Repopulation of Damaged Livers With Skin-Derived Stem Cells


Patients with severe liver disease must receive a liver transplant. This major procedure requires that the patient survives major surgery and then takes anti-rejection drugs for the rest of their lives. In general, liver transplant patients tend to fair pretty well. The one-year survival rate of liver transplant patients approaches 90% (see O’Mahony and Goss, Texas Heart Institute Journal 2012 39(6): 874-875).

A potentially better way to treat liver failure patients would be to take their own liver cells, convert them into induced pluripotent stem cells (iPSCs), differentiate them into liver cells, and use these liver cells to regenerate the patient’s liver. Such a treatment would contain a patient’s own liver cells and would not require anti-rejection drugs.

Induced pluripotent stem cells or iPSCs are made from genetically-engineered adult cells that have had four specific genes (Oct4, Klf4, Sox2, and c-Myc) introduced into them. As a result of the heightened expression of these genes, some of the adult cells dedifferentiate and are reprogrammed into cells that resemble embryonic stem cells. Normally, this procedure is relatively inefficient, slow, and induces new mutations into the engineered cells. Also, when iPSCs are differentiated into liver cells (hepatocytes), they do not adequately proliferate after differentiation, and they also fail to properly function the way adult hepatocytes do.

New work from laboratories at the University of California, San Francisco (UCSF), has differentiated human hepatocytes by means of a modified technique that bypasses the pluripotency stage. These cells were then used to repopulate mouse livers.

“I really like this paper. It’s a step forward in the field,” said Alejandro Soto-Gutiérrez, assistant professor of pathology at the University of Pittsburgh, who was not involved in the work. “The concept is reprogramming, but with a shortcut, which is really cool.”

Research teams led by Holger Willenbring and Sheng Ding isolated human skin cells called fibroblasts and infected them with engineered viruses that forced the expression of three genes: OCT4, SOX2, and KLF4. These transduced cells were grown in culture in the presence of proteins called growth factors and small molecules in order to induce reprogramming of the cells into the primary embryonic germ layer known as endoderm. In the embryo, the endoderm is the inner-most layer of cells that forms the gastrointestinal tract and its associated structures (liver, pancreas, and so on). Therefore, the differentiation of adult cells into endodermal progenitor cells provides a handy way to form a cell type that readily divides and can differentiate into liver cells.

“We divert the cells on their path to pluripotency,” explained coauthor Holger Willenbring, associate professor of surgery at UCSF. “We still take advantage of what is intrinsic to reprogramming, that the cells are becoming very plastic; they’ve become flexible in what kind of cell type they can be directed towards.”

The authors called these cells induced multipotent progenitor cells (iMPCs). The iMPCs were easily differentiated into endodermal progenitor cells (iMPC-EPCs). These iMPC-EPCs were grown in culture with a cocktail of small molecules and growth factors to increase iMPC-EPC colony size while concomitantly maintain them in an endodermal state. Afterwards, Willenbring and others cultured these cells with factors and small molecules known to promote liver cell differentiation. When these iMPC-Hepatocytes (Heps) were transplanted into mice with damaged livers, the iMPC-Hep cells continued to divide at least nine months after transplantation. Furthermore, the transplanted cells matured and displayed gene expression profiles very similar to that of typical adult hepatocytes. Transplantation of iMPC-Heps also improved the survival of a mouse model of chronic liver failure about as well as did transplantation of adult hepatocytes.

“It is a breakthrough for us because it’s the first time that we’ve seen a cell that can actually repopulate a mouse’s liver,” said Willenbring. Willenbring strongly suspects that iMPCs are better able to repopulate the liver because the derivation of iMPC—rather than an iPSC—eliminates some steps along the path to generating hepatocytes. These iMPCs also possess the ability to proliferate in culture to generate sufficient quantities of cells for therapeutic purposes and, additionally, can functionally mature while retaining that proliferative ability to proliferate. Both of these features are important prerequisites for therapeutic applications, according to Willenbring.

Before this technique can enter clinical trials, more work must be done. For example: “The key to all of this is trying to generate cells that are identical to adult liver cells,” said Stephen Duncan, a professor of cell biology at Medical College of Wisconsin, who was not involved in the study. “You really need these cells to take on all of the functions of a normal liver cell.” Duncan explained that liver cells taken directly from a human adult might be able to repopulate the liver in this same mouse model at levels close to 90 percent.

