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).

Accelerating Bone Regeneration with Combination Gene Therapy and Novel Scaffolds

A truly remarkable paper in the journal Advanced Healthcare Materials by Fergal J. O’Brien and his co-workers from the Tissue Engineering Research Group at the Royal College of Surgeons in Dublin, Ireland has examined a unique way to greatly speed up bone regeneration.

Mesenchymal stem cells from bone marrow (other locations as well) can differentiate into bone-making cells (osteoblasts) that will make architecturally normal bone under particular conditions. The use of mesenchymal stem cells and a variety of manufactured biomaterial matrices and administered growth factors enhance bone formation by mesenchymal stem cells (M. Noelle Knight and Kurt D. Hankenson, Adv Wound Care 2013; 2(6): 306–316; also see Marx RE, Harrell DB. Int J Oral Maxillofac Implants 2014 29(2)e201-9; and Kaigler D, et al., Cell Transplant 2013;22(5):767-77).

Protein growth factors tend to have rather short half-lives when applied to growth scaffolds. A better way to apply growth factors is to use the genes for these growth factors and apply them to “gene activated scaffolds.” Gene-activated scaffolds consist of biomaterial scaffolds modified to act as depots for gene delivery while simultaneously offering structural support and a matrix for new tissue deposition. A gene-activated scaffold can therefore induce the body’s own cells to steadily produce specific proteins providing a much more efficient alternative.

In this paper by O’Brien and his groups, the genes for two growth factors, VEGF and BMP2, were applied to a gene-activated scaffold that consisted of collagen-nanohydroxyapatite. VEGF drives the formation of new blood vessels, and this fresh vascularization, coupled with increase bone deposition, which is induced by BMP2, accelerated bone repair.

Mind you, the assays in the paper were conducted in cell culture systems. However, O’Brien and his colleagues implanted these gene-activated scaffolds with their mesenchymal stem cells into rats that had large gaps in their skulls. In this animal model system for bone repair, stem cell-mediated bone production, in addition to increased blood vessel formation accelerated bone repair in these animals. Tissue examinations of the newly-formed bone showed that bone made from gene-activated scaffolds with mesenchymal stem cells embedded in them made thicker, more vascularized bone than the other types of strategies.

This is not a clinical trial, but this preclinical trial shows that vascularization and bone repair by host cells is enhanced by the use of nanohydroxyapatite vectors to deliver a combination of genes, thus markedly enhancing bone healing.

Amniotic Fluid Stem Cells Aid Kidney Transplantation Success in a Pig Model

When a kidney patient receives a new kidney, the donated kidney undergoes a brief loss of blood supply followed by a restoration of the blood supply. This phenomenon is called ischemia/reperfusion (IR), and IR tends to cause cell death, followed by rather extensive scarring. Tissue scarring is called tissue fibrosis and a scarred kidney can lead to so-called transplant dysfunction, which means that the transplanted kidney does not work terrible well, and this can cause transplant failure.

Previous studies in laboratory rodents have shown that mesenchymal stem cells from amniotic fluid (afMSCs) are beneficial in protecting against transplant-induced fibrosis (Perin L, et al. PLoS One 2010;5:e9357; Hauser PV, et al. Am J Pathol 2010;177:2011-2021).

Now a research group at INSERM, France led by Thierry Hauet has developed a pig-based model of kidney autotransplantation that is comparable to the human situation with regards to the structure of the kidney and the damage that results from renal ischemia (for papers, see Jayle C, et al. Am J Physiol Renal Physiol 2007; 292: F1082-1093; and Rossard L, et al. Curr Mol Med 2012; 12: 502-505). On the strength of these previous experiments, Hauet’s group has published a new paper in Stem Cells Translational Medicine in which they report that porcine afMSCs can protect against IR-related kidney injuries in pigs.

