Enzyme Helps Stem Cells Improve Recovery From Limb Injury


Ischemia refers to the absence of oxygen in a tissue or organ. Ischemia can cause cells to die and organs to fail and protecting cells, tissues and organs from ischemia-based damaged is an important research topic.

Perfusion refers to the restoration of the blood flow to an organ or tissue that had experienced a cessation of blood flow for a period of time. Even though the restoration of circulation is far preferable to ischemia, perfusion has its own share of side effects. For example, perfusion heightens cells death and inflammation and this can greatly exacerbate the physical condition of the patient after a heart attack, traumatic limb injury, or organ donation.

“Think about trying to hold onto a nuclear power plant after you unplug the electricity and cannot pump water to cool it down,” said Jack Yu, Chief of Medical College of Georgia’s Section of Plastic and Reconstructive Surgery. “All kinds of bad things start happening,”

Earlier studies in the laboratory of Babak Baban have shown that stem cells can improve new blood vessel growth and turn down the severe inflammation after perfusion (see Baban, et al., Am J Physiol Regul Integr Comp Physiol. 2012 Dec;303(11):R1136-46 and Mozaffari MS, Am J Cardiovasc Dis. 2013 Nov 1;3(4):180-96). Baban is an immunologist in the Medical College of Georgia and College of Dental Medicine at Georgia Regents University.

The new study from the Baban laboratory shows that an enzyme called indolamine 2,3,-dioxygenase or IDO can regulate inflammation during perfusion. IDO is widely known to generate immune tolerance and dampen the immune response in the developing embryo and fetus, but it turns out that stem cells also make this enzyme.

In their study, Including IDO with bone marrow-derived stem cells increased the healing efficiency of injected stem cells.

 Treatment Effect on Toe Spread Ratio Averages (48–72 hours after treatment). The outcome of stem cell (SC) therapy indicates that IDO may improve recovery. IDO-KO mice treated with SC demonstrated an accelerated recovery compared with IDO-KO treated with PBS (p-value <0.05). However, the WT mice treated with SC showed the greatest recovery of intrinsic paw function when expressed as a ratio comparing it to the non-injured paw (p-value = 0.027). Functional recovery from ischemia-reperfusion (IR) injury in the different treatment groups was measured, using a modified version of walking track analysis. For each subject, toe spread was measured in the IR limb (Ti) and control contralateral limb (Tc). The ratio of the toe spread in the injured limb (Ti) to the control limb (Tc) was then calculated by Ti/Tc. A ratio of 1 indicates 100% recovery or equal width and thus normal intrinsic function. When comparing the WT group treated with stem cells to those treated with PBS, a 45% increase in recovery was seen demonstrating the efficacy of stem cell therapy alone in the presence of an environment where IDO expression is present. doi:10.1371/journal.pone.0095720.g001
Treatment Effect on Toe Spread Ratio Averages (48–72 hours after treatment).
The outcome of stem cell (SC) therapy indicates that IDO may improve recovery. IDO-KO mice treated with SC demonstrated an accelerated recovery compared with IDO-KO treated with PBS (p-value <0.05). However, the WT mice treated with SC showed the greatest recovery of intrinsic paw function when expressed as a ratio comparing it to the non-injured paw (p-value = 0.027). Functional recovery from ischemia-reperfusion (IR) injury in the different treatment groups was measured, using a modified version of walking track analysis. For each subject, toe spread was measured in the IR limb (Ti) and control contralateral limb (Tc). The ratio of the toe spread in the injured limb (Ti) to the control limb (Tc) was then calculated by Ti/Tc. A ratio of 1 indicates 100% recovery or equal width and thus normal intrinsic function. When comparing the WT group treated with stem cells to those treated with PBS, a 45% increase in recovery was seen demonstrating the efficacy of stem cell therapy alone in the presence of an environment where IDO expression is present.
doi:10.1371/journal.pone.0095720.g001

Also indicators of inflammation, swelling, and cell death decreased in animals that received bone marrow-derived stem cell injections and had IDO.  Baban’s group also showed that the injected stem cells increased endogenous expression of IDO in the perfused tissues.

