Donor Fat-Based Stem Cells May Provide Augmented Healing of Rectovaginal Fistulas of Crohn’s Disease Patients

Fistulas are openings in organ systems that connect with another system. They usually result from wounds or erosions in the lining of a tube or duct that gets deeper and deeper and eventually opens into another tube or duct. Physical injuries can cause fistulas, but so can diseases such as Crohn’s disease. Anal fistulas result from erosions of the rectum that open to the outside and are typically very painful and do not readily heal.

Damián García-Olmo and his colleagues at the Universidad Autónoma de Madrid have conducted several clinical trials that have examined the ability of adipose-derived stem cells (ASCs) to facilitate the healing of fistulas in Crohn’s disease patients. A phase I study, which primarily examines safety, was published in 2005 (see García-Olmo D., et al., Dis Colon Rectum 2005; 48:1416-1423). According to this study, “No adverse effects were observed in any patient at the end of the follow-up period (minimum follow-up, 12 months; maximum follow-up, 30 months; follow-up average, 22 months).” The Phase II study was published in 2009 (García-Olmo, D., et al., Dis Colon Rectum 2009; 52:79-86). According to the results of this study, fistula healing was observed in 71 percent of patients who were treated with ASCs in combination with fibrin glue compared with 16 percent of patients who received fibrin glue alone. Quality of life scores were also higher in patients who received ASCs than in those who received fibrin glue alone. Once again, the stem cell treatments were well tolerated. The third study was a multicenter, randomized, single-blind clinical trial that enrolled 200 adult patients from 19 centers that were randomly assigned to three groups. The first group received 20 million stem cells (group A, 64 patients). The second group received 20 million adipose-derived stem cells plus fibrin glue (group B, 60 patients). The third group received only fibrin glue (group C, 59 patients). In treatment of anal fistulas in Crohn’s disease patients, a dose of 20 or 60 million adipose-derived stem cells alone or in combination with fibrin glue were demonstrably safe and did promote healing. However, there were no statistically significant differences between the three groups once the 3 groups were compared.

These studies suggest that stem cells from fat might have a place in the treatment of fistulas in Crohn’s disease patients. The application of the stem cells is feasible and safe, and requires no new equipment or skill. The stem cells also might augment the healing of these fistulas.

Unfortunately, anal fistulas are not the only type of fistulas that Crohn’s disease patients can experience. Female Crohn’s disease patients can have fistulas that open from their rectum into their birth canal. These rectovaginal fistula can deposit the contents of the gastrointestinal tract into the lower reproductive tract. While Crohn’s disease is not the only cause rectovaginal fistulas, Crohn’s disease patients are at higher risks for complications, which include: loss of control over stool deposition (fecal incontinence), hygiene problems combined with recurrent vaginal or urinary tract infections, inflammation of the birth canal and skin around the anus (perineum), abscess formation, which can become life-threatening if not treated, and recurrence of the fistula. Surgical treatment of rectovaginal fistulas requires that the tissue be free of inflammation before surgery, which can take time and cause extensive amounts of patient suffering.

Garcia-Olmo and his colleagues have conducted a small phase I-IIa clinical trial to evaluate the possibility of banked fat-based stem cells to treat recto-vaginal fistulas in female Crohn’s patients. This study has several limitations because it is so small and they have to exclude at least half of the participants because of complications beyond their control. Therefore, this study is not statistically significant. However, it does show what might be the beginnings of a stem-based treatment for this horrid condition.

The design of the study included 11 subjects who were initially enrolled in the study, but one of those recruited patients did not meet the criteria for the study. Therefore, ten subjects, all of whom suffered from Crohn’s disease and had rectovaginal fistulas were treated with 20 million fat-based stem cells that had been donated by a healthy volunteer in addition to surgical repair of their fistulas. These donated fat-based stem cells were provided by a Spanish biotechnology company called Cellerix S.L. Three months after this stem cell treatment, two patients were healed and the other either were given an additional treatment of 40 million fat-based stem cells. Of this group, four were healed. However, five of these patients experienced severe flare-ups of their Crohn’s disease that required treatment with biological agents, which disqualified these patients from further consideration from this study. The biological agents used to treat the Crohn’s disease flare-ups are very powerful medicines and can significantly influence the outcome of this study. Thus half of the subjects in this study had to be excluded. Of the five subjects that remained, 3 showed healing of their fistulas, and 2 did not.