Willenbring and his colleagues observed repopulation levels of 2 percent by iMPC-Heps, which is substantially better than the 0.05 percent repopulation typically accomplished by hepatocytes derived from iPSCs or embryonic stem cells. However: “As good as this is, the field will need greater levels of expansion,” said Ken Zaret of the Institute for Regenerative Medicine at the University of Pennsylvania, who did not participate in the work. “But the question is: What is limiting the proliferative capacity of the cells?”

Zaret explained that it is not yet clear whether some aspect of how the cells were programmed that differed from how they normally develop could have an impact on how well the population expands after transplantation. “There still is a ways to go [sic],” he said, “but [the authors] were able to show much better long-term repopulation with human cells in the mouse model than other groups have.”

See S. Zhu et al., “Mouse liver repopulation with hepatocytes generated from human fibroblasts,” Nature, doi:10.1038/nature13020, 2014.

Sweat Glands Are A Source of Stem Cells for Wound Healing


Stem Cells from human sweat glands serve as a remarkable source for wound healing treatments according to a laboratory in Lübeck, Germany.

Professor Charli Kruse, who serves as the head of the Fraunhofer Research Institute for Marine Biotechnology EMB, Lübeck, Germany, and his colleagues isolated cultured pancreatic cells in the course of their research to look into the function of a protein called Vigilin. When the pancreatic cells were grown in culture, they produced, in addition to other pancreatic cells, nerve and muscle cells. Thus the pancreas contains a stem cell population that can differentiate into different cell types.

Kruse and his group decided to investigate other glands contained a similar stem cell population that could differentiate into other cell types.

Kruse explained: “We worked our way outward from the internal organs until we got to the skin and the sweat glands. Again, this yielded the same result: a Petri dish full of stem cells.”

Up to this point, sweat glands have not received much attention from researchers. Mice and rats only have sweat glands on their paws, which makes them rather inaccessible. Human beings, on the other hand, have up to three million sweat glands, predominantly on the soles of out feet, palms of the hand, armpits, and forehead.

Ideally, a patient could have stem cells taken from her own body to heal an injury, wound, or burn, Getting to these endogenous stem cell populations, however, represents a challenge, since it requires bone marrow biopsies or aspirations, liposuction, or some other invasive procedure.

Sweat glands, however, are significantly easier to find, and a short inpatient visit to your dermatologist that extracts three millimeters of underarm skin could provide enough stem cells to grow in culture for treatments.

Stem cells from sweat glands have the capacity to aid wound healing. Kruse and his group used sweat gland-based stem cells in laboratory animals. The Kruse group used skin biopsies from human volunteers and separated out the sweat gland tissues under a dissecting scope. Then the sweat gland stem cells were grown in culture and induced to differentiate into a whole host of distinct cell types.

Then Kruse’s team grew these sweat gland stem cells in a skin-like substrate that were applied to wounds on the backs of laboratory animals. Those animals that had received stem cell applications healed faster than those that received no stem cells.

If the stem cells were applied to the mice with the artificial substrate, the cells moved into the bloodstream and migrated away from the site of the injury. In order to help heal the wound the cells had to integrate into the skin and participate in the healing process.

“Not only are stem cells from sweat glands easy to cultivate, they are extremely versatile, too,” said Kruse.

Kruse and his team are already in the process of testing a treatment for macular degeneration using sweat gland-based stem cells. “In the long-term, we could possibly set up a cell bank for young people to store stem cells from their own sweat glands/ They would then be available for use should the person need new cells, following an illness,l perhaps, or in the event of an accident,” Kruse said.

Human Stem Cells Converted into Functional Lung Cells


Scientists from the Columbia University Medical Center have succeeded in transforming human stem cells into functional lung and airway cells. This finding has significant potential for modeling lung disease, screening lung-specific drugs, and, hopefully, generating lung tissue for transplantation.

Study leader, Hans-Willem Snoeck, professor of medicine and affiliated with the Columbia Center for Translational Immunology and the Columbia Stem Cell Initiative, said, “Researchers have had relative success in turning human stem cells into heart cells, pancreatic beta cells, intestinal cells, liver cells, and nerve cells, raising all sorts of possibilities for regenerative medicine. Now, we are finally able to make lung and airway cells. This is important because lung transplants have a particularly poor prognosis. Although any clinical application is still many years away, we can begin thinking about making autologous lung transplants – that is, transplants that use a patient’s own skin cells to generate functional lung tissue.”