Hauet and others showed that porcine afMSCs could be easily collected at birth and cultured. These cells showed the ability to differentiate into fat, and bone cells, made many of the same cell surface markers as other types of mesenchymal stem cells (e.g., CD90, CD73, CD44, and CD29), but showed a diminished ability to differentiate into blood vessel cells. When afMSCs are added to extirpated kidneys during the reperfusion (reoxygenation) process in an “in vitro” (fancy way of saying “in a culture dish”) model of organ-preservation, these stem cells significantly increased the survival of blood vessel (endothelial) cells. Endothelial cells are one of the main targets of ischemic injury, and the added cells bucked up these endothelial cells and rescued them from programmed cell death. In addition to these successes, Hauet and others showed that adding intact porcine afMSCs was not necessary, since addition of the culture medium used to grow the afMSCs (conditioned medium or CM) also rescued kidney endothelial cell death. The afMSC-treated kidneys survived because they had significantly larger numbers of blood vessels, and this seems to be the main factor that causes the extirpated kidney to survive intact.

While these experiments were successful, Hauet and others know that unless they were able to show that these cells improved kidney transplant outcomes in a living animal, their research would not be deemed clinically relevant. Therefore, Hauet and others injected afMSCs into the renal artery of pigs that had received a kidney transplant six days after the transplant. IR injuries following kidney transplants led to increased serum creatinine levels, but those pigs that had been infused with afMSCs showed reduced creatinine levels and lower protein levels in their urine (proteinuria). In fact, seven days after the stem cell infusion, the urine creatinine and protein levels had returned to pre-transplant levels. Three months after the transplant, the pigs were put down, and then the kidneys were subjected to tissue analyses. Microscopic examination of tissue slices from these kidneys showed that afMSC injection preserved the structural integrity of microscopic details of the kidneys and reduced the signs of inflammation. Control animals that were not treated with afMSCs showed disruption of the microscopic structures of the kidneys and extensive inflammation and scarring. Also, because the kidney controls blood chemistry, a comparison of the blood chemistries of these two groups of animals showed that the blood chemistries of the afMSC-treated animals were normal as opposed to the control animals.

Amniotic Fluid Stem Cells Aid Kidney Transplantation in Porcine Model

Molecular analyses also showed a whole host of pro-blood vessel molecules in the kidneys of the afMSC-treated pigs. VEGFA (pro-angiogenic growth factor), and Ang1 (capillary structure strengthening and maintenance of vessel stability), proteins were increased in the kidneys of afMSC animals compared to control animals. Thus the infused stem cells increased the expression of pro-blood vessel molecules, which led to the formation of larger quantities of blood vessels, reduced cell death and decreased inflammation.

These findings demonstrate the beneficial effects of infused afMSCs on kidney transplant. Since afMSCs are easy to isolate and grow in culture, secrete proangiogenic and growth factors, and can differentiate into many cell lineages, including renal cells (see Perin L, et al. Cell Prolif 2007; 40: 936-948; De Coppi P, et al. Nat Biotechnol 2007; 25: 100-106; and In ‘t Anker PS, et al. Stem Cells 2004;22:1338-1345). This makes these cells a viable candidate for clinical application. This study also highlights pigs as a preclinical model as a powerful tool in the assessment of stem cell-based therapies in organ transplantation.

Modified RNA Induces Vascular Regeneration After a Heart Attack

Regenerating the heart after a heart attack remains one of the Holy Grails of regenerative medicine. It is a daunting task. Even though text books may say, “the heart is just a pump,” this pump has a lot of tricks up its sleeve.

Stem cell treatments can certainly improve the structure and function of the heart after a heart attack, but getting the heart back to where it was before the heart attack is a whole different ball game. To truly regenerate, the heart, the organ or parts of it need to be reprogrammed to a time when the heart could regenerate itself. If that sounds difficult, it’s because it is. But some recent work suggests that it might at least partially possible.

Kenneth Chien and his colleagues from the Department of Stem Cell Biology and Regenerative Medicine at Harvard University have published a terrific paper in the journal Nature Biotechnology that tries to turn back to clock of the heart to augment its regenerative capabilities.

The outermost layer of the heart that surrounds the heart muscle is a layer called the “epicardium.”

epicardiumIn the epicardium are epicardial heart progenitors and these cells are activated within 48 hours after a heart attack in the mouse.  In the fetal heart, epicardial heart progenitors migrate into the heart and differentiate into heart muscle, blood vessels and smooth muscles.  In adults, these cells remain on the surface of the heart and differentiate largely into fibroblasts.  When it comes to regenerative medicine, can we take adult epicardial cells and reprogram them to act like fetal epicardial heart progenitors?

