammatory bowel diseases such as ulcerative colitis.

All tissues in out body probably contain a stem cell population of some sort, and these tissue-specific stem cells are responsible for the lifelong maintenance of these tissues, and, ultimately, organs. Organ-specific stem cells tend to be restricted in their differentiation abilities to the cell types within that organ. Therefore, stem cells from the digestive tract will tend to differentiate into cell types typically found in the digestive tract, and skin-based stem cells will usually form cell types found in the skin.

When this research team examined developing intestinal tissue in mouse fetuses, they discovered a stem cell population that differed from the adult stem cells that have already been described in the gastrointestinal tract. These new-identified cells actively divided and could be grown in the laboratory over a long period of time without terminally differentiating into adult cell types. When exposed to the right conditions, however, these cells could differentiate into mature intestinal tissue.


Could these cells be used to repair a damaged bowel? To address this question, this team transplanted these cells into mice that suffered from a type of inflammatory bowel disease, and within three hours the stem cells has attached to the damaged areas of the mouse intestine. integrated into the intestine, and contributed to the repair of the damaged tissue.

“We found that the cells formed a living plaster (British English for a bandage) over the damaged gut,” said Jim Jensen, a Wellcome Trust researcher and Lundbeck Foundation fellow, who led the study. “They seemed to response to the environment they had been placed in and matured accordingly to repair the damage. One of the risks of stem cell transplants like this is that the cells will continue to expand and form a tumor, but we didn’t see any evidence of that with this immature stem cell population from the gut.”

Because these cells were derived from fetal intestines, Jensen and his team sought to establish a new source of intestinal progenitor cells.  Therefore, Jensen and others isolated cells with similar characteristics from both mice and humans, and  made similar cells similar cells by reprogramming adult human cells in to induced pluripotent stem cells (iPSCs) and growing them in the appropriate conditions.  Because these cells grew into small spheres that consisted of intestinal tissue, they called these cells Fetal Enterospheres (FEnS).

Established cultures of FEnS expressed lower levels of Lgr5 than mature progenitors and grew in the presence of the Wnt antagonist Dkk1 (Dickkopf).  New cultures can be induced to form mature intestinal organoids by exposure to the signaling molecule Wnt3a. Following transplantation in a model for colon injury, FEnS contributed to regeneration of the epithelial lining of the colon by forming epithelial crypt-like structures that expressed region-specific differentiation markers.

“We’ve identified a source of gut stem cells that can be easily expanded in the laboratory, which could have huge implications for treating human inflammatory bowel diseases. The next step will be to see whether the human cells behave in the same way in the mouse transplant system and then we can consider investigating their use in patients,” Jensen said.

Porous Material Helps Deliver Molecules to Stem Cell-Derived Cells

A Swedish group has successfully tested a new porous material that allows for the efficient delivery of key molecules to transplanted cells that have been derived from stem cells. Such a material can dramatically improve the way stem cell-based treatments for neurodegenerative diseases.

This research project included a collaboration between Danish, Swedish and Japanese laboratories, and it tested a new type of porous material that efficiently delivers key molecules to transplanted cells derived from stem cells in an animal model.

Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p  .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.
Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p < .001. No statistically significant differences were observed between groups with direct or MesoMim administration of the factors (p > .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.

This potentially versatile and widely applicable strategy for the efficient differentiation and functional integration of stem cell derivatives upon transplantation, and it can serve as a foundation for improving stem cell-based neurodegenerative protocols, for example, Parkinson’s disease.

Alfonso Garcia-Bennett of Stockholm University, one of the lead authors of this study, said: “We are working to provide standard and reproducible methods for the differentiation and implementation of stem cell therapies using this type of approach, which coupled material science with regenerative medicine.”

Garcia-Bennett continued: “We demonstrated that delivering key molecules for the differentiation of stem cells in vivo with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also improved their long-term survival.”

This research group is already working together with two companies to speed up the commercialization of a standard differentiation kit that will allow other scientists and clinicians to reproduce their work in their own laboratories.

