Induced Pluripotent Stem Cell-Derived Kidney Progenitor Cells Heal Kidneys in Laboratory Animals

The kidney is a crucial organ for human survival and human flourishing. This organ filters metabolic wastes from the blood and if the kidney does not work, the body slowly poisons itself.

When the kidneys fail to work properly, they must be replaced by transplantation of a tissue-matched kidney from a donor. However, if the kidney is not completely damaged, then it might be possible to heal it by means of cell therapies. For example, if we could transplant renal progenitor cells into the kidney that then differentiate into kidney-specific tissues, then we could potentially replace damaged tissues in the kidney and help the kidney fully recover. The tough part of such a treatment strategy has been acquiring a sufficient number of kidney progenitor cells. However, scientists have considered using induced pluripotent stem cells (iPSCs), since these cells can be expanded in culture to very high numbers of cells that can be effectively differentiated into kidney progenitors.

Induced pluripotent stem cells are made from mature, adult cells by means of a combination of genetic engineering and cell culture techniques. These cells have the potency to differentiate into any cell type in the human body. Ideally, renal progenitors could be transplanted directly into the kidney parenchyma, but, again, this is not a simple-to-solve problem. “The kidney is a very solid organ, which makes it very difficult to bring enough number of cells upon transplantation,” explains Professor Kenji Osafune. Dr. Osafune’s laboratory is at the Center for iPS Cell Research and Application (CiRA) at Kyoto University, Japan, and is using iPSCs to investigate new treatments for kidney disease. Several studies have successfully transplanted adequate numbers of kidney progenitors to treat kidney disease.

In a new study, Dr. Osafune has collaborated with Astellas Pharma Inc., in order to potentially design a solution that can solve the problem of treating the kidney with exogenous cells. In this study, Osafune and his colleagues tried a different way to deliver the kidney progenitor cells. Instead of injecting cells directly into the kidney, they transplanted their iPSC-derived renal progenitors into the kidney subcapsule that is at the kidney surface.

Kidney Capsule

The mice that received the cells were suffering from acute kidney injury. Even though the transplanted cells never integrated with the host, mice that received this transplant showed better recovery, including less cell death (necrosis) and scarring (fibrosis) compared with mice that received transplants of other cell types.

Damaged kidney tissue (left) of an AKI model mouse shows high levels of fibrosis (blue). Treatment with Osr1+Six2+ cell therapy significantly ameliorates the fibrosis (right) of another AKI model mouse.
Damaged kidney tissue (left) of an AKI model mouse shows high levels of fibrosis (blue). Treatment with Osr1+Six2+ cell therapy significantly ameliorates the fibrosis (right) of another AKI model mouse.

Osafune attributed the improvement in his laboratory mice to the use of cells that expressed the Osr1 and Six2 genes. The Osr1 and Six2 proteins are known markers of renal progenitor cells, but until this particular study, researchers had not exclusively used cells that expressed both of these proteins for cell therapies.

Kidney Progenitor cells

Another conclusion from the study was that because the cells did not integrate into the kidney, their therapeutic effects were the result of secreted proteins that promoted kidney healing and protection. While most stem cell therapies aim for integration of the transplanted cells, the results of these experiments could have important clinical implications. In particular, this experiment is one of the first to show the benefits of using human iPS cell-derived renal lineage cells for cell therapy. Secondly, scarring of the kidney is a marker that indicated progression of the kidney to chronic kidney disease. Since scarring was significantly reduced in these experiments, these data suggest that the paracrine effects of the transplanted cells could act as preventative therapy for other serious ailments. Finally, Osafune believes these effects could provide valuable clues for drug discovery. “There is no medication for acute kidney injury. If we can identify the paracrine factor, maybe it will lead to a drug.”

From:  Takafumi Toyohara, et al., “Cell therapy using human induced pluripotent stem cell-derived renal progenitors ameliorates acute kidney injury in mice” Stem Cells Translational Medicine.

Mesenchymal Stem Cells Make Blood Vessel Cells and Improve Wound Healing

Mesenchymal stem cells from umbilical cord have the ability to differentiate into cartilage cells, fat cells, bone cells, and blood vessels cells. These cells also are poorly recognized by the immune system of the patient and are at a low risk of being rejected by the patient’s immune system.

