Getting Stem Cells to Engraft More Effectively – With A Little Help From My “Friends”


The old Beatles song, “With A Little Help from My Friends” begins:

What would you think if I sang out of tune
Would you stand up and walk out on me?
Lend me your ears and I’ll sing you a song
And I’ll try not to sing out of key
Oh I get by with a little help from my friends
Mm I get high with a little help from my friends
Mm going to try with a little help from my friends

For mesenchymal stem cells, a little help from circulating stem cells, that is, their “friends” can make all the difference.

Ruei-Zeng Lin, in the laboratory of Juan M. Melero-Martin at the Boston Children’s Hospital and Department of Surgery at Harvard Medical School, in Boston, Massachusetts, have made a profound discovery that was published in the Proceedings of the National Academy of Sciences USA. They have shown that cells called “endothelial colony-forming cells” or ECFCs that not only circulate throughout the bloodstream but also contribute to the formation of new blood vessels, can function as “nurse cells” that positively regulate the regenerative potential of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) secrete a whole cocktail of healing molecules, but these cells also respond to several different molecules made by other cells, and ECFCs make some of these pro-MSC molecules.

In their experiment, Lin and others injected human MSCs isolated from white fat and bone marrow aspirates underneath the skin of immunodeficient mice in the presence or absence of ECFCs derived from human umbilical cord blood. The results were quite telling.

The engraftment of the MSCs (engraftment means the ability of the implanted stem cells to survive, differentiate and integrate into existing tissues) was regulated by a protein secreted by ECFCs called “platelet-derived growth factor BB” or PDGF-BB. When MSCs and ECFCs were transplanted together, the ECFCs significantly enhanced MSC engraftment. The MSCs not only survived better, showed much less cell death, but they also preserved the stem cell-character of the MSCs. THis is was established by the fact that when the implanted MSCs were removed and reimplanted into another mouse, these cells could repopulate secondary grafts. However, if MSCs were implanted without ECFCs, MSC engraftment was negligible. Also, if a drug called Tyrphostin AG1296 was used, MSCs engraftment was also negligible. Tyrphostin AG1296 inhibits the receptor for PDGF-BB and completely abrogates any EFCF-related enhancement of MSC function.  This shows that the enhancement of MSC engraftment by ECFCs is largely dependent on PDGF-BB-mediated signaling.

Strangely, transplanted MSCs that had been co-transplanted with ECFCs displayed fate-restricted differentiation in animals.  This simply means that the fat-based stem cells differentiated into fat and the bone marrow-derived MSCs differentiated into bone.  It seems that with the increased growth and stem cell function comes a more restricted differentiation program as well.  This could potentially prevent the phenomenon of “out-of-place” differentiation also known as heterotypic differentiation, which can cause the formation things like bone during fat transplantation or other such things.

These experiments show that blood-derived ECFCs can amplify the regenerative potential of MSCs via PDGF-BB – based signaling.  These data also suggest that the systematic use of ECFCs can improve MSC transplantation, and provides new insights into the therapeutic capabilities of ECFCs.  The authors add: “We foresee the use of ECFCs as a means to improve the outcome of MSC transplantation.”

This is a remarkable preclinical trial, but before it can work in humans, it must prove its efficacy and safety in human clinical trials and in other preclinical trials as well.

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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.

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.

Inhibition of a Heart-Specific Enzyme After a Heart Attack Decreases Heart Damage and Prevents Remodeling


Cardiac Troponin I-interacting Kinase or TNNI3K is an enzyme that was initially identified in fetal and adult heart tissue, but was undetectable in other tissues. The function of this enzyme remains unknown, but Chinese scientists showed that overexpression of TNNI3K in cultured heart muscle cells causes them to blow up and get large (hypertrophy). Earlier this year, a research team from Peking Union Medical College showed that overexpression of TNNI3K in mice caused enlargement of the heart (Tang H., et al., J Mol Cell Cardiol 54 (2013): 101-111). These results suggested that TNNI3K is a potential therapeutic target for heart attack patients.

To that end, Ronald Vagnozzi and his colleagues in the laboratory of Thomas Force at Temple University School of Medicine and their collaborators designed small molecules that can inhibit TNNI3K activity, and these small molecules decrease cardiac remodeling after a heart attack in rodents. Large animal trials are planned next.

