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.

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.

Nanometer Scaffolds Regulate Neural Stem Cells


In the laboratory, stem cells can grow in liquid culture quite well in many cases, but this type of culture system, though convenient and rather inexpensive, does not recapitulate the milieu in which stem cells normally grow inside our bodies. Inside our bodies, stem cells stick to all kinds of surfaces and interact with and move over a host of complex molecules. Many of the molecules that stem cells contact have profound influences over their behaviors. Therefore, reconstituting or approximating these environments in the laboratory is important even though it is very difficult.

Fortunately nanotechnology is providing ways to build surfaces that approximate the kinds of surfaces stem cells encounter in our bodies. While this field is still in its infancy, stem cell-based nanotechnology may provide strategies to synthesize biologically relevant surfaces for stem cell growth, differentiation, and culture.

One recent contribution to this approach comes from Jihui Zhou and his team from the Fifth Hospital Affiliated to Qiqihar Medical University. Zhou and his co-workers prepared randomly oriented collagen nanofiber scaffolds by spinning them with an electronic device. Collagen is a long, fibrous protein that is found in tendons, ligaments, skin, basement membranes (the substratum upon which sheets of cells sit), bones, and is also abundant in cornea, blood vessels, cartilage, intervertebral disc, muscles, and the digestive tract. Collagen is extremely abundant in the human body; some 30% of all the proteins in our bodies are collagen. It is the main component in connective tissues.

There are many different types of collagen. Some types of collagen form fibers, while others for sheets. There are twenty-eight different types of collagen. Mutations in the genes that encode collagens cause several well-known genetic diseases. For example, mutations in collagen I cause osteogenesis imperfecta, the disease made famous by the Bruce Willis/Samuel T. Jackson movie, “Unbreakable.” Mutations in Collagen IV cause Alport syndrome, and mutations in either collagen III or V cause Ehlers-Danlos Syndrome.

Wen cells make fibrous collagen, they weave three collagen polypeptides together to form a triple helix protein that is also heavily crosslinked. This gives collagen its tremendous tensile strength.

Collagen fibers
Collagen fibers

In this experiment, electronic spinning technology made the collagen fibers and these fibers had a high swelling ratio when placed in water, high pore size, and very good mechanical properties.

Zhou grew neural stem cells from spinal cord on these nanofiber scaffolds and the proliferation of the neural stem cells was enhanced as was cell survival. Those genes that increase cell proliferation (cyclin D1 and cyclin-dependent kinase 2) were increased, as was those genes that prevent cells from dying (Bcl-2). Likewise, the expression of genes that cause cells to die (caspase-3 and Bax) decreased.

Thus novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit programmed cell death without inducing differentiation of the stem cells. These scaffolds do this by inducing the expression of proliferation- and survival-promoting genes.

BMP-2 Treatment Limits Infarct Size in After a Heart Attack in Mice


Bone Morphogen Protein 2 (BMP2) is a powerful signaling molecule that is made during development, healing, and other significant physiological events. During the development of the heart, BMP2 modulates the activation of cardiac genes. In culture, BMP2 can protect heart muscle cells from dying during serum starvation. Can BMP2 affect hearts that have just experienced a heart attack?

Scientists from the laboratories of Karl Werdan and Thomas Braun at the Max Planck Institute or Heart and Lung Research in Bad Nauheim, Germany have addressed this question in a publication in the journal Shock.

In this paper, Henning Ebelt and his colleagues Gave intravenous BMP2 to mice after a heart attack. CD-1 mice were subjected to LAD-ligation to induce a heart attack (LAD stands for left anterior descending coronary artery, which is tied shut to deprive the heart muscle of oxygen). 1 hour after the heart attack, mice were given 80 microgram / gram of body weight of intravenous recombinant BMP2. The hearts of some animals were removed 5-7 days after the heart attack, but others were examined 21 days after the heart attack to determine the physiological performance of the hearts. Control animals were given intravenous phosphate buffered saline.