Valeria Aguilera and her colleagues from the laboratory of Claudio AguayoWe at the University of Concepción, Chilee have evaluated the use of mesenchymal stem cells from umbilical cord in the formation of new blood vessels in damaged tissues. Wharton’s jelly mesenchymal stem cells of hWMSCs were used to potentially accelerate tissue repair in living animals.

Aguilera and her co-workers began by isolating mesenchymal stem cells from human Wharton’s jelly (a connective tissue in umbilical cord). Then they grew these cells in culture for 14 or 30 days. Interestingly, the longer the WMSCs grew in culture, the more they looked like blood vessel cells. They began to express blood vessel-specific genes and proteins. WMSCs cultured for 30 days were even more like blood vessels than those grown in culture for 14 days.

When these cells were injected in the mice with damaged skin, the results showed that the WMSCs cultured for 30 days significantly accelerated wound healing compared with animals injected with either undifferentiated hWMSCs or with no cells.

Effect of hWMSCs and endothelial-differentiated hWMSC transplantation in a wound-healing model. A) Representative images of wounds at day 1 (top panels) and 12 (lower panels) after injury and subcutaneous injection of hWMSCs, hWMSC trans-differentiated into endothelial cells for 14 days (hWMSC-End14d) or 30 days (hWMSC-End30d), or control (PBS). B) Wound healing quantified in PBS (○), hWMSC (•), hWMSC-End14d (□) or hWMSC-End30d (▪) treated mice (n = 5 independent experiments, in duplicate). Values are expressed as mean±S.E.M, +P<0.05 in hWMSC-End30d v/s hWMSC, hWMSC-End14d, at the corresponding time; **P<0.03 in hWMSC-End30d v/s PBS; *P<0.001 in hWMSC-End30d v/s PBS; # P<0.01 in hWMSC-End30d v/s PBS.
Effect of hWMSCs and endothelial-differentiated hWMSC transplantation in a wound-healing model.
A) Representative images of wounds at day 1 (top panels) and 12 (lower panels) after injury and subcutaneous injection of hWMSCs, hWMSC trans-differentiated into endothelial cells for 14 days (hWMSC-End14d) or 30 days (hWMSC-End30d), or control (PBS). B) Wound healing quantified in PBS (○), hWMSC (•), hWMSC-End14d (□) or hWMSC-End30d (▪) treated mice (n = 5 independent experiments, in duplicate). Values are expressed as mean±S.E.M, +P



The wounds of mice treated with the WMSCs cultured for 30 days looked healthier, but they had many more blood vessels.

Histologic analysis of wounds in the wound-healing model. A) Representative photographs of wounds (hematoxilin/eosin staining) 12 days after injury and subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d. Quantification of histological images, for blood vessels area (B) and histological score (C) for each group of mice. Values are mean ± S.E.M (n = 5 independent experiments, in duplicate), *P<0.001 in hWMSC-End30d or hWMSC-End14d v/s MSC; +P<0.05 in hWMSC-End30d or hWMSC-End14d v/s hWMSC. Magnification x40 (-). Ep, epidermis; D, dermis; H, hypodermis.
Histologic analysis of wounds in the wound-healing model.
A) Representative photographs of wounds (hematoxilin/eosin staining) 12 days after injury and subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d. Quantification of histological images, for blood vessels area (B) and histological score (C) for each group of mice. Values are mean ± S.E.M (n = 5 independent experiments, in duplicate), *P

When laboratory animals received the culture medium from the WMSCs cultured for 30-days also showed significant acceleration of their healing, which suggests that these cells secrete a host of healing molecules that induced the formation of new blood vessels.  One might also conclude that the implanted WMSCs did not contribute to the formation of new blood vessels, but simply directed the formation of new blood vessels by secreting healing molecules.  However, when WMSCs were detected in the healed tissue, they were predominantly found in the walls of new blood vessels.

Immunohistochemical detection of human mesenchymal cells in a wound-healing model. A. Immunohistochemical staining of human mitochondria was performed in permeabilized tissue sections obtained after 12 days of subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d in mice. Cell nuclei were stained with hematoxyline. In B. Number of positive cells per vessel. Representative images of 5 independent experiments, in duplicate. Magnification x40 and insert 100x. Bars 50 µm.
Immunohistochemical detection of human mesenchymal cells in a wound-healing model.
A. Immunohistochemical staining of human mitochondria was performed in permeabilized tissue sections obtained after 12 days of subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d in mice. Cell nuclei were stained with hematoxyline. In B. Number of positive cells per vessel. Representative images of 5 independent experiments, in duplicate. Magnification x40 and insert 100x. Bars 50 µm.