BMDScs can enhance IDO and regulatory T cells while reducing inflammatory cytokines in the hind limb IR injury. Immunohistochemical analysis of paraffin embedded tissues from murine model with IRI of hind limb showed that treating the animals with BMDSCs in an IDO sufficient microenvironment first: increased IDO and FOXP3 expression (panels A and B, red arrows), while decreased the inflammatory cytokines, IL-17 and IL-23 (panels C and D). Anti inflammatory cytokine, IL-10, was increased as demonstrated in panel E. All together, these analysis suggest a potential therapeutic role for BMDSCs, re-enforced by possible IDO dependent mechanisms. All pictures are 400X magnification
BMDScs can enhance IDO and regulatory T cells while reducing inflammatory cytokines in the hind limb IR injury.
Immunohistochemical analysis of paraffin embedded tissues from murine model with IRI of hind limb showed that treating the animals with BMDSCs in an IDO sufficient microenvironment first: increased IDO and FOXP3 expression (panels A and B, red arrows), while decreased the inflammatory cytokines, IL-17 and IL-23 (panels C and D). Anti inflammatory cytokine, IL-10, was increased as demonstrated in panel E. All together, these analysis suggest a potential therapeutic role for BMDSCs, re-enforced by possible IDO dependent mechanisms. All pictures are 400X magnification

Baban thinks that even though these experiments were performed in mice, because mice tend to be a rather good model system for limb perfusion/ischemia, these results might be applicable in the clinic.  “We don’t want to turn off the immune system, we want to turn it back to normal,” said Baban

According to Baban’s collaborator, Jack Yu, even a short period of inadequate blood supply and nutrients results in the rapid accumulation of destructive acidic metabolites, reactive oxygen species (also known as free radicals), and cellular damage.  The power plant of the cell, small structures called the mitochondria, tend to be one of the earliest casualties of ischemia/perfusion.  Since mitochondria require oxygen to make a chemical called ATP, which is the energy currency in cells, a lack of oxygen makes the mitochondria leaky, swollen and dysfunctional.

“The mitochondria are very sick,” said Yu. ” When blood flow is restored, it can put huge additional stress on sick powerhouses.  “They start to leak things that should not be outside the mitochondria.”

Without adequate energy production and a cellular power plant that leaks, the cells fill with toxic byproducts that cause the cells to commit a kind of cellular hari-kari.  Inflammation is a response to dying cells, since the role of inflammation is to remove dead or dying cells, but inflammation can give the coup de grace to cells that are already on the edge.  Therefore, inflammation can worsen the problem of cell death.

Even though these results were applied to limb ischemia perfusion, Baban and his colleagues think that their results are applicable to other types of ischemia perfusion events, such as heart attacks and deep burns.  Yu, for example, has noticed that in the case of burn patients, the transplantation of new tissue into areas that have undergone ischemia perfusion can die off even while the patient is still in the operating room.

“It cuts across many individual disease conditions,”  said Yu.  Transplant centers already are experimenting with pulsing donor organs to prevent reperfusion trauma.

The next experiments will include determining if more is better.  That is, if giving more stem cells will improve the condition of the injured animal.  In these experiments, which were published in the journal PLoS One, only one stem cell dose was given.  Also, IDO-enhancing drugs will be examined for their ability to prevent reperfusion damage.

Even though stem cells are not given to patients in hospitals after reperfusion, stem cell-based treatments are being tested for their ability to augment healing after reperfusion.  Presently, physicians reestablish blood flow and then give broad-spectrum antibiotics.  The results are inconsistent.  Hopefully, this work by Baban and others will pave the road for future work that leads to human clinical trials.

Human Neural Stem Cells Heal Damaged Limbs


The term “ischemia” refers to conditions under which a part of your body, organ, or tissue is deprived of oxygen. Without life-giving cells begin to die. Therefore, ischemia is usually a very bad thing.

Critical limb ischemia or CLI results when blood vessels to the legs, feet or arms are severely obstructed. The results of CLI are never pretty, and CLI remains a medical condition that presents few treatment options.

A study from a research team and the University of Bristol’s School of Clinical Sciences has used stem cells in a trial that uses laboratory mice to treat CLI. The success of this study provides a new direction and new hope for procedures that relieve symptoms and prolong the life of the limb.

Autologous stem cells treatments, or those stem treatments that utilize a patient’s own stem cells care subject to clear limitations. After collection from bone marrow, fat, or other source, the stem cells must be expanded in culture after stimulation with chemicals called cytokines. After growth in culture, the cells typically contain a collection of different types of stem cells of variable quality and potency. Also, if the patients has had a heart attack or has diabetes, then the quality and potency of their own stem cells are seriously compromised.

To circumvent this problem, Paulo Madeddu and his team at the Bristol Heart Institute have used an immortalized human neural stem cell line called CTX to treat animals who suffered from diabetes mellitus and CLI.

The CTX cell line comes from a biotechnology company called ReNeuron. This company is using this cell line in a clinical trial for stoke patients, and wants to use the CTX cell line in a clinical trial for CLI patients in the future.

When CTX cells are injected into the muscle of diabetic mice with CLI, the cells promote recovery from CLI. The CTX cells do so by promoting the growth of new blood vessels.