In the first experiments of this paper, Vagnozzi and others showed that the levels of TNNI3K in the heart increase after a heart attack. Measurements of TNNI3K protein levels failed to detect it in all tissue other than the heart. Furthermore, it was present throughout the heart, and mainly in heart muscle and not in blood vessels, fibroblasts, and other types of non-muscle heart tissues.

Next, Vagnozzi and others measured TNNI3K protein levels in heart transplant patients. The heart tissues of these patients, who had badly dysfunctional hearts showed higher than usual levels of TNNI3K protein. Thus, TNNI3K is associated with heart tissue and is up-regulated in response to heart dysfunction.

The next experiment examined the effects of overexpressing the human TNNI3K gene in mice. While the overexpression of TNNI3K did not affect heart function of structure under normal circumstances, under pathological conditions, however, this is not he case. If mice that overexpressed TNNI3K where given heart attacks and then “reperfused,” means that the blood vessel that was tied off to cause the heart attack was opened and blood flowed back into the infarcted area. In these cases, mice that overexpressed TNNI3K had a larger area of cell death in their hearts than their counterparts that did not overexpress TNNI3K. The reason for this increased cell death had to do with the compartment in the cell that generated most of the energy – the mitochondrion. TNNI3K causes the mitochondria in heart muscle cells to go haywire and kick out all kinds of reactive oxygen-containing molecules that damage cells.

Cell damage as a result of reactive oxygen-containing molecules (known as reactive oxygen species or ROS) activates a pathway in heart cells called the “p38” pathway, which leads to programmed cell death.

p38 signaling

Once Vagnozzi and his colleagues nailed down the function of TNNI3K in heart muscle cells after a heart attack, they deleted the gene that encodes TNNI3K and gave those TNNI3K-deficient mice heart attacks. Interestingly enough, after a heart attack, TNNI3K-deficient mice showed much small dead areas than normal mice. Also, the levels of the other mediators of TNNI3K-induced cell death (e.g., oxygen-containing molecules, p38, ect.) were quite low. This confirms the earlier observations that TNNI3K mediates the death of heart muscle cells after a heart attack, and inhibiting TNNI3K activity decreases the deleterious effects of a heart attack.

And now for the pièce de résistance – Vagnozzi and his crew synthesized small molecules that inhibited TNNI3K in the test tube. Then they gave mice heart attacks and injected these molecules into the bellies of the mice. Not only were the infarcts, or areas of dead heart muscle cells small in the mice injected with these TNNI3K inhibitors, but the heart of these same mice did not undergo remodeling and did not enlarge, showed reduced scarring, and better ventricular function. This is a proof-of-principle that inhibiting TNNI3K can reduce the pathological effects of a heart attack.

This strategy must be tested in large animals before it can move to human trials, but the strategy seems sound at this point, and it may revolutionize the treatment of heart attack patients.

Biphasic Electrical Stimulation Increases Stem Cell Survival


One of the challenges of stem cell-based therapies is cell survival. Once stem cells are implanted into a foreign site, many of them tend to pack up and die before they can do any good. For this reason, many scientists have examined strategies to improve stem cell survival.

A new technique that improves stem cells survival have been discovered by Yubo Fan and his colleagues at Beihang University School of Biological Science and Medical Engineering. This non-chemical technique, biphasic electrical stimulation (BES) might become important for spinal cord injury patients in the near future.

