Stem Cells Derived From Amniotic Tissues Have Immunosuppressive Properties

Ever since they were first isolated, amnion-based stem cells have been considered promising candidates for cell therapies because of their ease of access, plasticity, and absence of ethical issues in their derivation and use. However, a Japanese research team has discovered that stem cells derived from human female amnion also have the ability to suppress the inappropriate activation of the immune system and that there are straight-forward ways to enhance their immunosuppressive potential.

The amniotic membrane is a three-layered structure that surrounds the baby and suspends it in amniotic fluid. Amniotic fluid acts as a protective shock-absorber, a lubricant and an important physiological player in the life of the embryo and fetus. Because the fetus is a privileged entity that escapes attack from the mother’s immune system, researchers have been very interested in determining the immunological properties of the amnion cells.

“The human amniotic membrane contains both epithelial cells and mesenchymal cells,” said study co-author Dr. Toshio Nikaido, Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences at the University of Toyama. “Both kinds of cells have proliferation and differentiation characteristics, making the amniotic membrane a promising and attractive source for amnion-derived cells for transplantation in regenerative medicine. It is clear that these cells have promise, although the mechanism of their immune modulation remains to be elucidated.”

In this study by Nikaido and his coworkers, amnion-derived cells inhibited natural killer cell activity and induced white blood cell activation. Nikaido reported that he and his colleagues saw the amnion-derived cells increase production of a molecule called interleukin-10 (IL-10).

“We consider that IL-10 was involved in the function of amnion-derived cells toward NK cells,” explained Dr. Nikaido. “The immunomodulation of amnion-derived cells is a complicated procedure involving many factors, among which IL-10 and prostaglandin E2 (PGE2) play important roles.”

Molecules called “prostaglandins,” such as PGE2, mediate inflammation, smooth muscle activity, blood flow, and many other physiological process. In particular, PGE2 exerts important effects during labor and stimulates osteoblasts (bone-making cells) to release factors that stimulate bone resorption by osteoclasts. PGE2 also suppresses T cell receptor signaling and may play a role in the resolution of inflammation.

When Nikaido and others used antibodies against PGE2 and IL-10, they removed the immunosuppressive effects of the amnion-derived cells on natural killer cells. These data imply that these two factors contribute to the immunosuppressive abilities of amnion-derived cells.

“Soluble factors IL-10 and PGE2 produced by amnion-derived cells may suppress allogenic, or ‘other’ related immune responses,” concluded Dr. Nikaido. “Our findings support the hypothesis that these cells have potential therapeutic use. However, further study is needed to identify the detailed mechanisms responsible for their immodulatory effects. Amnion-derived cells must be transplanted into mouse models for further in vivo analysis of their immunosuppressive activity or anti-inflammatory effects.”

Given the levels of autoimmune diseases on the developed world, these results could be good news for patients who suffer from diseases like Crohn’s disease, systemic lupus erythematosus, or rheumatoid arthritis. While more work is needed, amnion-based cells certainly show promise as immunosuppressive agents.

The study will be published in a future issue of Cell Transplantation.

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.

Turning Stem Cells into Drug Factories

Wouldn’t it be nice to have cells that express the right molecules at the right place and the right time to augment or even initiate healing?

Researchers at the Brigham and Women’s Hospital and Harvard Stem Cell Institute have inserted modified messenger RNA to induce mesenchymal stem cells to produce adhesive proteins  (PSGL-1)and secrete interleukin-10, a molecule that suppresses inflammation. When injected into the bloodstream of mice, these modified stem cells home to the right location, stick to that site, and secrete interleukin-10 (IL-10) to suppress inflammation.

Improving MSC therapeutic potential viamRNA transfection with homing ligands and immunomodulatory factors. Illustration of (A) mRNA-engineered MSCs that express a combination of homing ligands (PSGL-1 and SLeX) and an immunomodulatory factor (IL-10), and (B) targeting mRNA-engineered MSCs to site of inflammation.
Improving MSC therapeutic potential viamRNA transfection with homing ligands and immunomodulatory factors. Illustration of (A) mRNA-engineered MSCs that express a combination of homing ligands (PSGL-1 and SLeX) and an immunomodulatory factor (IL-10), and (B) targeting mRNA-engineered MSCs to site of inflammation.

Jeffrey Karp, Harvard Stem Cell Institute principal faculty member and leader of this research, said this about this work: “If you think of a cell as a drug factory, what we’re doing is targeting cell-based, drug factories to damaged tissues, where the cells can produce drugs at high enough levels to have a therapeutic effect.”