These results, which were published in PLOS ONE, demonstrate that mesenchymal stem cells isolated from umbilical cord connective tissue or Wharton’s jelly can be successfully grown in culture in the laboratory and trans-differentiated into blood vessels-forming cells (endothelial cells).  These differentiated hWMSC-derived endothelial cells seem to promote the formation of new networks of blood vessels, which augments tissue repair in laboratory animals through the secretion of soluble pro-blood vessel-making molecules and, occasionally, by contributing to the formation of new vessels, themselves.

Meta Study Shows that Mesenchymal Stem Cells Promote Healing in Animal Models of Stroke

Two scientists from my alma mater, UC Irvine, have examined experiments that treated stroke with bone marrow-derived stem cells. Their analysis has shown that infusions of these stem cells trigger repair mechanisms and limit inflammation in the brains of stroke patients.

UC Irvine neurologist Dr. Steven Cramer and biomedical engineer Weian Zhao identified 46 studies that examined the use of a specific type of bone marrow stem cells called mesenchymal stromal cells to treat stroke. Mesenchymal stromal cells are a type of multipotent adult stem cells that are found in many locations in the body. The best-known examples of mesenchymal stem cells are from bone marrow. When purified from whole bone marrow and used to treat stroke in animal models of stroke, Cramer and Zhao found that mesenchymal stromal cells (MSCs) were significantly better than control therapy in 44 of the 46 studies that were examined.

Further data culling of these studies showed that functional recovery from stroke were robust regardless of the MSC dosage or the time when MSCs were administered relative to the onset of the stroke, or the method of administration (whether introduced directly into the brain or injected via a blood vessel).

“Stroke remains a major cause of disability, and we are encouraged that the preclinical evidence shows [MSCs’] efficacy with ischemic stroke,” said Cramer, a professor of neurology and leading stroke expert. “MSCs are of particular interest because they come from bone marrow, which is readily available, and are relatively easy to culture. In addition, they already have demonstrated value when used to treat other human diseases.”

Another theme of these studies is that MSCs do not differentiate into brain-specific. MSCs have the capacity to differentiate into bone, cartilage and fat cells. “But they do their magic as an inducible pharmacy on wheels and as good immune system modulators, not as cells that directly replace lost brain parts,” he said.

In an earlier Cramer and Zhao examined the mechanism by which MSCs promote brain repair after stroke. These cells have the ability to home to the damages areas in the brain and release chemicals that stimulate healing. By releasing their cornucopia of healing-promoting molecules, MSCs orchestrate blood vessel creation to enhance circulation, the protection of moribund cells on the verge of death, and the growth of existing brain cells. Additionally, when MSCs reach the bloodstream, they settle in those parts of the body that control the immune system and they suppress the inflammatory response that can augment tissue damage. In this way, MSCs foster an environment more conducive to brain repair.

“We conclude that MSCs have consistently improved multiple outcome measures, with very large effect sizes, in a high number of animal studies and, therefore, that these findings should be the foundation of further studies on the use of MSCs in the treatment of ischemic stroke in humans,” said Cramer, who is also clinical director of the Sue & Bill Gross Stem Cell Research Center.

When Is the Best Time to Treat Heart Attack Patients With Stem Cells?

Several preclinical trials in laboratory animals and clinical trials have definitively demonstrated the efficacy of stem cell treatments after a heart attack. However, these same studies have left several question largely unresolved. For example, when is the best time to treat acute heart attack patients? What is the appropriate stem cell dose? What is the best way to administer these stem cells? Is it better to use a patient’s own stem cells or stem cells from someone else?

A recent clinical trial from Soochow University in Suzhou, China has addressed the question of when to treat heart attack patients. Published in the Life Sciences section of the journal Science China, Yi Huan Chen and Xiao Mei Teng and their colleagues in the laboratory of Zen Ya Shen administered bone marrow-derived mesenchymal stromal cells at different times after a heart attack. Their study also examined the effects of mesenchymal stem cells transplants at different times after a heart attack in Taihu Meishan pigs. This combination of preclinical and clinical studies makes this paper a very powerful piece of research indeed.