The BES incubation system. (a) Schematic diagram of a longitudinal section of the incubation chamber including: the upper and lower electric conductive glass plates (FTO glass), a closed silicone gasket, the incubation chamber, and a pair of electrode wires; (b) Schematic diagram of a longitudinal section of the entire BES incubation system including the incubation chamber, the fluid inflow-outflow system, the air filter system, a pair of electrode wires, and a fixed cover and base. Conditions of BES: the NPCs were exposed to 12 h of BES at 25mV/mm and 50mV/mm electric field strengths with a pulse-burst pattern and 8ms pulses (20% duty cycle). Cells that were not exposed to BES served as controls. (A color version of this figure is available in the online journal)
The BES incubation system. (a) Schematic diagram of a longitudinal
section of the incubation chamber including: the upper and lower electric  conductive glass plates (FTO glass), a closed silicone gasket, the incubation
chamber, and a pair of electrode wires; (b) Schematic diagram of a longitudinal
section of the entire BES incubation system including the incubation chamber,
the fluid inflow-outflow system, the air filter system, a pair of electrode wires, and
a fixed cover and base. Conditions of BES: the NPCs were exposed to 12 h of
BES at 25mV/mm and 50mV/mm electric field strengths with a pulse-burst
pattern and 8ms pulses (20% duty cycle). Cells that were not exposed to BES
served as controls. 

Spinal cord injury affects approximately 250,000 Americans, with 52% being paraplegic and 47% quadriplegic. There are 11,000 new spinal cord injuries each year and 82% are male.

Stem cell transplantions into the spinal cord to regenerate severed neurons and associated cells provides a potentially powerful treatment. However, once stem cells are implanted into the injured spinal cord, many of them die. Cell death is probably a consequence of several factors such as a local immune response, hypoxia (lack of oxygen), and probably most importantly, limited quantities of growth factors.

Fan said of his work, “We’ve shown for the very first time that BES may provide insight into preventing growth factor deprivation-triggered apoptosis in olfactory bulb precursor cells. These findings suggest that BES may thus be used as a strategy to improve cell survival and prevent cell apoptosis (programmed cell death) in stem cell-based transplantation therapies.”

The olfactory bulb is in green in this mouse brain.
The olfactory bulb is in green in this mouse brain.

Since electrical stimulation dramatically accelerates the speed of axonal regeneration and target innervation and positively modulates the functional recovery of injured nerves, Fan decided to test BES. His results showed that BES upregulated all the sorts of responses in stem cells that you would normally see with growth factors. Thus BES can increase stem cell survival without exogenous chemicals or genetic engineering.

Fan and his team examined the effects of BES on olfactory bulb neural precursor cells and they found that 12 hours of BES exposure protected cells from dying after growth factor deprivation. How did BES do this? Fan and other showed that BES stimulated a growth factor pathway called the PI3K/Akt signaling cascade. BES also increase the output of brain-derived neurotrophic factor.

“What was especially surprising and exciting,” said Fan, “was that a non-chemical procedure can prevent apoptosis in stem cell therapy for spinal cord patients.” Fan continued: “How BES precisely regulates the survival of exogenous stem cells is still unknown but will be an extremely novel area of research on spinal cord injury in the future.”

BES alters the ultrastructure of NPCs. The ultrastructural morphological changes of cells were investigated by TEM. In the control group (unstimulated), cells had a necrotic appearance: most cells lost the normal cellular structure with a consequent release of cell contents. In the 25mV/mm and 50mV/mm BES groups, the NPCs showed an apoptotic morphology with nuclear fragmentation and condensation
BES alters the ultrastructure of NPCs. The ultrastructural morphological changes of cells were investigated by TEM. In the control group (unstimulated), cells had a necrotic appearance: most cells lost the normal cellular structure with a consequent release of cell contents. In the 25mV/mm and 50mV/mm BES groups, the NPCs showed an apoptotic morphology with nuclear fragmentation and condensation

BES can improve the survival of neural precursor cells and will provide the survival of neural precursor cells and will provide the basis or future studies that could lead to novel therapies for patients with spinal cord injury.

Tissue Kallikrein-Modified Human EPCs Improve Cardiac Function


When cells are implanted into the heart after a heart attack, the vast majority of them succumb to the hostile environment in the heart and die. Twenty-four hours after implantation there is a significant loss of cells (see Wu et al Circulation 2003 108:1302-1305). That fact that implanted bone marrow or fat-based stem cells benefit the heart despite their evanescence is a remarkable testimony to their healing power.

To mitigate this problem, stem cell scientists have used a variety of different strategies to increase the heartiness and survival of implanted stem cells. Two main strategies have emerged: preconditioning cells and genetically engineering cells. Both strategies increase the survival of implanted stem cells (see here, and here).

