Mesenchymal Stem Cell Transplantation Improves Atherosclerotic Lesions

Several animal studies have shown that transplantation of mesenchymal stem cells from several different sources is beneficial in myocardial infarction and hind limb ischemic. However, can these cells improved atherosclerosis, otherwise known as hardening of the arteries?

Shih-Chieh Hung and colleagues from National Yang-Ming University in Taipei, Taiwan tested this very hypothesis.

Hung and others used to lines of experimentation to address this question. First, they used cultured endothelial cells that had been treated with oxidized low-density lipoprotein particles. Secondly, they fed mice mutant for ApoE-deficient a high-fat diet.  ApoE-deficient humans and mice develop atherosclerotic plaques rather quickly.

In the cultured endothelial cells, oxidized LDL turned off the production of nitric oxide (NO). NO is a signaling molecule produced by several cell types, but in particular, endothelial cells use NO to dilate blood vessels. NO also is a good signal of endothelial health. Therefore, when oxidized LDL causes cultured endothelial to decrease NO production, it is affecting endothelial cell health. However, when cultured endothelial cells that had been treated with oxidized LDL were cocultured with mesenchymal stem cells, NO production and the enzymes that synthesize NO increased precipitously. Thus in a cultured system, MSCs have the ability to prevent the deleterious of oxidized LDL.

In ApoE-deficient mice fed a high fat diet, the arteries of the mice showed extensive plaque formation. However, if these animals were implanted with bone-marrow-derived mesenchymal stem cells, plaque formation was greatly decreased. Further work showed that a protein secreted by mesenchymal stem cells called macrophage inflammatory protein-2 (MIP-2) was responsible for these ameliorative effects. If MIP-2 was applied without mesenchymal stem cells, plaque formation was limited, and if antibodies that neutralize MIP-2 were co-administered with mesenchymal stem cells, the cells failed to reduced plaque formation.

Thus, this interesting study shows that transplantation of mesenchymal stem cells can limit plaque formation in atherosclerotic animals and they do this through secretion of MIP-2. Secondly, mesenchymal stem cells can improve the health of endothelial cells, which are the cells that form the inner layer of blood vessels, which are so adversely affected by atherosclerosis. By utilizing the encore of proteins secreted by mesenchymal stem cells, scientists should be able to develop a cocktail of proteins that can ameliorate atherosclerosis in human patients.

Differentiation of Induced Pluripotent Stem Cells Decreases Immune Response Against Them

The goal of regenerative medicine is to replace dead or damaged cells, tissues and even organs with living, properly functioning cells tissues and organs. However, this goal has a few genuine barriers that include tumor formation in the case of pluripotent stem cells, poor cell survival, or even immunological rejection of the transplanted cells before they can render any long-term benefits. Induced pluripotent stem cells (iPSCs), which are made from adult cells by a combination of genetic engineering and cell culture techniques, can be made from a patient’s own mature cells and the differentiated into almost any tissue in the adult body. However, research with mouse iPSCs has shown that even stem cells produced from the subject’s own tissues can be rejected by the subject’s own immune system.

Immune rejection of iPSCs is a legitimate concern, but research from the Stanford University School of Medicine has shown that differentiation of iPSCs into more mature cells before transplantation into mice allows them to be tolerated by the immune system.

Joseph Wu, MD, PhD, director of the Stanford Cardiovascular Institute, said, “Induced pluripotent stem cells have tremendous potential as a source for personalized cellular therapeutics for organ repair. This study shows that undifferentiated iPS cells are rejected by the immune system upon transplantation in the same recipient, but that fully differentiating these cells allows for acceptance and tolerance by the immune system without the need for immunosuppression.”

Wu is the senior author of this publication, which appeared online on May 30th in Nature Communications. Lead authorship of this paper is shared by Patricia Almeida, PhD, and Nigel Kooreman, MD, and assistant professor of medicine Everett Meyer, MD, PhD.

Several other studies have suggested that differentiation of iPSCs can reduce their tendency to activate the immune system after transplantation. However, this study of Wu and others is the first to closely examine, at the molecular and cellular level, how this works.