The authors present the data as a “final efficacy rate of 60%.” However, given the high rate of exclusion and the very low numbers of subjects in this study, all we can say with any confidence is that based on the previous successes of this treatment in other studies, there is precedent for such a technique to be safe and somewhat effective, and that the data in this study are in a favorable direction. However, that’s about it.

One feature of this study that differs from the other clinical trials done by this same group is that the previous studies utilized the patient’s own fat-based stem cells, whereas this study used stem cells from a healthy donor. The authors stress that this modification greatly simplifies the procedure and decreases its expense. Because of the ease of the treatments, it reduces postoperative hospitalization and is minimally invasive. This new trial suggests that further work is warranted and the results or even minimally hopeful.

This work was published in the journal Stem Cells Translational Medicine 2016; 5(11): 1441-1446.

ASTIC Clinical Trial Fails to Show Clear Advantage to Hematopoietic Stem Cell Transplantation as a Treatment for Crohn’s Disease

Patients with Crohn’s disease (CD) sometimes suffer from daily bouts of stomach pain and diarrhea. These constant gastrointestinal episodes can prevent them from absorbing enough nutrition to meet their needs, and, consequently, they can suffer from weakness, fatigue, and a general failure to flourish.

To treat Crohn’s disease, physicians use several different types of drugs. First there are the anti-inflammatory drugs, which include oral 5-aminosalicylates such as sulfasalazine (Azulfidine), which contains sulfur, and mesalamine (Asacol, Delzicol, Pentasa, Lialda, Apriso). These drugs, have several side effects, but on the whole are rather well tolerated. If these don’t work, then corticosteroids such as prednisone are used. These have a large number of side effects, including a puffy face, excessive facial hair, night sweats, insomnia and hyperactivity. More-serious side effects include high blood pressure, diabetes, osteoporosis, bone fractures, cataracts, glaucoma and increased chance of infection.

If these don’t work, then the stronger immune system suppressors are brought out. These drugs have some very serious side effects. Azathioprine (Imuran) and mercaptopurine (Purinethol) are two of the most widely used of this group. If used long-term, these drugs can make the patient more susceptible to certain infections and cancers including lymphoma and skin cancer. They may also cause nausea and vomiting. Infliximab (Remicade), adalimumab (Humira) and certolizumab pegol (Cimzia) are the next line of immune system suppressors. These drugs are TNF inhibitors that neutralize an immune system protein known as tumor necrosis factor (TNF). These drugs are also associated with certain cancers, including lymphoma and skin cancers. The next line of drugs include Methotrexate (Rheumatrex), which is usually used to treat cancer, psoriasis and rheumatoid arthritis, but methotrexate also quells the symptoms of Crohn’s disease in patients who don’t respond well to other medications. Short-term side effects include nausea, fatigue and diarrhea, and rarely, it can cause potentially life-threatening pneumonia. Long-term use can lead to bone marrow suppression, scarring of the liver and sometimes to cancer. You will need to be followed closely for side effects.

Then there are specialty medicines for patients who do not respond to other medicines or who suffer from openings in their lower large intestines to the outside world (fistulae). These include cyclosporine (Gengraf, Neoral, Sandimmune) and tacrolimus (Astagraf XL, Hecoria). These have the potential for serious side effects, such as kidney and liver damage, seizures, and fatal infections. These medications are definitely cannot be used for long period of time as their side effects are too dangerous.

If the patient still does not experience any relief, then two humanized mouse monoclonal antibodies natalizumab (Tysabri) and vedolizumab (Entyvio). Both of these drugs bind to and inhibit particular cell adhesion molecules called integrins, and in doing so prevent particular immune cells from binding to the cells in the intestinal lining. Natalizumab is associated with a rare but serious risk of a brain disease that usually leads to death or severe disability called progressive multifocal leukoencephalopathy. In fact, so serious are the side effects of this medicine that patients who take this drug must be enrolled in a special restricted distribution program. The other drug, vedolizumab, works in the same way as natalizumab but does not seem to cause this brain disease. Finally, a drug called Ustekinumab (Stelara) is usually used to treat psoriasis. Studies have shown it’s useful in treating Crohn’s disease and might useful when other medical treatments fail. Ustekinumab can increase the risk of contracting tuberculosis and an increased risk of certain types of cancer. Also there is a risk of posterior reversible encephalopathy syndrome. More common side effects include upper respiratory infection, headache, and tiredness.