The research builds on Snoeck’s earlier discoveries in 2011 that a set of chemical factors could induce the differentiation of embryonic or induced pluripotent stem cells into “anterior foregut endoderm,” which is the embryo in the tissue from which the lungs form (Green MD, et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat Biotechnol. 2011 Mar;29(3):267-72).

Human Embryological Development - one month

In his new study, Snoeck and his colleagues found new factors that can transform anterior foregut endoderm cells into lung and airway cells. In particular, Snoeck and his co-workers were able to establish the presence of “type 2 alveolar epithelial cells,” which secrete the lung surfactant that maintains the lung alveoli (those tiny sacs in the lung where all the oxygen exchange takes place).

lung alveolus

With these techniques, lung researchers hope to study diseases like idiopathic pulmonary fibrosis (IPF), in which type 2 epithelial cells seem to divide and produce scarring in the lungs.

“No one knows what causes the disease, and there’s no way to treat it,” said Snoeck. “Using this technology, researchers will finally be able to create laboratory models of IPF, study the disease at the molecular level, and screen drugs for possible treatments or cures. In the longer term, we hope to use this technology to make an autologous lung graft. This would entail taking a lung from a donor, removing all the lung cells, leaving only the lung scaffold; and seeding the scaffold with new lung cells derived from the patient. In this way, rejection problems could be avoided.”

Snoeck is investigating this approach in collaboration with researchers in the Columbia University Department of Biomedical Engineering.

The Therapeutic Potential of Fat-Based Stem Cells Decreases With Age


Fat is a rich source of stem cells for regenerative medicine.  Treating someone with their own stem cells from their own fat certainly sounds like an attractive option.  However, a new study shows that demonstrates that the therapeutic value of fat-based stem cells declines when those cells come from older patients.

“This could restrict the effectiveness of autologous cell therapy using fat, or adipose-derived mesenchymal stromal cells (ADSCs), and require that we test cell material before use and develop ways to pretreat ADSCs from aged patients to enhance their therapeutic potential,” said Anastasia Efimenko, M.D., Ph.D.  Dr Efimenko and Nina Dzhoyashvili, M.D., were first authors of the study, which was led by Yelena Parfyonova, M.D., D.Sc., at Lomonosov Moscow State University, Moscow.

Heart disease remains the most common cause of death in most countries.  Mesenchymal stromal cells (MSCs) collected from either bone marrow or fat are considered one of the most promising therapeutic agents for regenerating damaged tissue because of their ability to proliferate in culture and differentiate into different cell types.  Even more importantly they also have the ability to stimulate the growth of new blood vessels (angiogenesis).

In particular, fat is considered an ideal source for MSCs because it is largely dispensable and the stem cells are easily accessible in large amounts with a minimally invasive procedure.  ADSCs have been used in several clinical trials looking at cell therapy for heart conditions, but most of the studies used stem cells from relatively healthy young donors rather than sick, older ones, which are the typical patients who suffer from heart disease.

“We knew that aging and disease itself may negatively affect MSC activities,” Dr. Dzhoyashvili said. “So the aim of our study was to investigate how patient age affects the properties of ADSCs, with special emphasis on their ability to stimulate angiogenesis.”

The Russian team analyzed age-associated changes in ADSCs collected from patients of different age groups, including some patients who suffered from coronary artery disease and some without.  The results showed that ADSCs from the older patients in both groups showed some of the characteristics of aging, including shorter telomeres (the caps on the ends of chromosomes that protect them from deterioration), which confirms that ADSCs do age.

“We showed that ADSCs from older patients both with and without coronary artery disease produced significantly less amounts of angiogenesis-stimulating factors compared with the younger patients in the study and their angiogenic capabilities lessened,” Dr. Efimenko concluded. “The results provide new insight into molecular mechanisms underlying the age-related decline of stem cells’ therapeutic potential.”

“These findings are significant because the successful development of cell therapies depends on a thorough understanding of how age may affect the regenerative potential of autologous cells,” said Anthony Atala, M.D., director of the Wake Forest Institute for Regenerative Medicine, and editor of STEM CELLS Translational Medicine, where this research was published.