The results of the clinical trial came from 42 heart attack patients who were treated 3 hours after suffering a heart attack, or 1 day, 3 days, 2 weeks or 4 weeks after a heart attack. The patients were evaluated with echocardiogram to ascertain heart function and magnetic resonance imaging of the heart to determine the size of the heart scar, the thickness of the heart wall, and the amount of blood pumped per heart beat (stroke volume).

When the data were complied and analyzed, patients who received their stem cell transplants 2-4 weeks after their heart attacks fared better than the other groups. The heart function improved substantially and the size of the infarct shrank the most. 4 weeks was better than 2 weeks,

The animal studies showed very similar results.

Eight patients were selected to receive additional stem cell transplants. These patients showed even greater improvements in heart function (ejection fraction improved to an average of 51.9% s opposed to 39.3% for the controls).

These results show that 2-4 weeks constitutes the optimal window for stem cell transplantation. If the transplant is given too early, then the environment of he heart is simply too hostile to support the survival of the stem cells. However, if the transplant is performed too late, the heart has already experiences a large amount of cell death, and a stem cell treatment might be superfluous. Instead 2-4 weeks appears to be the “sweet spot” when the heart is hospitable enough to support the survival of the transplanted stem cells and benefit from their healing properties. Also, this paper shows that multiple stem cell transplants a two different times to convey additional benefits, and should be considered under certain conditions.

Umbilical Cord Stem Cells Preserve Heart Function After a Heart Attack in Mice

A consortium of Portuguese scientists have conducted an extensive examination of the effects of mesenchymal stromal cells from umbilical cord on the heart of mice that have suffered a massive heart attack. Even more remarkable is that these workers used a proprietary technique to harvest, process, and prepare the umbilical cord stem cells in the hopes that this technique would give rise to a commercial product that will be tested in human clinical trials,

Human umbilical cord tissue-derived Mesenchymal Stromal Cells (MSCs) were obtained by means of a proprietary technology that was developed by a biomedical company called ECBio. Their product,, UCX®, consists of clean, high-quality, umbilical cord stem cells that are collected under Good Manufacturing Practices. The use of Good Manufacturing Practice means that UCX is potentially a clinical-grade product. Thus, this paper represents a preclinical evaluation of UCX.

This experiments in this paper used standard methods to give mice heart attacks that were later received injections of UCX into their heart muscle. The same UCX cells were used in experiments with cultured cells to determine their effects under more controlled conditions.

The mice that received the UCX injections into their heart muscles after suffering from a large heart attack showed preservation of heart function. Also, measurements of the numbers of dead cells in the heart muscle of heart-sick mice that did and did not receive injections of umbilical cord cells into their hearts showed that the umbilical cord stem cells preserved heart muscle cells and prevented them from dying. Additionally, the implanted umbilical cord MSCs induced the growth and formation of many small blood vessels in the infarcted area of the heart. This prevented the heart from undergoing remodeling (enlargement), and preserved heart structure and function.

When subjected to a battery of tests on cultured cells, UCX activated cardiac stem cells, which are the resident stem cell population in the heart. Implanted UCX cells activated the proliferation of cardiac stem cells and their differentiation into heart muscle cells. There was no evidence that umbilical cord MSCs differentiated into heart muscle cells and engrafted into the heart. Rather UCX seems to help the heart by means of paracrine mechanisms, which simply means that they secrete healing molecules in the heart and help the heart heal itself.

In conclusion, Diana Santos Nascimento, the lead author of this work, and her colleagues state that, “the method of UCX® extraction and subsequent processing has been recently adapted to advanced therapy medicinal product (ATMP) standards, as defined by the guideline on the minimum quality data for certification of ATMP. Given that our work constitutes a proof-of-principle for the cardioprotective effects UCX® exert in the context of MI, a future clinical usage of this off-the-shelf cellular product can be envisaged.”

Preclinical trials with larger animals should come next, and after that, hopefully, the first human clinical trials will begin.

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.

A New Way to Treat Kidney Disease and Heart Failure

St. Michael’s Hospital in Toronto, Ontario is the site of new research that uses bone marrow stem cells to treat chronic kidney disease and heart failure in rats.

Darren Yuen and Richard Gilbert of St. Michael’s Hospital were the first to show in 2010 that enriched stem cells improved heart and kidney functions in rats afflicted with both diseases. Their work generated concerns about the side effects of returning such stem cells to the body.