If this litany of side effects sounds undesirable, then maybe a cell-based treatment can help Crohn’s patients. To that end, a clinical trial called the Autologous Stem Cell Transplantation International Crohn’s Disease or ASTIC trial was conducted and its results were published in the December 15th, 2015 edition of the Journal of the American Medical Association.

The ASTIC trial enrolled 45 Crohn’s disease patients, all of whom underwent stem cell mobilization with cyclophosphamide and filgrastim, and were then randomly assigned to immediate stem cell transplantation (at 1 month) or delayed transplantation (at 13 months; control group).  Blood samples were drawn and mobilized stem cells were isolated from the blood.  In twenty-three of these patients, their bone marrow was partially wiped out and reconstituted by means of transplantations with their own bone marrow stem cells. The other 22 patients were given standard Crohn disease treatment (corticosteroids and so on) as needed.

The bad news is that hematopoietic stem cell transplantations (HSCT) were not significantly better than conventional therapy at inducing sustained disease remission, if we define remission as the patient not needing any medical therapies (i.e. drugs) for at least 3 months and no clear evidence of active disease on endoscopy and GI imaging at one year after the start of the trial. All patients in this study had moderately to severely active Crohn’s disease that was resistant to treatment, had failed at least 3 immunosuppressive drugs, and whose disease that was not amenable to surgery.  All participants in this study had impaired function and quality of life.  Also, the stem cell transplantation procedure, because it involved partially wiping out the bone marrow, cause considerable toxicities.

Two patients who underwent HSCT (8.7%) experienced sustained disease remission compared to one control patient (4.5%). Fourteen patients undergoing HSCT (61%) compared to five control patients (23%) had discontinued immunosuppressive or biologic agents or corticosteroids for at least 3 months. Eight patients (34.8%) who had HSCTs compared to two (9.1%) patients treated with standard care regimens were free of the signs of active disease on endoscopy and radiology at final assessment.

However, there were 76 serious adverse events in patients undergoing HSCT compared to 38 in controls, and one patient undergoing HSCT died.

So increased toxicities and not really a clear benefit to it; those are the downsides of the ASCTIC study.  An earlier report of the ASTIC trial in 2013, while data was still being collected and analyzed was much more sanguine.  Christopher Hawkey, MD, from the University of Nottingham in the United Kingdom said this: “Some of the case reports are so dramatic that it’s reasonable to talk about this being a cure in those patients.”  These words came from a presentation given by Dr. Hawkey at Digestive Disease Week 2013.  Further analysis, however, apparently, failed to show a clear benefit to HSCT for the patients in this study.  It is entirely possible that some patients in this study did experience significant healing, but statistically, there was no clear difference between HSCT and conventional treatment for the patients in this study.

The silver lining in this study, however, is that compared to the control group, significantly more HSCT patients were able to stop taking all their immunosuppressive therapies for the three months prior to the primary endpoint. That is a potential upside to this study, but it is unlikely for most patients that this upside is worth the heightened risk of severe side effects. An additional potential upside to this trial is that patients who underwent HSCT showed greater absolute reduction of clinical and endoscopic disease activity. Again, it is doubtful if these potential benefits are worth the higher risks for most patients although it might be worth it for some patients.

Therefore, when HSCT was compared with conventional therapy, there was no statistically significant improvement in sustained disease remission at 1 year. Furthermore, HSCT was associated with significant toxicity. Overall, despite some potential upside to HSCT observed in this study, the authors, I think rightly, conclude that their data do not support the widespread use of HSCT for patients with refractory Crohn’s disease.

Could HSCT help some Crohn’s patients more than others? That is a very good question that will need far more work with defined patient populations to answer.  Perhaps further work will ferret out the benefits HSCT has for some Crohn’s disease patients relative to others.