Coronary arteries

The extirpated hearts were analyzed for cell death, and the size of their heart scars. Also, protein expression analyses showed the different proteins expressed in the heart muscle cells as a result of BMP2 treatment. Also, the effects of BMP2 on cultured heart muscle cells was ascertained.

The results showed that BMP2 could protect cultured heart muscle cells from dying in culture if they when they were exposed to hydrogen peroxide. Hydrogen peroxide mimics stressful conditions and under normal circumstances, cultured heart muscle cells pack up and die in the presence of hydrogen peroxide (200 micromolar for those who are interested). However, if cultured with 80 ng / mL BMP2, the survival of cultured heart muscle cells greatly increased.

When it came to the hearts of mice that were administered iv BMP2, the BMP2-administered mice survived better and had a smaller infarct size (almost 50% of the heart in the controls and less than 40% in the BMP2-administered hearts). When the degree of cell death was measured in the mouse hearts, those hearts from mice that were administered BMP2 showed less cell death (as determined by the TUNEL assay). BMP2 also increased the beat frequency and contractile performance of isolated heart muscle cells.

FInally, the physiological parameters of the BMP2-treated animals were slightly better than in the control animals. The improvements were consistent, but not overwhelming.

Interestingly, when the proteins made by the hearts of BMP2- and PBS-administered animals were analyzed, there were some definite surprises. BMP2 normally signals to cells by binding a two-part receptor that sticks phosphates on itself, and in doing so, recruits “SMAD” proteins to it that end up getting attached to them. The SMAD proteins with phosphates on them stick together and go to the nucleus where they activate gene expression.

BMP signaling

However, the heart muscle cells of the BMP2-administered mice did not contain heavily phosphorylated SMAD2, even though they did show phosphorylated SMAD1, 5, & 8.  I realize that this may sound like Greek to you, but it means this:  Different members of the BMP superfamily signal to cells by utilizing different combinations of phosphorylated SMADs.  The related signaling molecule, TGF-beta (transforming growth factor-beta), increases scar formation in the heart after a heart attack.  TGF-beta signals through SMAD2.  BMP2 does not signal through SMAD2, and therefore, elicits a distinct biological response than TGF-beta.

These results show that BMP2 administration after a heart attack decreases cell death and decreases the size of the heart scar.  There might be a clinical use for BMP2 administration after a heart attack.

See Henning Ebelt, et al., Shock 2013 Apr;39(4):353-60.

Mesenchymal Stem Cells Engineered to Express Tissue Kallikrein Increase Recovery After a Heart Attack


Julie Chao is from the Department of Biochemistry and Molecular Biology, at the Medical University of South Carolina. Dr. Chao and her colleagues have published a paper in Circulation Journal about genetically modified mesenchymal stem cells and their ability to help heal a heart that has just experienced a heart attack.

Several laboratories have used mesenchymal stem cells (MSCs), particularly from bone marrow, to treat the hearts of laboratory animals that have recently experienced a heart attack. However, heart muscle after a heart attack is a very hostile place, and implanted MSCs tend to pack up and die soon after injection. Therefore, such injected cells do little good.

To fix this problem, researchers have tried preconditioning cells by growing them in a harsh environment or by genetically engineering them with genes that can increase their tolerance of harsh environments. Both procedures have worked rather well. In this paper, Chao and her group engineered bone marrow-derived MSCs to express the genes that encode “tissue kallikrein” (TK). TK circulates throughout our bloodstream but several different types of cells also secrete it. It is an enzyme that degrades the protein “kininogen” into small bits that have several benefits. Earlier studies from Chao’s own laboratory showed that genetically engineering TK into the heart improved heart function after a heart attack and increased the ability of MSCs to withstand harsh conditions (see Agata J, Chao L, Chao J. Hypertension 2002; 40: 653 – 659; Yin H, Chao L, Chao J. Journal of  Biol Chem 2005; 280: 8022 – 8030). Therefore, Chao reasoned that using MSCs engineered to express TK might also increase the ability of MSCs to survive in the post-heart attack heart and heal the damaged heart.