Since 2010, Yuen and Gilbert have found that enriched bone marrow stem cells secrete stromal cell–derived factor-1α (SDF-1α), a chemokine that is made by ischemic tissue but is rapidly degraded by dipeptidyl peptidase-4 (DPP-4), in culture dishes.  Injection of SDF-1α into rats has many of the same positive effects as when the stem cells themselves are injected into rats.  Even more remarkably, if a drug that inhibits the enzyme DPP-4 is given (sitagliptin) produced many improvements as well.

“We’ve shown that we can use these ‘hormones’ to replicate the beneficial effects of the stem cells in treating animals with chronic kidney disease and heart failure,” said Yuen, who practices as a nephrologist. “In our view, this is a significant advance for stem cell therapies because it gets around having to inject stem cells.”

Yuen said that they do not yet know what kind of hormone the cells are secreting, but identifying the hormone would be the first step toward the goal of developing a synthetic drug.

Chronic kidney disease (CKD) is much more prevalent than was once believed, with recent estimates suggesting that up to five percent of the Canadian population may be affected with this condition.

The number of people with CKD and end-stage renal failure is expected to rise as the population ages and more people develop Type 2 diabetes. People with kidney disease often develop heart disease, and many of them die from heart failure rather than kidney failure.

Regenerating Injured Kidneys with Exosomes from Human Umbilical Cord Mesenchymal Stem Cells

Zhou Y, Xu H, Xu W, Wang B, Wu H, Tao Y, Zhang B, Wang M, Mao F, Yan Y, Gao S, Gu H, Zhu W, Qian H: Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro. Stem Cell Res Ther 2013, 4:34.

Ying Zhou and colleagues from Jiangsi University have provided helpful insights into how adult stem cell populations – in particular, mesenchymal stem cells (MSCs) isolated from human umbilical cord (hucMSCs) – are able to regulate tissue repair and regeneration. Adult stem cells, including MSCs from different sources, confer regenerative effects in animal models of disease and tissue injury. Many of these cells are also in phase I and II trials for limb ischemia, congestive heart failure, and acute myocardial infarction (Syed BA, Evans JB. Nat Rev Drug Discov 2013, 12:185–186).

Despite the documented healing capabilities of MSCs, in many cases, even though the implanted stem cells produce genuine, reproducible therapeutic effects, the presence of the transplanted stem cells in the regenerating tissue is not observed. These observations suggest that the predominant therapeutic effect of stem cells is conferred through the release of therapeutic factors. In fact, conditioned media from adult stem cell populations are able to improve ischemic damage to kidney and heart, which confirms the presence of factors released by stem cells in mediating tissue regeneration after injury (van Koppen A, et al., PLoS One 2012, 7:e38746; Timmers L, et al., Stem Cell Res 2007, 1:129–137). Additionally, the secretion of factors such as interleukin-10 (IL-10), indoleamine 2,3-dioxygenase (IDO), interleukin-1 receptor antagonist (IL-1Ra), transforming growth factor-beta 1 (TGF-β1), prostaglandin E2 (PGE2), and tumor necrosis factor-alpha-stimulated gene/protein 6 (TSG-6) has been implicated in conferring the anti-inflammatory effects of stem cells (Pittenger M: Cell Stem Cell 2009, 5:8–10). These observations cohere with the positive clinical effects of MSCs in treating Crohn’s disease and graft-versus-host disease (Caplan AI, Correa D. Cell Stem Cell 2011, 9:11–15).

Another stem cell population called muscle-derived stem/progenitor cells, which are related to MSCs, can also extend the life span of mice that have the equivalent of an aging disease called progeria. These muscle-derived stem/progenitor cells work through a paracrine mechanism (i.e. the release of locally acting substances from cells; see Lavasani M, et al., Nat Commun 2012, 3:608). However, it is unclear what factors released by functional stem cells are important for facilitating tissue regeneration after injury, disease, or aging and the precise mechanism through which these factors exert their effects. Recently, several groups have demonstrated the potent therapeutic activity of small vesicles called exosomes that are released by stem cells (Gatti S, et al., Nephrol Dial Transplant 2011, 26:1474–1483; Bruno S, et al., PLoS One 2012, 7:e33115; Lai RC, et al., Regen Med 2013, 8:197–209; Lee C, et al., Circulation 2012, 126:2601–2611; Li T, et al., Stem Cells Dev 2013, 22:845–854). Exosomes are a type of membrane vesicle with a diameter of 30 to 100 nm released by most cell types, including stem cells. They are formed by the inverse budding of the multivesicular bodies and are released from cells upon fusion of multivesicular bodies with the cell membrane (Stoorvogel W, et al., Traffic 2002, 3:321–330).