“We’ve demonstrated definitively that, once the cells are differentiated, the immune response to iPS-derived cells is indistinguishable from its response to unmodified tissue derived from elsewhere in the body,” said lead author Nigel Kooreman.

Pluripotent stem cells have the capacity to differentiate into any cell in the adult body. Of the two types of pluripotent stem cells, embryonic stem cells are made from embryos and iPSCs are made in the laboratory from existing adult cells (e.g., skin or blood). Induced pluripotent stem cells are easier to come by than embryonic stem cells, they match the genetic background of the person from whom they were obtained, and they are not as ethically dubious as embryonic stem cells. Thus, in theory, iPSCs are a good option for any physician who wants to make patient-specific stem cells for potential therapies.

Previous studies in mice have shown, however, that even genetically identicaliPSCs can trigger an immune response after transplantation. Thus, Wu and his colleagues have, for the past six years, been investigating how to use immunosuppressive medications to dampen the body’s response to both embryonic andiPSCs and render them more amenable for clinical use (see AS Lee, et al., J Biol Chem 2011 286(37):32697-704; Durruthy-Durruthy L, et al.,PLoS One, 2014 9(4):e94231 and others).

In this recent study, Kooreman and his co-lead authors decided to examine the immune response against transplanted stem cells. They first transplanted undifferentiated iPS cells into the leg muscles of genetically identical recipient mice. These grafts were rejected and no iPSCs were detected six weeks after transplantation.

Next, Wu and his co-workers differentiated the iPSCs into blood vessel-making endothelial cells that line the interior of the heart and blood vessels and then transplanted them into genetically-identical mice. Kooreman, Almeida, and Meyer then compared the acceptance by the immune system of these iPSC-derived endothelial cells with that of naturally occurring endothelial cells derived from the aortic lining of genetically-identical donor mice. To emphasize once again, all the transplanted cells were genetically identical to the mice in which they were injected. Unlike the undifferentiated iPS cells, both the iPS-derived endothelial cells and the aortic endothelial cells survived for at least nine weeks after transplantation.

Next, Wu and his group repeated the experiment, but they removed the grafts 15 days after transplantation. They observed immune cells called lymphocytes in all grafts, but these immune cells were much more prevalent in the grafts of undifferentiated iPS cells. When the lymphocytes that infiltrated the grafts of undifferentiated iPSCs were compared with those in the differentiated iPSC-derived grafts and the endothelial grafts, their gene expression profiles differed significantly. Those lymphocytes in the undifferentiated iPSC grafts expressed high levels of genes known to be involved in robust immune responses, but lymphocytes in both types of endothelial cell grafts expressed higher levels of genes known to be involved in dampening the immune response and inducing self-tolerance.

Finally, Wu and others directly examined a specific type of lymphocyte called a T cell. Grafts of undifferentiated iPS cells harbored large numbers of T cells that were largely homogeneous, which is characteristic of a robust immune response. Conversely, T cell from grafts of the two types of endothelial cells were more diverse, which suggests a more limited immune response which is typically associated with a phenomenon known as self-tolerance.

“The immune response to the iPS-derived endothelial cells and the aortic endothelial cells, and the longevity of the grafts, was very similar,” said Kooreman. “If we specifically look at the T cells, we see they’re also very similar and that they look much different from grafts that are rejected.”

Wu, who is also a professor of cardiovascular medicine and of radiology, said, “This study certainly makes us optimistic that differentiation — into any nonpluripotent cell type — will render iPS cells less recognizable to the immune system. We have more confidence that we can move toward clinical use of these cells in humans with less concern than we’ve previously had.”

Making Heart Muscle from Skeletal Muscle Stem Cells

Several experiments in animals and a few clinical trials in human patients have shown that implanting skeletal muscle cells isolated from muscle biopsies into the heart after a heart attack can help the heart to some degree, but the implanted skeletal muscle cells do not integrate into the existing heart muscle mass and the skeletal muscle cells do not differentiate into heart muscle cells.