Mechanism that Prevents Stem Cell Aging

A research group at the University of Valencia, Spain, led by Isabel Fariñas Gómez, at the Molecular Neurobiology Unit, has discovered a mechanism that maintains stem cell populations in the brain and prevents these stem cells from overproliferating early in life and burning out.

Gómez’s group has discovered that the product of the CDKn1a/p21 gene is essential for maintaining brain stem cells.  By keeping these stem cells active and functional, the brain dynamically changes as it learns and remembers, and maintains its good state of health.  In the absence of p21, brain stem cell populations deplete and this prevents the formation of new neurons toward the end of life.

Stem cells require p21 to replicate themselves in a controlled fashion.  In other cell types, p21 acts as a “tumor suppressor” gene.  Tumor suppressor genes encode proteins that tend to put the brakes on cell proliferation.  Loss-of-function mutations in tumor suppressor genes causes uncontrolled group and predisposes that cell and its descendants to become cancer cells.

p21 function

However, in neural stem cells, p21 functions differently.  Depletion of p21 from neural stem cells causes their depletion rather than their overgrowth.  In short, an absence of p21 causes these cells to age.

This research, conducted in collaboration with Anxo Vidal from the University of Santiago de Compostela, shows that p21 in neural stem cells restrains the production of molecules that induce the depletion of these this stem cell population.  Thus p21 restricts aging.  According to Fariñas Gómez, “The research allows us to understand better how stem cells get lost in our brains as we age, and opens the possibility to try to alleviate this deterioration.”

Stomach Cells Naturally Revert to Stem Cells

George Washington University scientists from St. Louis, Missouri have found that the stomach naturally produces more stem cells than previously realized. These stem cells probably repair stomach damage from infections, the foods we eat, and the constant tissue insults from stomach acid.

The reversion of adult cells to a stem cell fate is one of the goals of stem cell research. Shinya Yamanaka’s research group at the Center for iPS Cell Research and Application and the Institute for Frontier Medical Sciences at Kyoto University won the Nobel Prize in 2012 for his work on reprogramming adult cells into embryonic-like stem cells, otherwise known as induced pluripotent stem cells (iPSCs) that was initially published in 2006.

A collaborative research effort between scientists from Washington University School of Medicine in St. Louis and Utrecht Medical Center in the Netherlands have shown that this reversion from adult cells to stem cells occurs naturally in the stomach on a regular basis.

Jason Mills, associate professor of medicine at Washington University, said, “We already knew that these cells, which are called chief cells, can change back into stem cells to make temporary repairs in significant stomach injuries in significant stomach injuries, such as a cut to damage from infection. The fact that they’re making this transition more often, even in the absence of noticeable injuries, suggests that it may be easier than we realized to make some types of mature, specialized adult cells revert to stem cells.”

Chief cells normally produce a protein called pepsinogen. In the presence of stomach acid, pepsinogen activates itself and once active, the new protein product, pepsin, degrades proteins. Pepsin in an enzyme that is most active in the acidic environment of the stomach. Another enzyme released by chief cells is chymosin, which is also known as rennet. Chymosin curdles the proteins in milk and makes them easier to degrade.


Mills and his groups are in the process of studying the transformation of chief cells into stem cells, for injury repair. Mills would also like to investigate the possibility that the potential for growth unleashed by this change may contribute to stomach cancers.

Mills and his collaborator Hans Clevers from the Netherlands have identified stomach stem cell marker proteins that show that chief cells become stem cells even in the absence of serious injury. In the case of serious injury, either in cell culture of in animal models, more chief cells become stem cells, making it possible to repair the damage in the stomach.