When it comes to genetically engineering stem cells, Lee and Julie Chao from the Medical University of South Carolina in Charleston, South Carolina have used endothelial progenitor cells (EPCs) from human umbilical cord blood to treat mice that had suffered heart attacks, except that these cells were genetically engineered to express “Tissue Kallikrein” or TK. TK is encoded by a gene called KLKB1, which is on chromosome 4 at region q34-35 (in human genetics, the long arm of a chromosome is the “q” arm and the small arm is the “p” or petite arm). TK is initially synthesized as an inactive precursor called prekallikrein. Prekallikrein must be clipped in order to be activated and the proteases (proteases are protein enzymes that cut other proteins into smaller fragment) that do so are either clotting factor XII, which plays a role in blood clotting, and PRCP, which is also known as Lysosomal Pro-X carboxypeptidase.

TK is a protease that degrades a larger protein called kininogen in two smaller peptides called bradykinin and kallidin, both of which are active signaling molecules. Bradykinin and kallidin cause relaxation of smooth muscles, thus lowering blood pressure, TK can also degrade plasminogen to form the active enzyme plasmin.

So why engineer EPCs to express TK? As it turns out, TK activates an internal protein in cells called Akt, and activated Akt causes cells to survive and prevents them from dying (see Krankel et al., Circulation Research 2008 103:1335-1343; Yao YY, et al., Cardiovascular Research 2008 80: 354-364; Yin H et a., J Biological Chem 2005 280: 8022-8030).

The first experiments were test tube experiments in which TK EPCs were incubated with cultured heart muscle cells to determine their ability to prevent cell death. When cultured heart muscle cells were exposed to hydrogen peroxide, they died left and right, but when they were incubated with the TK-EPCs and hydrogen peroxide, far fewer of them died.

Upper panel consists of cells stained with a TUNEL stain, which designates those cells that are dead or dying.  The bottom panel are DAPI stained cells, which is a nuclear stain that marks all available cells dead or live. From left to right, normal cells, cell exposed to hydrogen peroxide, cells exposed to hydrogen peroxide plus the genes for TK, and finally, cells exposed to hydrogen peroxide and TK-EPCs.
Upper panel consists of cells stained with a TUNEL stain, which designates those cells that are dead or dying. The bottom panel are DAPI stained cells, which is a nuclear stain that marks all available cells dead or live.
From left to right, normal cells, cell exposed to hydrogen peroxide, cells exposed to hydrogen peroxide plus the genes for TK, and finally, cells exposed to hydrogen peroxide and TK-EPCs.

When these cells were exposed to low levels of oxygen, a similar result was observed, expect that the cells co-incubated with TK-EPCs showed significantly less cell death.

When TK-EPCs were injected into the infarct border zones of the heart just after they had heart attacks, the results seven days after the heart attacks were striking. The heart function of the control mice was lousy to say the least. The heart walls had thinned, their ejection fractions were in the tank (~23%) and their echocardiograms were far from normal. However, the TK-EPC-injected mice had a relatively normal echocardiogram, thick heart wall, pretty good ejection fractions (52% and oppose to the 76% of mice that had never had a heart attack), and good heart function in general. Also, the size of the infarcts was reduced in those animals whose hearts had been injected with TK-EPCs.

Representative Masson’s trichrome staining. Original magnification is 10. (f) Echocardiographic measurements for determination of LV function from M-mode measurements. (g) MDA in the ischemic mouse heart at day 7 after MI. Values are expressed as mean±s.e.m. (n¼6, *Po0.05 vs Ad.Null-hEPC- and medium-treated group; #Po0.05 vs medium-treated group).
Representative Masson’s trichrome staining. Original magnification is 10. (f) Echocardiographic measurements for determination of LV function from M-mode measurements. (g) MDA in the ischemic mouse heart at day 7 after MI. Values are expressed as mean±s.e.m. (n¼6, *Po0.05 vs Ad.Null-hEPC- and medium-treated group; #Po0.05 vs medium-treated group).

There were two other bonuses to using TK-EPCs. First, as expected, the density of new blood vessels was substantially higher in hearts that received injections of TK-EPCs. Secondly, the TK-EPCs definitely survived better than their non-genetically engineered counterparts.