A few experiments have suggested that we can.  In 2011, Smart and others used a small peptide called thymosin β4 to reprogram epicardial cells in mice to form heart muscle and other heart-specific tissues.  Even though the reprogramming was not terribly robust, Smart and others convincingly showed that it was real (Nature 474,640–644).

The Chien group used modified RNA molecules made with unusual nucleotides that encoded the protein vascular endothelial growth factor-A (VEGF-A) to reprogram the epicardium of mice.  VEGF-A is very good and reprogramming the epicardium, and this modified RNA technique does not induce and immune response the way injecting DNA does and the RNA causes bursts of VEGF-A activity that efficiently reprograms the epicardium.

After giving mice heart attacks, Chien and others injected the VEGF-A modified RNAs into the border of the infarcted area of the heart. The modified RNAs induced new gene expression that is normally seen during the establishment of blood vessels.  VEGF-A expression was elevated for up to 6 days after the injections, and animals that had their hearts injected with modified VEGF-A RNA had smaller scars in their hearts, less cell death, and greater tissue volume in their hearts than animals that received either injections of VEGF-A DNA, buffer, or modified RNA that expressed a glowing protein.  Also, the effects of the modified VEGF-A RNA could be abrogated with co-administrating the drug Avastin, which is an antagonist of VEGF-A

Tests with cultured heart cells showed that VEGF-A modified RNA induced blood vessel-specific genes.  These inductions were sensitive to drugs that blocked the VEGF-A receptor, which shows that it is indeed the VEGF-A protein that is inducing these trends.  Finally, a heart muscle gene, Tnnt2 is also induced by the modified VEGF-A RNA.  When the efficacy of the modified VEGF-A RNA was tested in living animals, if was clear that the most numerous cells induced by the modified VEGF-A RNA was endothelial cells, which line blood vessels, followed by smooth muscle cells, and then by heart muscle cells.

Thus, the growth factor VEGF-A can signal to epicardial heart progenitor cells to heal the heart after a heart attack in mice.  It works through the VEGF-A receptor (KDR), and it induces epicardial derived cells (EPDCs) to differentiate into blood vessels, heart muscle cells, and smooth muscle cells, all of which are required to heal the heart.  If VEGF-A signaling can be used to augment heart healing after a heart attack, it might provide a new strategy for healing the heart after a heart attack in a manner that helps the heart heal itself from the inside rather than placing something from the outside into it.

New US Phase IIa Trial and Phase III Trial in Kazakhstan Examine CardioCell’s itMSC Therapy to Treat Heart Attack Patients

The regenerative medicine company CardioCell LLC has announced two new clinical trials in two different countries that utilize its allogeneic stem-cell therapy to treat subjects with acute myocardial infarction (AMI), which is a problem that faces more than 1.26 million Americans annually. The United States-based trial is a Phase IIa AMI clinical trial that is designed to evaluate the clinical safety and efficacy of the CardioCell Ischemia-Tolerant Mesenchymal Stem Cells or itMSCs. The second clinical trial in collaboration with the Ministry of Health in Kazakhstan is a Phase III AMI clinical trial on the intravenous administration of CardioCell’s itMSCs. This clinical trial is proceeding on the strength of the efficacy and safety of itMSCs showed in previous Phase II clinical trials.

CardioCell’s itMSCs are exclusively licensed from CardioCell’s parent company Stemedica Cell Technologies Inc. Normally, when mesenchymal stem cells from fat, bone marrow, or some other tissue source are grown in the laboratory, the cells are provided with normal concentrations of oxygen. However, CardioCell itMSCs are grown under low oxygen or hypoxic conditions. Such growth conditions more closely mimic the environment in which these stem cells normally live in the body. By growing these MSCs under these low-oxygen conditions, the cells become tolerant to low-oxygen conditions (ischemia-tolerant), and if transplanted into other low-oxygen environments, they will flourish rather than die.

Another advantage of itMSCs for regenerative treatments over other types of MSCs is that itMSCs secrete higher levels of growth factors that induce the formation of new blood vessels and promote tissue healing. These clinical trials have been designed to help determine if CardioCell’s itMSC-based therapies stimulate a regenerative response in acute heart attack patients.