The ASTIC trial was a collaborative project between the European Society for Blood and Marrow Transplantation (EBMT) and the European Crohn’s and Colitis Organization (ECCO) and was funded by the Broad Medical Foundation and the Nottingham Digestive Diseases Centers.

Pre-treatment of MSCs Can Reduce Their Regenerative Properties

Mesenchymal stem cells (MSCs) are excellent suppressors of unwanted inflammation.  This anti-inflammatory activity has been established for systemic inflammatory diseases in animal experiments (Klinker MW, Wei CH. World J Stem Cells. 2015 Apr 26;7(3):556-67), and in clinical trials with human patients (Dulamea A. J Med Life. 2015 Jan-Mar;8(1):24-7; Simonson OE et al., Stem Cells Transl Med. 2015 Oct;4(10):1199-213. doi: 10.5966/sctm.2015-0021).  Stem cell researchers have also shown that MSCs can suppress inflammation in the bowel (see Swenson E and Theise N. Clinical and Experimental Gastroenterology 2010;3:1-10; Chen Z, et al., Biochem Biophys Res Commun. 2014 Aug 8;450(4):1402-8).

After being introduced into the body of a patient, MSCs to move to the site where they are needed (a phenomenon known as “homing”) and promote tissue repair and healing.  Sometime MSC homing works quite well, but other times, it is so-so.  Therefore, several inventive scientists have devised ways to beef up homing to specific sites in order to improve MSC-based tissue healing.  Also, investigators are equally interested in increasing the ability of MSCs to stick to tissues once they arrive there to ensure that the homed MSCs stay where they are needed (see Kavanagh DP, Robinson J, and Kalia N. Stem Cell Rev 2014;10:587-599).  Unfortunately, at the moment, the whole homing process is a bit of a black box and while artificially increasing homing might help in the laboratory, whether or not it increases the therapeutic benefit of MSCs is even less well understood.

A new report from the laboratory of Neena Kalia, who works at the University of Birmingham, UK, has examined the effect of artificial enhancement on the therapeutic capacity of MSCs to treat inflammation in the bowel.  This is an important study because pre-treatment strategies have been suggested as ways to boost MSC homing and retention to various tissues.  The Kalia study suggests such pre-treatment strategies should be viewed with a degree of skepticism.

In this study, Kalia her coworkers induced inflammation in the gastrointestinal tracts of mice by clamping off the blood supply to the this tissue for a time and then releasing the clamps and letting the blood flow anew.  This type of damage, known as ischemia/reperfusion (IR) injury deprives cells of vital oxygen and nutrients for a short period of time, which causes some cells to die.  When the blood is allowed to flow into the tissue, inflammation is induced in the damaged tissue.  Therefore, this technique can efficiently induce  inflammation in tissues in the gastrointestinal tract.

Two groups of mice were treated with bone marrow-derived MSCs.  One group had experiences IR injury to their gastrointestinal tracts, and the other group did not.  In these experiments, administered MSCs showed similar levels of and cell adhesion in both injured and non-injured guts.  In general, cell adhesion levels were nothing to write home about:  as reported in the paper, “limited cell adhesion observed.”  Despite these initial observations, those MSCs that found their way to the gut were able to help heal the tissues to some degree.  There were fewer white blood cells in the middle part of the small intestine (jejunum), and the degree of blood flow seemed to have improved.  Unfortunately, the lower part of the small intestine (ileum) was not helped to the same degree, and the paper reports that a fair number of MSCs got stuck in small blood vessels, which suggests that these vessels got stuck on their way to the intestine.

If these results seem underwhelming, it might be because they are.  Undaunted, Kalia and her crew tried to boost the regenerative abilities of their isolated MSCs by pretreating them.  Kalia’s laboratory and other laboratories as well have used a variety of chemical agents to augment the healing abilities of MSCs.  These agents include things like tumor necrosis factor (TNF)-α, CXCL12 (also known as stromal cell-derived factor 1 or SDF1, which strongly activates white blood cells), interferon (IFN)-γ, or hydrogen peroxide.  When these pre-treated MSCs were administered to mice whose guts were damaged by means of IR injury, the pretreatment not only did not enhance their intestinal recruitment, but actually decreased the healing capacities of MSCs.  Pretreatment of MSCs with tumor necrosis factor (TNF)-α, CXCL12, interferon (IFN)-γ, or hydrogen peroxide did not enhance their intestinal recruitment.  Pretreatment with TNFα and IFNγ abrogated ability of transplanted MSCs to reduce white blood cells infiltration and improve blood flow in the jejunum.