Ex-vivo optical imaging study. (a, b) Representative NIR fluorescent images in explanted organs at days 2 or 7 following implantation of DiDlabeled hEPCs into the ischemic myocardium of nude mice. Bars represent maximum radiance. (a: 2 days after cell delivery; b: 7 days after cell delivery). (c) Quantitative analysis of NIR fluorescent signals in explanted hearts among each group at two time points. All values are expressed as mean±s.e.m. (n¼3–4, *Po0.01 vs control group).
Ex-vivo optical imaging study. (a, b) Representative NIR fluorescent images in explanted organs at days 2 or 7 following implantation of DiDlabeled hEPCs into the ischemic myocardium of nude mice. Bars represent maximum radiance. (a: 2 days after cell delivery; b: 7 days after cell delivery). (c) Quantitative analysis of NIR fluorescent signals in explanted hearts among each group at two time points. All values are expressed as mean±s.e.m. (n¼3–4, *Po0.01 vs control group).

These results also confirm that TK works in heart muscle cells by activating the Akt protein inside the cells.  This establishes that TK works through the Akt pathway.

Once again, we see that transplantation of stem cells after a heart attack can improve the function and structure of the heart after a heart attack.  Indeed this strategy seems to work again and again.  These experiments were done in mice and therefore, they must be successful in a larger animal, like a pig before they can be deemed efficacious and safe for use in human clinical trials.  Even so, these results are hopeful.

Culture Medium from Endothelial Progenitor Cells Heals Hearts


Endothelial Progenitor Cells or EPCs have the capacity to make new blood vessels but they also produce a cocktail of healing molecules. EPCs typically come from bone marrow, but they can also be isolated from circulating blood, and a few other sources.

The laboratory of Noel Caplice at the Center for Research in Vascular Biology in Dublin, Ireland, has grown EPCs in culture and shown that they make a variety of molecules useful to organ and tissue repair. For example, in 2008 Caplice published a paper in the journal Stem Cells and Development in workers in his lab showed that injection of EPCs into the hearts of pigs after a heart attack increased the mass of the heat muscle and that this increase in heart muscle was due to a molecule secreted by the EPCs called TGF-beta1 (see Doyle B, et al., Stem Cells Dev. 2008 Oct;17(5):941-51).

In other experiments, Caplice and his colleagues showed that the culture medium of EPCs grown in the laboratory contained a growth factor called “insulin-like growth factor-1” or IGF1. IGF1 is known to play an important role in the healing of the heart after a heart attack. Therefore, Caplice and his colleagues tried to determine if IGF1 was one of the main reasons EPCs heal the heart.

To test the efficacy of IGF1 from cultured EPCs, Caplice’s team grew EPCs in the laboratory and took the culture medium and tested the ability of this culture medium to stave off death in oxygen-starved heart muscle cells in culture. Sure enough, the EPC-conditioned culture medium prevented heart muscle cells from dying as a result of a lack of oxygen.

When they checked to see if IGF1 was present in the medium, it certainly was. IGF1 is known to induce the activity of a protein called “Akt” inside cells once they bind IGF1. The heart muscle cells clearly had activated their Akt proteins, thus strongly indicating the presence of IGF1 in the culture medium. Next they used an antibody that specifically binds to IGF1 and prevents it from binding to the surface of the heart muscle cells. When they added this antibody to the conditioned medium, it completely abrogated any effects of IGF1. This definitively demonstrates that IGF1 in the culture medium is responsible for its effects on heart muscle cells.

Will this conditioned medium work in a laboratory animal? The answer is yes. After inducing a heart attack, injection of the conditioned medium into the heart decreased the amount of cell death in the heart and increased the number of heart muscle cells in the infarct zone, and increased heart function when examined eight weeks after the heart attacks were induced. The density of blood vessels in the area of the infarct also increased as a result of injecting IGF1. All of these effects were abrogated by co-injection of the antibody that specifically binds IGF1.

From this study Caplice summarized that very small amounts of IGF1 (picogram quantities in fact) administered into the heart have potent acute and chronic beneficial effects when introduced into the heart after a heart attack.

These data are good enough grounds for proposing clinical studies. Hopefully we will see some in the near future.