Karp’s paper reports a proof-of-principle study has piqued the interest of several biotechnology companies, since it has the potential to target biological drug to disease sites. While ranked as the top sellers in the drug industry, biological drugs are still challenging to use. Karp’s approach might improve the clinical applications of biological drugs and improve the somewhat mixed results of clinical trials with mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have emerged as one of the favorite sources for stem cell therapies. The attractiveness of MSCs largely lies with their availability, since they are found in bone marrow, fat, liver, muscle, and many other places. Secondly, MSCs can be grown in culture for a limited period of time without a great deal of difficulty. Third, MSCs tend to be ignored by the immune system when injected. For these reasons, MSCs have been used in many clinical trials, and they appear to be quite safe to use.

To genetically modify MSCs, Karp and his co-workers made chemically modified messenger RNAs (mRNAs) whose bases differed slightly from natural mRNAs. These chemical modifications did not affect the recognition of the mRNA by the protein synthesis machinery of the MSCs, but did affect the recognition of these mRNAs by those enzymes that degrade mRNAs. Therefore, these synthetic mRNAs are very long-lived and the transfected cells end up making the proteins encoded by these mRNAs for a very long time. RNA transfection does not modify the genome of the host cells, and this makes it a very safe procedure, since the engineered cells will express the desired protein for some time, but not indefinitely.

The mRNAs introduced into the cultured MSCs included mRNAs that encode the IL-10 protein, which is cytokine that suppresses inflammation, the PSGL-1 protein, a cell-surface protein that sticks to the P-and E-selectin receptors, and the Fut7 gene product.  FUT7 encodes an enzyme called fucosyltransferase 7, which adds a sugar called “fucose” to the PSGL-1 protein and without this sugar, PSGL-1 cannot bind to the selectins.  Selectins are stored by cells and during inflammation, they are sent to the cell surface where they can bind cells and keep them there to mediate inflammation.  By expressing PSGL-1 in the MSCs, Karp and his group hoped to that the engineered MSCs would bind to the surfaces of blood vessels and not be washed out.


Oren Levy, lead author of this paper, said, “This opens the door to thinking of messenger RNA transfection of cell populations as next generation therapeutics in the clinic, as they get around some of the delivery challenges that have been encountered with biological agents.”

A problem that constantly troubles clinical trials that use MSCs is that they are rapidly cleared from the bloodstream within a few hours or days after they are introduced. The Harvard team showed that rapid targeting of MSCs to inflamed tissue produced a therapeutic effect despite rapid clearance of the MSCs.

Karp and his colleagues would like to extend the lifespan of these cells in the bloodstream and they are presently experimenting with new synthetic mRNAs that encode pro-survival factors.

“We’ve interested to explore the platform nature of this approach and see what potential limitations it may have or how far we can actually push it. Potentially we can simultaneously deliver proteins that have synergistic therapeutic impacts,” said Weian Zhao, another author of this paper.

Treating a Rare Immune Disorder with Mesenchymal Stem Cells

In the journal Stem Cells and Development, there is a case report from the University Hospital at Karolinska Institutet in Stockholm, Sweden of a 21-year-old man who suffered from a rare immune disorder and was treated with an infusion of mesenchymal stem cells (MSCs) from a donor.

This patient was seen in October, 2010 and had been suffering from a fever for 2 months. He had had a previous gastrointestinal infection that had resolved, but the inflammation that resulted from the infection refused to go away. He was diagnosed with hemophagic lymphohistiocytosis (HLH). This is a mouthful, but it is a relatively rare immune disorder that results in pronounced systemic hyperinflammation. This hyperinflammation essentially results from some sort of infection that causes inflammation, but the inflammation does not turn off when the infection resolves. The condition causes the spleen to enlarge and the number of blood cells to decrease to abnormally low levels and the patient has a constant, burning fever.

The medical team that treated this poor soul used steroids, and that worked from about a week. Then they tried the HLH-94 treatment protocol, which involves treating the patient with a combination of powerful immunosuppressive drugs; etoposide, (VP-16), corticosteroids, CyclosporinA, and, in some patients, intrathecal methotrexate, before the patient is given a bone marrow transplant. The HLH-94 protocol returned the patient to normal – for about 2 months, and then the patient was back to square one.

At this point, the medical team needed a Hail Mary, if you will. Therefore, they decided to use MSCs from a healthy donor. The patient was given a total of 124 million bone marrow-derived MSCs, and within 24 hours, the patient’s fever was gone and his blood work normalized.

Unfortunately, the poor chap contracted a nasty fungal infection that, in his weakened state, spread throughout his whole body and killed him. However, postmortem examinations showed that the MSCs had mobilized a whole gaggle of special white blood cells called macrophages, and these MSC-recruited macrophages suppressed the over-active immune response of this HLH patient. The fungal infection was contracted before the administration of the MSCs, therefore, the stem cell treatment had no causal relationship to the fungal infection.

However, this case study suggests that MSCs have a future in the treatment of immune disorders. Furthermore, the use of MSCs from donors can also provide therapeutic material for the treatment of immune disorders.