Stem Cell Transplants for Non-Hodgkin’s Lymphoma


In patients with aggressive non-Hodgkin’s lymphoma, early stem cell transplants do not improve the overall survival in high-risk patients, but are beneficial in those patients who are at the highest risk.

Lymphomas are cancers of the lymphocytes, which are a specific group of white blood cells. A particular type of lymphoma known as Non-Hodgkin’s lymphoma is more common than the other general type of lymphoma — Hodgkin lymphoma. There are several different subtypes of non-Hodgkin’s lymphoma. The most common non-Hodgkin’s lymphoma subtypes include diffuse large B-cell lymphoma and follicular lymphoma.

The symptoms of non-Hodgkin’s lymphoma include Non-Hodgkin’s lymphoma symptoms may include: swollen lymph nodes in the neck, armpits or groin, swelling of the abdomen and abdominal pain, Chest pain, coughing or trouble breathing, fatigue (tiredness), fever, night sweats, and weight loss.

The usual treatment for aggressive non-Hodgkin’s lymphoma is a combination of four different chemotherapeutic agents designated as “CHOP,” which stands for Cyclophosphamide (alkylating agent that rituximabdamages DNA), Hydroxydaunorubicin (also called doxorubicin or Adriamycin, also a DNA-damaging agent), Oncovin (vincristine, which binds to microtubules and prevents cells from dividing duplicating by binding to the protein tubulin), and Prednisone or prednisolone (corticosteroids). Recently, many oncologists are adding Rituximab to this drug regimen (but only if the lymphoma is of B-cell origin). Rituximab is a monoclonal antibody that binds to the surface of B-lymphocytes (the very cells that have become cancerous) and facilitates their destruction. This new five-drug regimen, R-CHOP, can drive many patients into remission. However, some relapse and go on to receive stem cell transplants.

This present study, which was directed by Patrick Stiff from the Loyola University Medical Center’s Cardinal Bernardin Cancer Center, was designed to determine if an early stem cell transplant before the patient relapsed increase patient survival. This study examined patients from 40 different clinical sites in the United States and Canada.

397 patients who were in defined groups of high risk or intermediate-high risk of relapsing. After initial chemotherapy treatment, those patients who responded to treatment were randomly assigned to receive an autologous stem cell transplant (125 patients) or to a control group (128 patients) who received three additional cycles of the R-CHOP regimen.

After two years, 69 percent of the transplantation patients had no disease progression, compared with 55 percent of the control group. This is a statistically significant difference, but the two-year survival rates in the transplantation group was 74 percent versus 71 percent in the control group, which was not statistically significant. However, patients in the control group who relapsed were later offered stem cell transplants, which is probably why the differences are not statistically significant.

However, mining the data further reveals something even more interesting. While the stem cell transplants did not improve overall survival among the entire group of high-risk and high-intermediate risk patients, the high-risk patients as an isolated subset rather clearly received a remission and survival benefit from the early stem cell transplants. The two-year survival rate was 82 percent in the stem cell transplant group and 64 percent in the control group, which is statistically significant.

Patrick Stiff and his colleagues concluded: “Early transplantation and late transplantation achieve roughly equivalent overall survival in the combined risk groups.” However, “early transplantation appears to be beneficial for the small group of patients presenting with high-risk disease.”

Stiff hopes that this finding will “trigger discussions between such patients and their physicians as to the feasibility of doing early transplants.”

Patients who receives doses of their own stem cells (so-called autologous stem cell transplants), can tolerate very high doses of chemotherapy and/or radiation. This high-dose treatment kills off many cancer cells, but it also destroys the patient’s immune system. Therefore, prior to the treatment, stem cells are removed from the blood or bone marrow of he patient and infused back into the patient. These stem cells then form a new immune set of immune cells that replace the ones destroyed by the chemotherapy.

Previous studies have shown that patients who undergo autologous stem cell transplants have a higher risk of developing secondary cancers that are caused by the chemotherapy or the radiation. However, this new study did not find a statistically significant difference (11 percent in the control group and 12 percent in the stem cell transplant group) in secondary tumor formation between the two groups.

Stiff and his crew are continuing to crunch the numbers and mine the data. “As years go by, there may be additional analysis that may help fine-tune the results so that we will be able to more carefully and concisely define any potential benefit,” said Stiff.

See Patrick J. Stiff, et al, New England Journal of Medicine 2013; 369(18):1681.