Kalia and her colleagues utilized a technique called “intravital” microscopy for this study.  Intravital microscopy can track individual cells in a living animals (Kavanagh DP, Yemm AI, Zhao Y, et al. PLoS One 2013;8:e59150). With this technique, they were able to efficiently monitor adhesion in the tinyu blood vessels in the injured intestinal tissue.  They documented poor MSC adhesion to the gut lining and that pre-treatment with various factors hopes failed to enhance adhesion of MSCs to the gut.

This study successfully demonstrated that MSCs can rapidly limit white blood cells recruitment to the inflamed gut, and improve tissue perfusion if they are administered after intestinal IR injury. However, Kalia’s study also shows that strategies to improve MSC therapeutic efficacy by means of pretreatment of MSCs may not be all it’s cracked up to be.  They suggest that in the future, cytokine or chemical pretreatments designed to enhance MSC recruitment and function will require more than just successful experiments in a cell culture system.  Instead, pretreatment strategies will need to be carefully validated in living organisms in order the confirm that such protocols help rather than hinder the therapeutic function of implanted stem cells.

This paper was published in the journal Stem Cells – Kavanagh DP, Suresh S, Newsome PN, et al. Stem Cells 2015;33:2785-2797

Laboratory-Grown Intestine Shows Promise in Mice and Dogs

David Hackam is a pediatric surgeon at the Johns Hopkins Children’s Center. Unfortunately, Dr. Hackam spends a good deal of his time removing dead sections of intestine from sick babies, but he would deeply love to be able to do more than just take out intestines but actually replace the dead or dying intestinal tissue. It is that desire that has driven Hackam and his colleagues to grow intestines in the laboratory.

They begin with stem cells taken from the small intestines of human infants and mice and apply them to intestine-shaped scaffolds. The stem cells dig in, grow and form mini-intestines that just might be able to treat disorders like necrotizing enterocolitis and Crohn’s disease someday. Transplantation experiments in laboratory animals have shown that this laboratory-grown tissue and scaffolding are not rejected, but integrate into the tissues of the animals. Experiments in dogs have shown that the scaffold allowed dogs to heal from damage to the colon lining, essentially restoring healthy bowel function.

The study is a “great breakthrough,” says Hans Clevers, a stem cell biologist at the Hubrecht Institute in Utrecht, the Netherlands, who was not involved in the new research. Clevers and his colleagues were the first to identify stem cells in the intestine, and his lab developed the technique Hackam’s team used to grow intestinal tissue.

Making replacement organs by growing cells on scaffolds molded into the shape of the organ is not a new idea, since other researchers have used exactly this technique to make bladders and blood vessels. However, the laboratory-grown intestines made by Hackam and his group come closer to the shape and structure of a natural intestine than anything created in the laboratory before. In previous experiments carried out in other laboratories, the gut lining has been grown on flat scaffolds or in culture flasks. Under these conditions, the tissue tends to roll up into little balls that have the absorptive surface on the inside. Hackam and his coworkers, however, overcame this problem by using a scaffold fabricated from materials similar to surgical sutures. This material can be molded into any desired intestinal size and shape, and in Hackam’s hands, the scaffolds formed a true tube-shaped (like a real gut), with tiny projections on the inner surface that can help the tissue form functional small intestinal villi (the small fingers of tissue that increase the surface area of the intestine to increase nutrient absorption. “They can now make sheets of cells that can be clinically managed,” Clevers says. “Surgeons can handle these things and just stick them in.”

To grow the gut lining in the lab, the researchers painted the scaffold with a sticky collagen-rich substance and then dripped onto it a solution of stem cells from the small intestine. This concoction was grown in a culture system for a week. Interestingly, Hackam and his team found that if they added connective tissue cells, immune cells, and probiotics (bacteria that help maintain a healthy gut), all of these things helped the stem cells mature and differentiate.