“CardioCell’s new Phase IIa AMI study is built on the excellent safety data reported in previous Phase I clinical trials using our unique, hypoxically grown stem cells,” says Dr. Sergey Sikora, Ph.D., CardioCell’s president and CEO. “We are also pleased to report that the Ministry of Health in Kazakhstan is proceeding with a Phase III CardioCell-therapy study following its Phase II study that was highly promising in terms of efficacy and safety. Our studies target AMI patients who have depressed left ventricular ejection fraction (LVEF), which makes them prone to developing extensive scarring and therefore to the development of chronic heart failure. CardioCell hopes our itMSC therapies will inhibit the development of extensive scarring and, thus, the occurrence of chronic heart failure in these patients.”

The United States-based Phase IIa clinical trial will take place at Emory University, Sanford Health and Mercy Gilbert Medical Center. The CardioCell Phase IIa AMI trial is a double-blinded, multicenter, randomized study designed to assess the safety, tolerability and preliminary clinical efficacy of a single, intravenous dose of allogeneic mesenchymal bone-marrow cells infused into subjects with ST segment-elevation myocardial infarction (STEMI).

“While stem-cell therapy for cardiovascular disease is nothing new, CardioCell is bringing to the field a new, unique type of stem-cell technology that has the possibility of being more effective than other AMI treatments,” says MedStar Heart Institute’s Director of Translational and Vascular Biology Research and CardioCell’s Scientific Advisory Board Chair Dr. Stephen Epstein. “Evidence exists demonstrating that MSCs grown under hypoxic conditions express higher levels of molecules associated with angiogenesis and healing processes. There is also evidence indicating they migrate with greater avidity to various cytokines and growth factors and, most importantly, home more robustly to ischemic tissue. Studies like those underway using CardioCell’s technology are designed to determine if we can evoke a more potent healing response that will reduce the extent of myocardial cell death occurring during AMI and thereby decrease the amount of scar tissue resulting from the infarct. A therapy that could achieve this would have a major beneficial impact in reducing the occurrence of chronic heart failure.”

Kazakhstan’s National Scientific Medical Center is conducting a Phase III AMI clinical trial using CardioCell’s itMSCs, which are sponsored by local licensee Altaco. This clinical trial is entitled, “Intravenous Administration of itMSCs for AMI Patients,” and is proceeding based on a completed Phase II efficacy and safety study. However, the results of this previous Phase II study are preliminary because the sample group was so small. Despite these limitations, the findings demonstrated statistically significant elevation (more than 12 percent over the control group) in the ejection fraction of the left ventricle of the heart in patients who had received itMSCs. Also, a significant reduction in inflammation was also observed, as ascertained by lower CRP (C-reactive protein) levels in the blood of treated patients in comparison to control groups. Thus, Dr. Daniyar Jumaniyazov, M.D., Ph.D., principal investigator in Kazakhstan clinical trials states: “In our clinical Phase II trial for patients with AMI, treatment using itMSCs improved global and local myocardial function and normalized systolic and diastolic left ventricular filling, as compared to the control group. We are encouraged by these results and look forward to confirming them in a Phase III study.”

CardioCell’s treatment is the first to apply itMSC therapies for cardiovascular indications like AMI, chronic heart failure and peripheral artery disease. Manufactured by CardioCell’s parent company Stemedica and approved for use in clinical trials, itMSCs are manufactured under Stemedica’s patented, continuous-low-oxygen conditions and proprietary media, which provide itMSCs’ unique benefits: increased potency, safety and scalability. itMSCs differ from competing MSCs in two key areas. itMSCs demonstrate increased migratory ability towards the place of injury, and they show increased secretion of growth and transcription factors (e.g., VEGF, FGF and HIF-1), as demonstrated in a peer-reviewed publication (Vertelov et al., 2013). This can potentially lead to improved regenerative abilities of itMSCs. In addition, itMSCs have significantly fewer HLA-DR receptors on the cell surface than normal MSCs, which might reduce the propensity to cause immune responses. As another benefit, itMSCs are highly scalable. A single donor specimen can currently yield about 1 million patient treatments, and this number is expected to grow to 10 million once full robotization of Stemedica’s facility is complete.