Experiments like those mentioned above utilized muscle satellite cells. Muscle satellite cells are a resident stem cell population that respond to muscle damage and divide to form skeletal muscle cells form new muscle. Satellite cells are a perfect example of a unipotent stem cell, which is to say a cell that makes one type of terminally differentiated cell type.

Skeletal muscles, however, have another cell population called muscle-derived stem cells or MDSCs. MDSCs express an entirely different set of cell surface proteins than satellite cells, and have the capacity to differentiate into skeletal muscle, smooth muscle, bone, tendon, nerve, endothelial and hematopoietic cells. MDSCs grow well in culture, tolerate low oxygen conditions quite well, and show excellent regenerative potential.

Other laboratories have managed to culture MDSCs in collagen and produce beating heart muscle cells. Others have observed MDSCs forming a proper myocardium under certain conditions. Several studies have established the ability to MDSCs to treat laboratory animals that have suffered a heart attack. The most recent work from Sekiya and others has established that cell sheets made from MDSCs can reduce dilation of the left ventricle, increased capillary density, and promoted recovery without causing erratic heat beat patterns.

Despite their obvious efficacy. MDSCs remain difficult to isolate in high enough numbers to therapeutic purposes. None of the cell surface molecules sported by MDSCs are unique to those cells. Therefore, getting clean cultures of MDSCs remains a challenge. Still, these cells represent some of the best hopes for regenerative medicine in the heart. These cells do form heart muscle cells and heal ailing hearts. They can be grown in bioreactors to high numbers and can also be combined with engineered materials to shore up a damaged heart and mediate its regeneration. While the use of MDSCs is still in its infancy, the promise certainly is there.

Reversing Lung Diseases By Directing Stem Cell Differentiation

Lung diseases can scar the respiratory tissues necessary for oxygen exchange. Without proper oxygen exchange, our cells lack the means to make the energy they so desperately need, and they begin to shut down or even die. Lung diseases such as asthma, emphysema, chronic obstructive pulmonary disease and others can permanently diminish lung capacity, life expectancy and activity levels.

Fortunately, a preclinical study in laboratory animals has suggested a new strategy for treating lung diseases. Carla Kim and Joo-Hyeon Lee of the Stem Cell Research Program at Boston Children’s have described a new lung-specific pathway that is activated by lung injury and directs a resident stem cell population in the lung to proliferate and differentiate into lung-specific cell types.

When Kim and Lee enhanced this pathway in mice, they observed increase production of the cells that line the alveolar sacs where gas exchange occurs. Alveolar cells are irreversibly damaged in emphysema and pulmonary fibrosis.

Inhibition of this same pathway increased stem cell-mediated production of airway epithelial cells, which line the passages that conduct air to the alveolar sacs and are damaged in asthma and bronchiolitis obliterans.

For their experiments, Kim and Lee used a novel culture system called a 3D culture system that mimics the milieu of the lung. This culture system showed that a single bronchioalveolar stem cell could differentiate into both alveolar and bronchiolar epithelial cells. By adding a protein called TSP-1 (thrombospondin-1), the stem cells differentiated into alveolar cells.

Next, Kim and Lee utilized a mouse model of pulmonary fibrosis. However, when they cultured the small endothelial cells that line the many small blood vessels in the lung, which naturally produce TSP-1, and directly injected the culture fluid of these cells into the mice, the noticed these injections reverse the lung damage.

When they used lung endothelial cells that do not produce TSP-1 in 3D cultures, lung-specific stem cells produce more airway cells. in mice that were engineered to not express TSP-1, airway repair was enhanced after lung injury.

Lung Stem Cell Repair of Lung Damage

Lee explained his results in this way: “When the lung cells are injured, there seems to be a cross talk between the damaged cells, the lung endothelial cells and the stem cells.”

Kim added: “We think that lung endothelial cells produce a lot of repair factors besides TSP-1. We want to find all these molecules, which could provide additional therapeutic targets.”

Even though this work is preclinical in nature, it represents a remarkable way to address the lung damage that debilitates so many people. Hopefully this work is easily translatable to human patients and clinical trials will be in the future. Before that, more confirmation of the role of TSP-1 is required.