Hackam’s group also sutured intestines grown from mouse stem cells into the tissue surrounding the abdominal organs of the mouse. The lab-grown intestines developed their own blood supply and normal gut structures despite the fact that they were not connected to the animals’ digestive tract. “Using the mouse’s own stem cells, we can actually create something that looks just like the native intestine,” Hackam says. The next step, he says, is “to hook it up.”

Before “hooking it up,” Hackam needed to be sure that the scaffold could be tolerated in living animals. Therefore he tested the new scaffold in dogs. He removed sections of large intestinal lining and replaced it with pieces of scaffolding. The dogs made a complete recovery: their gut lining regrew onto the scaffold and functioned normally to absorb water from the colon. After a few weeks, the scaffolding had completely dissolved and was replaced with normal connective tissue. “The scaffold was well tolerated and promoted healing by recruiting stem cells,” Hackam says. “[The dogs] had a perfectly normal lining after 8 weeks.”

This technique could help more than just dogs and mice, but could aid human patients. According to Hackam, scaffolds could be custom-designed for individual human patients to replace a portion of an intestine or the entire organ. This could be a revolutionary treatment for patients with necrotizing enterocolitis, a condition that destroys intestinal tissue in about 12% of premature babies in the United States. It could also potentially repair the intestines of patients with Crohn’s disease, an inflammatory bowel disorder that can have life-threatening complications and that affects more than 500,000 people in the United States. However, these lab-grown intestines must pass several other tests before they are ready for human clinical trials, Hackam cautions.

The first test that these laboratory-grown intestines must pass is the absorption test. Laboratory-grown small intestines must be transplanted into live animals and they must properly absorb food. Also, the technology that is used will also require some adjustments. For example, Mari Sogayar, a molecular biologist at the University of São Paulo in Brazil, points out that the collagen product that helps the stem cells stick to the scaffold is not meant for use in people. In the next experiments, Hackam says, the researchers plan to use a surgical-grade alternative.

“I take care of children who have intestinal deficiencies, eating deficiencies, and they are very much at wits’ end,” Hackam says. “I think what we can offer in the scientific community is a path toward something that one day will help a child.”

Mesenchymal Stem Cells Heal Gastrointestinal Ulcers

Stomach ulcers are a complication of routine use of aspirin, Advil, or other non-steroidal anti-inflammatory drugs. Additionally, radiation therapy, or inflammatory bowel disease can also cause stomach ulcers, and these are painful and potentially dangerous for patients. Trying to get our heads around ulcers is not easy, but a new study by Manieri and colleagues have provided some understanding of ulcer formation and ways that mesenchymal stem cells (MSCs) might help heal these painful lesions.

Manieri and others used prostaglandin-deficient mice as a model system for ulcer formation. In these mice, their stomachs do not produce the prostaglandins that protect the layers of the stomach from being digested by its own acid and enzymes. Consequently, these mice are subject to so-called “penetrating ulcer formation,” or ulcers that penetrate the underlying muscular layer (muscularis propria). When Manieri and his colleagues took biopsies of the colon of these prostaglandin-deficient mice, they observed extensive necrosis of the upper and lower layers of the colon.

When these mice were treated with stable prostaglandin-I2 (PGI2) analogs, Manieri and others showed that they could ameliorate the damage to the colon. However, when this research group analyzed the ulcer beds in these mutant mice, they noticed that CD31+ endothelial cells, which form blood vessels, were found in very low numbers. This suggested that reduced blood vessel formation could be a key driver of penetrating ulcer formation. To confirm their hypothesis, the authors stained the wound sites for vascular endothelial growth factor (VEGF). They saw fewer VEGF+ cells in the mutant mice compared with wild-type animals, which suggests that impaired blood vessel production contributes to ulceration. To further test this hypothesis, Manieri and others treated wild-type mice with tivozanib (a VEGF receptor antagonist), which also caused smooth muscle necrosis in the colon.

Next Manieri and others injected MSCs from the colons of mice that showed increased expression of VEGF into the ulcerated colon of mutant mice. The MSCs dutifully migrated to the ulcer beds, and rescued the muscle necrosis phenotype. These results show that MSC administration can provide a soothing treatment prospect for patients who are dealing with gastrointestinal ulceration.