Exosomes are distinct from larger vesicles, termed ectosomes, which are released by shedding from the cell membrane. The protein content of exosomes depends on the cells that release them, but they tend to be enriched in certain molecules, including adhesion molecules, membrane trafficking molecules, cytoskeleton molecules, heat-shock proteins, cytoplasmic enzymes, and signal transduction proteins. Importantly, exosomes also contain functional mRNA and microRNA molecules. The role of exosomes in vivo is hypothesized to be for cell-to-cell communication, transferring proteins and RNAs between cells both locally and at a distance.

To examine the regenerative effects of MSCs derived from human umbilical cord, Zhou and colleagues used a rat model of acute kidney toxicity induced by treatment with the anti-cancer drug cisplatin. After treatment with cisplatin, rats show increases in blood urea nitrogen and creatinine levels (a sign of kidney dysfunction) and increases in apoptosis, necrosis, and oxidative stress in the kidney. If exosomes purified from hucMSCs, termed hucMSC-ex are injected underneath the renal capsule into the kidney, these indices of acute kidney injury decrease. In cell culture, huc-MSC-exs promote proliferation of rat renal tubular epithelial cells in culture. These results suggest that hucMSC-exs can reduce oxidative stress and programmed cell death, and promote proliferation. What is not clear is how these exosomes pull this off. Zhou and colleagues provide evidence that hucMSC-ex can reduce levels of the pro-death protein Bax and increase the pro-survival Bcl-2 protein levels in the kidney to increase cell survival and stimulate Erk1/2 to increase cell proliferation.

Another research group has reported roles for miRNAs and antioxidant proteins contained in stem cell-derived exosomes for repair of damaged renal and cardiac tissue (Cantaluppi V, et al., Kidney Int 2012, 82:412–427). In addition, MSC exosome-mediated delivery of glycolytic enzymes (the pathway that degrades sugar) to complement the ATP deficit in ischemic tissues was recently reported to play an important role in repairing the ischemic heart (Lai RC, et al., Stem Cell Res 2010, 4:214–222). Clearly, stem cell exosomes contain many factors, including proteins and microRNAs that can contribute to improving the pathology of damaged tissues.

The significance of the results of Zhou and colleagues and others is that stem cells may not need to be used clinically to treat diseased or injured tissue directly. Instead, exosomes released from the stem cells, which can be rapidly isolated by centrifugation, could be administered easily without the safety concerns of aberrant stem cell differentiation, transformation, or recognition by the immune system. Also, given that human umbilical cord exosomes are therapeutic in a rat model of acute kidney injury, it is likely that stem cell exosomes from a donor (allogeneic exosomes) would be effective in clinical studies without side effects.

These are fabulously interesting results, but Zhou and colleagues have also succeeded in raising several important questions. For example: What are the key pathways targeted by stem cell exosomes to regenerate injured renal and cardiac tissue? Are other tissues as susceptible to the therapeutic effects of stem cell exosomes? Do all stem cells release similar therapeutic vesicles, or do certain stem cells release vesicles targeting only specific tissue and regulate tissue-specific pathways? How can the therapeutic activity of stem cell exosomes be increased? What is the best source of therapeutic stem cell exosomes?

Despite these important remaining questions, the demonstration that hucMSCderived exosomes block oxidative stress, prevent cell death, and increase cell proliferation in the kidney makes stem cell-derived exosomes an attractive therapeutic alternative to stem cell transplantation.

See Dorronsoro and Robbins: Regenerating the injured kidney with human umbilical cord mesenchymal stem cell-derived exosomes. Stem Cell Research & Therapy 2013 4:39.