Faulty Stem Cell Regulation Contributes to Down Syndrome Deficits

People who have three copies of chromosome 21 have a genetic condition known as Down Syndrome (DS). In particular, patients who have an extra copy of a small portion of chromosome 21 (q22.13–q22.2) known as the Down Syndrome Critical Region or DSCR have the symptoms of DS. The DSCR contains at least 30 genes or so and some of them tightly correlate to the pathology of DS. For example, the APP (amyloid protein precursor) gene accounts for the accumulation of amyloid protein in the brains of DS patients. DS patients develop Alzheimer disease-like pathology by the fourth decade of life, and the APP protein is overexpressed in the adult Down syndrome brain. Another gene found in the DSCR called DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) encodes a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and this protein participates in various cellular processes. Overproduction of DYRK1A seems to cause the abnormal brain development observed in DS babies.

Another gene found in the DSCR is called USP16 and this gene encodes a protein that removes small peptides called ubiquitin from other proteins. Ubiquitin attachment marks a protein for degradation, but it can also mark a protein to do a specific job. USP16 removes ubiquitin an either stops the protein from acting or prevents the proteins from being degraded. Overexpression of UPS16 occurs in DS patients, and too much UPS16 protein affects stem cell function.

Michael Clarke, professor of cancer biology at the Stanford University School of Medicine, said, “There appear to be defects in the stem cells in all the tissues we tested, including the brain.” Clarke continued, “We believe USP16 overexpression is a major contributor to the neurological deficits seen in Down Syndrome.” Clarke’s laboratory conducted their experiments in mouse and human cells.

Additional work by Clarke and his colleagues showed that downregulation of USP16 partially rescues the stem cell proliferation defects found in DS patients.

Clarke’s study suggests that drugs that reduce the activity of USP16 could reduce the some of the most profound deficits in DS patients.

This paper also details some of the pathological mechanisms of DS. DS patients age faster and exhibit early Alzheimer’s disease. The reason for this seems to rely on the overexpression of UPS16, which accelerates the rate at which stem cells are used during early development. This accelerated rate of stem cell use burns out and exhausts the stem cell reserves and, consequently, the brains age faster and are susceptible to the early onset of neurodegenerative diseases.

After examining laboratory mice that had a rodent form of DS, Clarke and his coworkers turned their attention to USP16 overexpression in human cells. Clarke collaborated with a Stanford University neurosurgeon named Samuel Cheshier and their study showed that skin cells from normal volunteers grew much more slowly when the Usp16 gene was overexpressed. Furthermore, neural stem cells, which normally clump into little balls of cells called neurospheres, no longer formed these structures when Usp16 was overexpressed in them.

a, Proliferation analysis, as well as SA-βgal and p16Ink4a staining, of three control and four Down’s syndrome (DS) human fibroblast cultures show growth impairment and senescence of Down’s syndrome cells. b, c, Lentiviral-induced overexpression of USP16 decreases the proliferation of two different control fibroblast lines (b), whereas downregulation of USP16 in Down’s syndrome fibroblasts promotes proliferation (c). d, Overexpression of USP16 reduces the formation of neurospheres derived from human adult SVZ cells. The right panel quantifies the number of spheres in the first and second passages. P < 0.0001. All the experiments were replicated at least twice. Luc, luciferase.
a, Proliferation analysis, as well as SA-βgal and p16Ink4a staining, of three control and four Down’s syndrome (DS) human fibroblast cultures show growth impairment and senescence of Down’s syndrome cells. b, c, Lentiviral-induced overexpression of USP16 decreases the proliferation of two different control fibroblast lines (b), whereas downregulation of USP16 in Down’s syndrome fibroblasts promotes proliferation (c). d, Overexpression of USP16 reduces the formation of neurospheres derived from human adult SVZ cells. The right panel quantifies the number of spheres in the first and second passages. P < 0.0001. All the experiments were replicated at least twice. Luc, luciferase.

Conversely, when cultured cells from DS patients had their USP16 activity levels knocked down, their proliferation defects disappeared. In Clarke’s words, “This gene is clearly regulating processes that are central to aging in mice and humans, and stem cells are severely compromised. Reducing Usp16 expression gives an unambiguous rescue at the stem cell level. The fact that it’s also involved in this human disorder highlights how critical stem cells are to our well-being.”