See N. A. Manieri et al., Mucosally transplanted mesenchymal stem cells stimulate intestinal healing by promoting angiogenesis. J. Clin. Invest. 10.1172/JCI81423 (2015).

Nusse Laboratory at Sanford Identifies Elusive Mouse Liver Stem Cell

Stanford University life science researchers have managed to successfully identify a stem cell population that has eluded many laboratories for some time. Essentially, the Stanford team has discovered a previously unknown population of liver cells in mice that function as liver stem cells. Such a find could aid drug testing and increase our present understanding of liver biology and disease.

Researchers in the laboratory of Roel Nusse at the Stanford University School of Medicine have identified a cell type in the liver of laboratory mice that can both self-renew and make new liver cells. This discovery by Nusse and others settles a long-standing conundrum of how the liver maintains itself when liver cells grow old and die.

“There’s always been a question as to how the liver replaces dying hepatocytes,” said professor of developmental biology Roel Nusse, PhD. “Most other tissues have a dedicated population of cells that can divide to make a copy of themselves, which we call self-renewal, and can also give rise to the more-specialized cells that make up that tissue. But there never was any evidence for a stem cell in the liver.”

It was assumed for some time that mature hepatocytes (liver cells) would themselves divide to replace a dying neighbor. However, hepatocytes have an abnormal amount of DNA, which would make cell division extremely difficult.

Nusse and his team published this research in the Aug. 5 edition of the journal Nature. The first author of this paper, Bruce Wang, MD, an assistant professor of gastroenterology and hepatology at the University of California-San Francisco, led the research while serving as a visiting scholar in Nusse’s lab.

The liver is a large, multi-lobed organ that filters toxins from the blood, synthesizes blood proteins, and makes digestive enzymes and bile. It is involved in many important metabolic processes. The liver contains a central vein that carries blood through it. The stem cells identified by Wang and Nusse are found adjacent to this vein.

Classically, hepatocytes were largely thought to be all alike. Most mature hepatocytes are “polyploid,” which means that they have more than the normal two copies of each chromosome. With all this extra DNA, it makes it difficult or even impossible, for these cells to divide normally, but this extra DNA might confer other benefits.

“If it’s not necessary for a cell to maintain the capacity to divide, it can do whatever it wants with its genome,” said Nusse. “Red blood cells, for instance, have no DNA. Muscle cells have many copies of each chromosome.” Having extra copies of chromosomes might allow these cells to make large amounts of particular proteins quickly, for example.

However, the cell population identified by Wang and Nusse in the livers of laboratory mice is diploid, and have only two copies of each chromosome. These cells can divide to make carbon copies of themselves, or to make cells that begin their lives as diploid but then acquire additional copies of their chromosomes as they move away from the central vein into the main body of the liver.

“People in the field have always thought of hepatocytes as a single cell type,” said Wang. “And yet the cell we identified is clearly different from others in the liver. Maybe we should accept that there may be several subtypes of hepatocytes, potentially with different functions.”

To identify liver-specific stem cells, Wang and Nusse identified cells that express the protein Axin2. They found these axin2-expressing cells surrounding the central vein. Axin2 is produced by cells in response to the presence of members of the Wnt signaling protein family, and decades of research by Nusse and other laboratories have shown that the Wnt proteins play a critical role in embryonic development, and in the growth and maintenance of stem cells throughout the body.

Interestingly, Wang and Nusse, and others showed that the endothelial cells that line the interior surface of the central vein in the liver produce Wnt2 and Wnt9b. These secreted Wnt proteins confer stem cell properties on the neighboring hepatocytes that surround the central vein.

Finally, Nusse’s team discovered that a portion of the descendants of the Axin2-expressing cells move outward from the central vein over time, become polyploid and begin to express other, hepatocyte-specific genes. About one year after being born, these descendant cells had effectively replaced about 30 percent of the entire mouse liver, and made up about 40 percent of all hepatocytes in the liver.

These newly identified liver stem cells also express genes associated with very early embryonic development, which may give a clue as to when and where they arise.