Adult Stem Cells Help Build Human Blood Vessels in Engineered Tissues

University of Illinois researchers have identified a protein expressed by human bone marrow stem cells that guides and stimulates the construction of blood vessels.

Jalees Rehman, associate professor of cardiology and pharmacology at the University of Illinois at Chicago College of Medicine and lead author of this paper, said: “Some stem cells actually have multiple jobs.”

As an example, stem cells from bone marrow known as mesenchymal stem cells can form bone, cartilage, or fat, but they also have a secondary role in that they support other cells in bone marrow.

Rehman and others have worked on developing engineered tissues for use in cardiac patients, and they noticed that mesenchymal stem cells were crucial for organizing other cells into functional stem cells.

Workers from Rehman’s laboratory mixed mesenchymal stem cells from human bone marrow with endothelial cells that line the inside of blood vessels. The mesenchymal stem cells elongated and formed a kind of scaffold upon which the endothelial cells adhered and organized to form tubes.

“But without the stem cells, the endothelial cells just sat there,” said Rehman.

When the cell mixtures were implanted into mice, blood vessels formed that were able to support the flow of blood. Then Rehman and his colleagues examined the genes expressed when their stem cells and endothelial cells were combined. They were aided in this venture by two different bone marrow stem cell lines, one of which supported the formation of blood vessels, and the other of which did.

Their microarray experiments showed that the vessel-supporting mesenchymal stem cells expressed high levels of the SLIT3 protein. SLIT3 is a blood vessel-guidance protein that directs blood vessel-making cells to particular places and induces them to make blood vessels. The cell line that do not stimulate blood vessel production made little to no SLIT3.

Rehman commented, “This means that not all stem cells are created alike in terms of their SLIT3 production and their ability to encourage blood vessel formation.”

Rehman continued: “While using a person’s own stem cells for making blood vessels is ideal because it eliminates the problem of immune rejection, it might be a good idea to test a patient’s stem cells to make sure they are good producers of SLIT3. If they aren’t, the engineered vessels may not thrive or even fail to grow.

Mesenchymal stem cells injections are being evaluated in clinical trials to see if their can help grow blood vessels and improve heart function in patients who have suffered heart attacks.

So far, the benefits of stem cell injection have been modest, according to Rehman. Evaluating the gene and protein signatures of stem cells from each patient may allow for a more individualized approach so that every patient receives mesenchymal stem cells that are most likely to promote blood vessel growth and cardiac repair. Such pre-testing might substantially improve the efficacy of stem cell treatments for heart patients.

Repairing Damaged Organs with New Blood Vessel-Making Stem Cells

A damaged organ usually needs to be removed (spleen or single kidney) or a new organ must be transplanted to replace the damaged organ (liver, heart, lungs, kidney). Wouldn’t it be terrific to inject blood vessel-making stem cells and let the organ heal itself? Such a strategy would render organ transplantation obsolete.

Studies by scientists at the Weill Cornell Medical College in New York have shown that endothelial cells – the cells that line the inside of blood vessels – can drive the regeneration of organ by releasing beneficial, organ-specific molecules. These organ-specific molecules were identified in a genome-wide screen that uncovered all the genes actively expressed in endothelial cells. Many of these genes found in this screen were previously not known to be expressed in endothelial cells. Researchers also found that organ dictate the structure and function of their own blood vessels and this includes the organ-specific repair molecules they elicit from endothelial cells.


Shahin Rafii, principal investigator of this work, is a professor of genetic medicine and co-director of the medical college’s Ansary Stem Cell Institute and the Tri-SCI Stem Center. Rafii is also a Howard Hughes Medical Institute investigator.

According to Rafii, when an organ in injured, its blood vessels may not have the ability to repair the organ on their own because of the damage to the blood vessels themselves, and the inflammation these same blood vessels might be experiencing.

“Our work suggests that an infusion of engineered endothelial cells could engraft into the injured tissue and acquire the capacity to repair the organ. These studies – along with the first molecule atlas of organ-specific blood vessel cells reported in the Developmental Cell paper (Developmental Cell 26, 204–219, July 29, 2013) – will open up a whole new chapter in translational vascular medicine and will have a major therapeutic application.”