Nanotubules Link Damaged Heart Cells With Mesenchymal Stem Cells to Both of Their Benefit

Mesenchymal stem cells are found throughout the body in bone marrow, fat, tendons, muscle, skin, umbilical cord, and many other tissues. These cells have the capacity to readily differentiate into bone, fat, and cartilage, and can also form smooth muscles under particular conditions.

Several animal studies and clinical trials have demonstrated that mesenchymal stem cells can help heal the heart after a heart attack. Mesenchymal stem cells (MSCs) tend to help the heart by secreting a variety of particular molecules that stimulate heart muscle survival, proliferation, and healing.

Given these mechanisms of healing, is there a better way to get these healing molecules to the heart muscle cells?

A research group from INSERM in Creteil, France has examined the use of tunneling nanotubes to connect MSCs with heart muscle cells. These experiments have revealed something remarkable about MSCs.

Florence Figeac and her colleagues in the laboratory of Ann-Marie Rodriguez used a culture system that grew fat-derived MSCs and with mouse heart muscle cells. They induced damage in the heart muscle cells and then used tunneling nanotubes to connect the fat-based MSCs.

They discovered two things. First of all, the MSCs secreted a variety of healing molecules regardless of their culture situation. However, when the MSCs were co-cultured with damaged heart muscle cells with tunneling nanotubes, the secretion of healing molecules increased. The tunneling nanotubes somehow passed signals from the damaged heart muscle cells to the MSCs and these signals jacked up secretion of healing molecules by the MSCs.

The authors referred to this as “crosstalk” between the fat-derived MSCs and heart muscle cells through the tunneling nanotubes and it altered the secretion of heart protective soluble factors (e.g., VEGF, HGF, SDF-1α, and MCP-3). The increased secretion of these molecules also maximized the ability of these stem cells to promote the growth and formation of new blood vessels and recruit bone marrow stem cells.

After these experiments in cell culture, Figeac and her colleagues used these cells in a living animal. They discovered that the fat-based MSCs did a better job at healing the heart if they were previously co-cultured with heart muscle cells.

Exposure of the MSCs to damaged heart muscle cells jacked up the expression of healing molecules, and therefore, these previous exposures made these MSCs better at healing hearts in comparison to naive MSCs that were not previously exposed to damaged heart muscle.

Thus, these experiments show that crosstalk between MSCs and heart muscle cells, mediated by nanotubes, can optimize heart-based stem cells therapies.

New Liver Stem Cell Might Aid in Liver Regeneration

For patients with end-stage liver disease, a liver transplant is the only viable option to stave off death. Liver failure is the 12th leading cause of death in the United States, and finding a way to regenerate failing livers is one of the Holy Grails of liver research. New research suggests that one it will be feasible to use a patient’s own cells to regenerate their liver.

Researchers at the Icahn School of Medicine at Mount Sinai have discovered that a particular human embryonic stem cell line can be differentiated into a previously unknown liver progenitor cell that can differentiate into mature liver cells.

“The discovery of the novel progenitor represents a fundamental advance in this field and potentially to the liver regeneration field using cell therapy,” said Valerie Gouon-Evans, the senior author of this study and assistant professor of medicine at the Icahn School of Medicine. “Until now, liver transplantation has been the most successful treatment for people with liver failure, but we have a drastic shortage of organs. This discovery may help circumvent that problem.”

Gouon-Evans collaborated with the laboratory of Matthew J. Evans and showed that the liver cells that were made from the differentiating liver progenitor cells could be infected with hepatitis C virus. Since this is a property that is exclusive to liver cells, this result shows that these are bona fide liver cells that are formed from the progenitor cells.

One critical step in this study was the identification of a new cell surface protein called KDR, which is the vascular endothelial growth factor 2. KDR was thought to be restricted to blood vessels, blood vessels progenitor cells (EPCs), and blood cells.  However, the Evans / Gouon-Evans study showed that activation of KDR in liver progenitor cells caused them to differentiate into mature liver cells (hepatocytes).  KDR is one of the two receptors for VEGF or vascular endothelial growth factor.  Mutations of this gene are implicated in infantile capillary hemangiomas.

KDR Protein Crystal Structure
KDR Protein Crystal Structure

The next step in this work is to determine if liver cells formed from these embryonic stem cells could potentially facilitate the repair of injured livers in animal models of liver disease.