“Perhaps these stem cells in the adult liver actually arise very early in development,” said Nusse, “when the embryo sets aside a certain population of cells to maintain the organ during adult life.”

Although the current research was conducted in mice, it is possible that there are more than just one kind of hepatocyte in humans as well, and this realization could transform the study of liver biology. For example, hepatocytes have proven notoriously difficult to grow in laboratory culture for study or for use in drug testing.

“The most common reason that promising new drugs for any type of condition fail is that they are found to be toxic to liver,” said Wang. “Researchers have been trying for decades to find a way to maintain hepatocytes in the laboratory on which to test the effects of potential medications before trying them in humans. Perhaps we haven’t been culturing the right subtype. These stem cells might be more likely to fare well in culture.”

There’s also an opportunity to better understand human disease.

“Does liver cancer arise from a specific subtype of cells?” said Wang. “This model also gives us a way to understand how chromosome number is controlled. Does the presence of the Wnt proteins keep the stem cells in a diploid state? These are fundamental biological questions we can now begin to address.”

Pregnancy and Delivery Unaffected in Women Patients With Crohn’s Disease Who Were Treated With Fat-Based Stem Cells

Fat is a readily accessible source of mesenchymal stem cells (MSCs). When fat is extracted by liposuction, the result is a so-called stromal vascular fraction (SVF) that contains a mishmash of mast cells (important in allergies), blood vessel-making cells, blood vessel-associated cells, fibroblasts, and MSCs. These adipose-derived stem cells (ASCs) as they are called, can be relatively easily prepared once the SVF is digested by enzymes, and centrifuges. The living adult MSCs are then rather easily identified because they adhere to plastic tissue culture plates.

Fat-based MSCs have been used in clinical studies to help heal patients with Crohn’s disease who have “fistulas.”  For a picture of a fistula, see here.  Crohn’s disease (CD) is one of a group of gastrointestinal diseases known as IBDs or inflammatory bowel diseases. CD features inflammation of any part of the GI tract, and this inflammation can affect multiple layers of the GI tract. Fistulas form when a hole is eroded through the GI tract and into another organ system. For example, in women, the rectum and erode and form an opening in the vagina. Alternatively, an opening can appear in some place other than the anus. Because of the repeated irritation and extensive inflammation of these lesions, they tend to not heal.

Beginning in 2003, Damián García-Olmo and his team at the Jiménez Diaz Foundation University Hospital in Madrid, Spain have tested the efficacy of fat-based stem cells in treating patients with CD-based fistulas.  The results have been encouraging and highly positive, since ASCs promote healing of the fistulas and decrease recovery time (see de la Portilla F, et al. (2013) Int J Colorectal Dis 28:313–323; García-Olmo D, et al. (2003) Int J Colorectal Dis 18:451–454; García-Olmo D, et al. (2005) Dis Colon Rectum 48:1416–1423; Garcia-Olmo D, et al. (2009) Dis Colon Rectum 52:79–86).

Recently, Garcia-Olmo and his colleagues examined data from several their patients who went on to become pregnant after their treatment with fat-based stem cells and even given birth. This study, which was published in the June 2015 edition of Stem Cells Translational Medicine, examined six patients from these previous clinical trials who were successfully treated with fat-based stem cells, had satisfactory resolution and healing of their lesions, and then went on to become pregnant and give birth.

Of the five women examined in this study, one was treated for rectovaginal and perinatal fistulas, two for rectovaginal fistulas only, and two others for perianal fistulas only. All women received 2 doses of 20 million and 40 million stem cells at three-four-month intervals. One patient, however, received 2 doses of 6.6 million and 20 million stem cells nine months apart.

The fertility of these women and their pregnancies were unaffected by their previous cell therapies. There were no signs of treatment-related malformations in the babies they delivered, and their bodies did not show any identifiable signs of structural abnormalities as a result of the stem cell treatments. It must be said, that all four women who delivered healthy babies (one of them even had twins) elected for Caesarian sections. The fifth woman, unfortunately, miscarried twice, both times during the first trimester.

However, even though this represents a small data set, this study does strongly suggest that injection of a patient’s own fat-based stem cells does not negatively affect a woman’s ability to conceive, the course of her pregnancy, or the health of her baby.