Rafii continued: “Scientists had thought blood vessels in each organ are the same, that they exist to deliver oxygen and nutrients. But they are very different.” According to Rafii, different organs are endowed with blood vessels with unique shape and function and delegated with the difficult task of complying with the metabolic demands of that organ.

In one study from the Rafii lab, nine different tissues were examined, in addition to bone marrow and liver that had undergone a traumatic injury. To examine the blood vessels from each of these tissues, Rafii’s laboratory development a very efficient way to make endothelial cells from embryonic stem cells. Daniel Nolan, the lead author of this work, said that this protocol produced a “a pure population of endothelial cells in a very rapid time frame.”

ECs Derived from hESCs Phenocopy Adult Mouse Tissue-Specific Capillaries (A) Schema of in vitro conditions to support the differentiation and identification of hESC-derived vasculature. hESCs are grown on an E4-ORF1 EC feeder layer and transduced with a VE-Cadherin-Orange reporter gene. VE-Cadherin-Orange+ vascular networks are readily identifiable by day 10. (B) Flow cytometry data depicting the expression of VPR-Orange on hESC-derived CD31+ ECs. These VPR+ ECs have distinct populations based on the expression of either CXCR4 (teal) or CD133 (purple). (C) VPR+CXCR4+CD133− and VPR+CD133+CXCR4− ECs are capable of forming distinct clusters of ECs in hESC cultures. (D) Heat maps of the genes, which were common in their statistically significant differential expression (Benjamini-Hochberg adjusted p < 0.05) between hESC-derived vasculature and adult mouse heart and brain tissues. (E) VPR+CXCR4+CD133− and VPR+CD133+CXCR4− ECs were analyzed for cKit and CD36 levels via flow cytometry. Validation of the higher expression of CD36 and Kit in the CXCR4+ ECs is shown. (F) Heat map of K-Mean clusters depicting the results of de novo motif discovery among non-ECs, CXCR4+VPR+ ECs, and CD133+VPR+ ECs. Candidate binding partners to the motifs are listed.
ECs Derived from hESCs Phenocopy Adult Mouse Tissue-Specific Capillaries.  (A) Schema of in vitro conditions to support the differentiation and identification of hESC-derived vasculature. hESCs are grown on an E4-ORF1 EC feeder layer and transduced with a VE-Cadherin-Orange reporter gene. VE-Cadherin-Orange+ vascular networks are readily identifiable by day 10.  (B) Flow cytometry data depicting the expression of VPR-Orange on hESC-derived CD31+ ECs. These VPR+ ECs have distinct populations based on the expression of either CXCR4 (teal) or CD133 (purple).  (C) VPR+CXCR4+CD133− and VPR+CD133+CXCR4− ECs are capable of forming distinct clusters of ECs in hESC cultures.  (D) Heat maps of the genes, which were common in their statistically significant differential expression (Benjamini-Hochberg adjusted p < 0.05) between hESC-derived vasculature and adult mouse heart and brain tissues.  (E) VPR+CXCR4+CD133− and VPR+CD133+CXCR4− ECs were analyzed for cKit and CD36 levels via flow cytometry. Validation of the higher expression of CD36 and Kit in the CXCR4+ ECs is shown.  (F) Heat map of K-Mean clusters depicting the results of de novo motif discovery among non-ECs, CXCR4+VPR+ ECs, and CD133+VPR+ ECs. Candidate binding partners to the motifs are listed.

From these laboratory-made endothelial cells (ECs), Rafii and his colleagues were able to take snapshots of all the genes expressed in various populations of ECs can compose the different vascular beds of the body. From these studies, Raffi and others discovered that ECs possess specific genes that code for unique growth factors, adhesion molecules, and factors regulating metabolism.

“We knew that these gene products were critical to the health of a particular tissue, but before our study it was not appreciated that these factors originate in the endothelial cells,” said Nolan.

Olivier Elemento, who performed much of the complex computational studies in this paper, noted, “We also found that the healing, or regeneration of tissue, in the liver and in the bone marrow were unexpectedly different – including the repair molecules, known as angiocrine growth factors, that were expressed by the endothelial cells.”