Implantation of Irradiated Embryonic Stem Cells into the Heart Improves Heart Function After a Heart Attack

Adult stem cell transplantation has been used to treat heart attack patients in several different clinical trials. While the results have not been consistent, adult stem cells, it is clear that adult stem cells, primarily from bone marrow, and in some cases fat, help improve heart function. However, a major criticism of the use of adult stem cells is that they do not differentiate into heart muscle cells, but only improve the heart through “paracrine mechanisms,” which means that they secrete molecules that help heal the heart. This criticism is only represent part of the picture, since bone marrow stem cells transdifferentiate into heart muscle and blood vessel cells, albeit at a rather low rate, and fuse with endogenous cells to form hybrid cells that show improved function (Strauer BE, Steinhoff G. J Am Coll Cardiol. 2011 Sep 6;58(11):1095-104. doi: 10.1016/j.jacc.2011.06.016). In addition, adult stem cells activate endogenous cardiac stem cells to divide and replace lost heart muscle cells and make new blood vessels (Loffredo FS, et al., Cell Stem Cell. 2011 Apr 8;8(4):389-98. doi: 10.1016/j.stem.2011.02.002).

Embryonic stem cells, on the other hand, are thought to differentiate into heart muscle cells that integrate into the heart and directly replace the dead heart muscle cells, Animal studies do show such improvements (Caspi O, et al., J Am Coll Cardiol. 2007 50(19):1884-93). However, there is a caveat to all this: Most of the animal experiments with heart muscles derived from embryonic stem cells have only analyzed heart function for up to four weeks after transplantation. Experiments that examined heart function for longer than four weeks have not been able to show that these improvements are sustained after four weeks (van Laake LW, et al., Stem Cell Res. 2007 Oct;1(1):9-24. doi: 10.1016/j.scr.2007.06.001). Therefore, could it be possible that embryonic stem cell-derived cells also help the heart mainly through paracrine mechanisms?

A new paper from Piero Anversa’s and Richard Burt’s laboratories has shown that implantation of embryonic stem that were hit with radiation so that they cannot divide significantly improves heart function after a heart attack.

Experiments were conducted with mice and rhesus monkeys, and mouse and human embryonic stem cells (ESCs) were used. The ESCs were treated with 20 to 100 Grays of radiation, which completely abolished their ability to divide (a gray is the absorption of one joule of energy, in the form of ionizing radiation, per kilogram of matter).

The irradiated ESCs or iESCs were implanted into mice and Rhesus monkeys that had suffered a heart attack. Control animals were implanted with conditioned culture media from the ESC culture dishes.

In the mice and the Rhesus monkeys, the control animals showed little improvement and their hearts continued to deteriorate after the heart attack. However, the animals that had been implanted with the iESCs showed significant improvement of their heart function.

The authors in the discussion suggest that the iESCs might have suppressed the inflammatory response that occurs in the heart after a heart attack, but tissue sections of the hearts after the experiment showed that the iESC-implanted hearts had just as many immune cells infiltrating the tissue as the hearts of the control animals. Mesenchymal stem cells, however, do a very fine job of suppressing inflammation in the heart after a heart attack (see the recent paper by van den Akker et al., Biochimica et Biophysica Acta 1830 (2013): 2449-58). Therefore, the mechanisms by which ESCs improve heart function might be more paracrine-based than anything else. If this is the case, then why are embryonic stem cells being pursued for clinical purposes? Adult stem cells heal by means of paracrine mechanisms and can also sidestep the problem of immune rejection. Also, adult stem cells treatments do not require the dismemberment of young human beings at the embryo stage of their existence. Therefore, even though the present ESC lines are certainly appropriate for clinical and biological research, deriving more of them for clinical treatments is inappropriate, and even murderous.

Mesenchymal Stem Cells Rarely Engraft But Work in a “Hit and Run” Manner

Even though this paper was published in 2012, it is a very important study that deserves a wide reading and lots of discussion.

The paper is “Analysis of Tissues Following Mesenchymal Stromal Cell Therapy in Humans Indicates Limited Long-Term Engraftment and No Ectopic Tissue Formation” from Kathleen Le Blanc’ s laboratory, which was published in Stem Cells 2012;30:1575–1578.

In this paper, Le Blanc and her colleagues examined autopsies from patients who had received mesenchymal stem cell transplants. Since many scientists consider mesenchymal stromal cells (MSCs) a novel treatment for a variety of medical conditions, it is crucial that the fate of MSCs after infusion is better understood. Also, the long-term safety profile of MSCs is also quite important. DO they cause malignant transformation and ectopic tissue formation? Autopsies are an excellent way to address this questions.