Madeddu said, “There are not effective drug interventions to treat CLI. The consequences are a very poor quality of life, possible major amputation and a life expectancy of less than one year from diagnosis in 50 percent of all CLI patients.”

Dr. Madeddu continued: “Our findings have shown a remarkable advancement towards more effective treatments for CLI and we have also demonstrated the importance of collaborations between universities and industry that can have a social and medical impact.”

Blood Vessel-Making Stem Cells From Fat


Blood vessel obstruction deprives tissues of life-giving oxygen and leads to the death of cells. If enough cells within a tissue die, the organ in which whose tissues reside could experience organ failure.

To quote the Sound of Music, “How does one solve a problem like blood vessel obstruction?” The obvious answer is to make new blood vessels to replace the blocked ones. Scientists have identified growth factors that are important in blood vessel formation during development. Therefore, injecting these growth factors should lead to the formation of new blood vessels, right? Unfortunately, such a strategy does not work very well (see Collison and Donnelly, Eur J Vasc Endovasc Surg 2004 28:9-23). Therefore, vascular specialists have focused on the ability of stem cells make new blood vessels, and this approach has yielded some very definite successes.

During development, the same stem cell gives rise to blood vessels and blood cells. This stem cell, the hemangioblast is found in a structure known as the yolk sac (even though it never functions as a yolk sac). In the yolk sac, during the third week of development, little specs form called “blood islands. These blood islands are small clusters of hemangioblasts with the cells at the center of the cluster forming blood cells and the cells at the periphery of the blood island forming blood vessels.

In adults, blood cell-making stem cells are found in the bone marrow. Blood vessel-making stem cells are endothelial progenitor cells or EPCs can be rather easily isolated from peripheral blood, however they are thought to originate from bone marrow. EPCs are not a homogeneous group of cells. There are different types with different surface molecules found in different locations.

Recently another cell from circulating blood called an “endothelial colony forming cell” or ECFC has been discovered, and this cell can attach to uncoated plastic surfaces in a growth medium. These cells can be grown to high numbers, even though it takes a rather long time to expand them. Once the ECFC culture system is further perfected, ECFCs will be excellent candidates for therapeutic trials (Reinisch et al., Blood 2009 113: 6716-25).

Fat tissue is also a reservoir of EPCs and mesenchymal stem cells. Fat-based mesenchymal stem cells help induce blood vessel formation and stimulate fat-based EPCs form blood vessels. Because of this remarkable “one-two punch” in fat, with cells that stimulate blood vessel formation and cells that actually form blood vessels, fat is a source of blood vessel-forming cells that can be used for therapeutic purposes.

Stem cells from fat.
Stem cells from fat.

Several pre-clinical experiments and presently ongoing clinical trials have examined the ability of fat-based stems to treat patients with conditions that result from insufficient circulation to various tissues. In rodents, experimental obstruction of the blood vessels in the hindlimb create a condition called “hindlimb ischemia.” In a rodent model of hindlimb ischemia, human fat-based stem cell applications not only improve the use of the limb and decrease limb damage, but also induce the formation of new blood vessels that definitely come from the applied stem cells (Miranville, et al., Circulation 2004 110: 349-55; Planat-Bernard, et al., Circulation 2004 109: 656-63 & Moon et al., Cell Physiol Biochem 2006 17: 279-90). Several clinical trials have been conducted with bone marrow-based EPCs for limb-based ischemia in humans, and these trials have been largely successful(see Szoke and Brinchmann, Stem Cells Translational Medicine 2012: 658-67 for a list of these trials). Adding mesenchymal stem cells from fat might improve the results of these trials.

In the heart, obstructed blood vessels can cause intense chest pain, a condition known as “angina pectoris.” EPCs have been used in clinical trials to treat patients with angina pectoris, and these trials have all been successful and have all used EPCs from bone marrow. These experiments, despite their success, have used bone marrow-based cells that were not fractionated and EPCs are less than 1% of the total number of cells. Also, the vast majority of cells introduced into heart migrate into the lungs, spleen and other organs. Also, those cells that remain tend to die off. A way to improve the survival of these implanted cells might be to combine them with mesenchymal stem cells from fat with EPCs from fat. Presently, the MyStromalCell trial is underway to test the efficacy of fat-based stem cells on the heart.

Fat provides an incredible treasure-trove of healing cells that have been demonstrated in animal experiments to relieve tissue ischemia and generate new blood vessels (for a summary of pre-clinical experiments in laboratory animals, see Qayyum AA, et al., Regen Med. 2012 May;7(3):421-8). Clinical trials with these cells are also underway. We have almost certainly only begun to tap to potential of these exciting cells that can be extracted so easily for our bodies.