Blood vessels differ among the various organs because the ECs have to constantly adapt to the metabolic, biomechanical, inflammatory, and immunological needs of that particular organ, said Michael Ginsberg, a senior postdoctoral research associate in Rafii’s lab. “And we have now found how endothelial cells have learned to behave differently in each organ and to adjust to the needs of those organs,” he said.

This work from Raffii’s laboratory raises the question as to how ECs have the capacity to adapt to the biological demands of each organ. Is it possible to design “immature” ECs that could allow scientists to identify the means by which particular microenvironmental cues educate these cells to become more specialized endothelial cells?

To address this question, Rafii and his army of graduate students, postdoctoral researchers, technicians, and visiting scientists made ECs from mouse embryonic stem cells and discovered that these cells were responsive to microenvironmental cues, and were also transplantable and functional.

Sina Rabbany, adjunct associate professor of genetic medicine and bioengineering at Weill Cornell Medical College said that embryonic stem cell derived ECs are “very versatile, so they can be transplanted into different tissues, become educated by the tissue, and acquire the characteristics of the native endothelial cells.” These ECs can also be grown in the lab into large numbers.

“We now know what it takes to keep these cells healthy, stable, and viable for transplantation,” said Rabbany.

When the ECs made by Rabbany were transplanted into the livers of laboratory mice, they integrated into the host tissue and become indistinguishable from the native tissue. Similar results were observed when these laboratory-derived ECs were transplanted into kidneys.

The Use of Synthetic Messenger RNAs Augment Heart Regeneration and Healing After a Heart Attack

A collaborative effect between researchers at Harvard University and Karolinska Institutet has shown that the application of particular factors to the heart after a heart attack can heal the heart and induce the production of new heart muscle.

Kenneth Chien, who has a dual appointment at the medical university Karolinska Institutet and Harvard University, led this research teams said this about this work: “This is the beginning of using the heart as a factory to produce growth factors for specific families of cardiovascular stem cells, and suggests that it may be possible to generate new heart parts without delivering any new cells to the heart itself.”

This study builds upon previous work by Chien and his colleagues in which the growth factor VEGFA, which is known to activate the growth of endothelial cells in the adult heart (endothelial cells line blood vessels), also serves as a switch that converts heart stem cells away from making heart muscle to forming coronary vessels in the fetal heart.

To drive the expression of VEGFA in the heart, Chien and others made synthetic messenger RNAs that encoded VEGFA and injected them into the heart cells. Injections of these synthetic VEGFA messenger RNAs produced a short burst of VEGFA.

Chien induced a heart attack in mice and then administered the synthetic VEGFA messenger RNAs to some mice and buffer to others 48 hours after the heart attacks. Chien and his crew was sure to inject the synthetic VEGFA mRNAs into the regions of the heart known to harbor the resident cardiac stem cell populations.

Not only did the VEGFA-mRNA-injected mice survive better than the other mice, but their hearts had smaller heart scars, and had clear signs of the growth of new heart muscle that had been made by the resident cardiac stem cell populations. One pulse of VEGFA had long-term benefits and those cells that would have normally made the heart scar ended up making heart muscle instead as a result of one pulse of VEGFA.

Chien said of this experiment, “This moves us very close to clinical studies to regenerate cardiovascular tissue with a single chemical agent without the need for injecting any additional cells into the heart.”

At the same time, Chien also noted that this technology is in the early stages of development. Even though these mice had their chests cracked open and their hearts injected, for human patients, the challenge is to adapt heart catheter technologies to the delivery of synthetic messenger RNAs. Also, to demonstrate the safety and efficacy of this technology to humans, Chien and others will need to repeat these experiments in larger animals that serve as a better model system for the human heart than rodents. Chien’s laboratory is presently in the process of doing that.

To adapt catheter technology to deliver these reagents, Chien had co-founded a company called Moderna Therapeutics to research this problem and develop the proper platform technology for clinical use. Chien is also collaborating with the biotechnology company AstraZeneca to help expedite moving the synthetic RNA technology into a clinical setting.