The Le Blanc laboratory examined autopsy material from 18 patients who had received MSC transplants from people other than themselves. They analyzed 108 tissue samples from 15 patients by means of polymerase chain reaction (PCR) to search for the DNA of MSCs from donors in the tissue. If such foreign DNA was present in the tissues of the stem cell recipients, this would indicate that the MSCs had engrafted into the tissues of the patient. Unfortunately, MSC donor DNA was detected in only one or several tissues including lungs, lymph nodes, and intestine in eight patients at very low levels (from 1/100 to <1/1,000). Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection, which simply means that the more time had elapsed since the time of the MSc transplant, the less likely it was that MSC DNA was found in the patient. For example, MSC DNA was detected in nine of 13 patients whose MSC infusions had been given within 50 days before sampling, in only two of eight of those infusions that had been given earlier.

On a more positive note, there were no signs of ectopic tissue formation or malignant tumors of MSC-donor origin upon macroscopic or histological examination of the tissues of the autopsied individuals.

What does all this mean? MSCs appear to mediate their healing capacities through the molecules that they secrete. This is called a “paracrine” mechanism. and MSCs seem to engraft into host tissues only very rarely. Instead MSCs come to a damaged tissue and stimulate the endogenous healing mechanisms already present. After doing this job, MSCs do not typically stick around. Thus, MSCs seem to work in, what Le Blanc calls a “hit and run” mechanism.

Because MSCs do not seem to engraft over a long period of time, the potential adverse reactions to these cells seems to be largely limited. Thus these cells are quite safe, but their effects are almost certainly indirect to some extent.

Big Strides in Stem Cell Treatments for Neonatal Lung Diseases

Bernard Thébaud works at the Ottawa Hospital Research Institute (OHRI) and Children’s Hospital of Eastern Ontario (CHEO), and is also a member of the Ottawa Stem Cell Initiative. Dr. Thébaud has proposed a new therapy that utilizes umbilical cord stem cells to treat a lung disease called bronchopulmonary dysplasia (BPD), which was previously thought to be untreatable.

Thébaud described BPD in this way: “BPD is a lung disease described 45 years ago in which we have made zero progress. And now, with these cord-derived stem cells there is a true potential for a major breakthrough. I am confident that we have the talent and the tools here at CHEO and OHRI to find a treatment for BPD. These findings published today are helping us get there.”

Every year, BPD affects ~10,000 premature newborns in Canada and the US. The lungs of infants with BPD are not developed enough to function properly, and consequently the baby has to be placed on a ventilator in order to receive sufficient quantities of oxygen. Mechanical respirators, however, are very hard on such young, friable lungs, and the lungs then to fray and this prevents them from developing properly. The longer the baby stays in the neonatal intensive care unit, the greater the degree of multiorgan damage (retina, kidneys, and the brain). Therefore, the baby needs oxygen to survive, but the very act of giving them oxygen eventually hastens their death.

Thébaud’s research team used new-born rats that were given oxygen soon after their premature birth. Some were given stem cell treatments and others were not. These experiments produced five new findings:

1) Mesenchymal stem cells (MSCs) from human umbilical cord can protect the lungs when injected into the lungs as the animals were put on oxygen.
2) MSCs had a tendency to stimulate repair of the damaged lungs when injected two weeks after the animals were put on oxygen.
3) The medium in which the MSCs were grown (conditioned medium) was injected into the lungs instead of the cells, this medium had the same reparative and protective effects as the cells themselves.  This suggests that it is the cocktail of growth factors and other supportive molecules secreted by the MSCs that provide their healing properties.  Such a mechanism, in which the cells secrete molecules that affect nearby cells and tissue, is known as a “paracrine” mechanism.
4) When examined six months after treatment (the equivalent of 40 human years), the treated animals had better exercise performance and more normal lung structure.
5) MSC administration did not adversely affect the long-term health of the laboratory animals. None of the MSC-treated animals had any tumors and MSCs given to control animals that did not have BPD were also normal six months later.

Thébaud would like to conduct a pilot clinical (Phase I) study within two years with around 20 human patients in order to determine if this treatment is feasible and safe. If the treatment turns out to be safe, Thébaud would like to initiate a randomized controlled (Phase II) clinical trial.

See Maria Pierro et al., “Short-term, long-term and paracrine effect of human umbilical cord-derived stem cells in lung injury prevention and repair in experimental bronchopulmonary dysplasia,” Thorax 2012: DOI:10.1136/thoraxjnl-2012-202323.