In this paper, Chao and others made adenoviruses that expressed the TK gene. Adenoviruses place genes inside cells, but they do not integrate those genes into the genome of the host cell. Therefore, they are safer to use than retroviruses. Chao and others used these TK-expressing adenoviruses to infect tissue and MSCs.

When TK-expressing MSCs were exposed to low-oxygen conditions, like what cells might experience in a post-heart attack heart, the TK-expressing cells were much heartier than their non-TK-expressing counterparts. When injected into rat hearts 20 minutes after a heart attack had been induced, the TK-expressing MSCs showed good survival and robust TK expression. Control hearts that had been injected with non-TK-expression MSCs or had not been given a heart attack showed no such elevation of TK expression.

There were also added bonuses to TK-expressing MSC injections. The amount of inflammation in the hearts was significantly less in the hearts injected with TK-expressing MSC injections compared to the controls. There were fewer immune cells in the heart 1 day after the heart attack and the genes normally expressed in a heart that is experiencing massive inflammation were expressed at lower levels relative to controls, if they were expressed at all.

Reduced inflammation by TK-MSC administration was determined by (C) ED-1 immunohistochemical staining, (D) monocyte/macrophage quantification, (E) neutrophil quantification, and gene expression of (F) TNF-α, (G) ICAM-1, and (H) MCP-1. ED-1-positive cells are indicated by arrows. Original magnification, ×200. Data are mean ± SEM (n=5–8). *P<0.05 vs. other MI groups; **P<0.05 vs. MI/Control group. MSC, mesenchymal stem cell.
Reduced inflammation by TK-MSC administration was determined by (C) ED-1 immunohistochemical staining, (D) monocyte/macrophage quantification, (E)
neutrophil quantification, and gene expression of (F) TNF-α, (G) ICAM-1, and (H) MCP-1. ED-1-positive cells are indicated by arrows.
Original magnification, ×200. Data are mean ± SEM (n=5–8). *P

Another major bonus to the injection of TK-expressing MSCs into the hearts of rats was that these cells protected the heart muscle cells from programmed cell death. To make sure that this was not some kind of weird artifact, Chao and her team placed the TK-expressing MSCs in culture with heart muscle cells and then exposed them to low-oxygen tension conditions. Sure enough, the heart muscle cells co-cultured with the TK-expressing MSCs survived better than those co-cultured with non-TK-expressing MSCs.

TK-MSCs protect against cardiac cell apoptosis at 1 day after myocardial infarction (MI) and in vitro. TK-MSC administration reduced apoptosis in the infarct area at 1 day after MI, as determined by (A) TUNEL staining, (B) quantification of apoptotic cells, and (C) caspase-3 activity. Original magnification, ×200. Data are mean ± SEM (n=5–8). *P<0.05 vs. other MI groups. Cultured cardiomyocytes treated with 0.5 ml of TK-MSC-conditioned medium exhibit higher tolerance to hypoxia-induced apoptosis, as evidenced by (D) Hoechst staining,
TK-MSCs protect against cardiac cell apoptosis at 1 day after myocardial infarction (MI) and in vitro. TK-MSC administration
reduced apoptosis in the infarct area at 1 day after MI, as determined by (A) TUNEL staining, (B) quantification of apoptotic
cells, and (C) caspase-3 activity. Original magnification, ×200. Data are mean ± SEM (n=5–8). *Pcardiomyocytes treated with 0.5 ml of TK-MSC-conditioned medium exhibit higher tolerance to hypoxia-induced apoptosis, as
evidenced by (D) Hoechst staining,

Finally, when the hearts of the rats were examined 2 weeks after the heart attack, it was clear that the enlargement of the heart muscle (so-called “remodeling”) occurred in animals that had received non-TK-expressing MSCs or had received no MSCs at all, but did not occur in the hearts of rats that had received injections of TK-expressing MSCs. The heart scar was also significantly smaller in the hearts of rats that had received injections of TK-expressing MSCs, and had a greater concentration of new blood vessels. Apparently, the TK-expressing MSCs induced the growth of new blood vessels by recruiting EPCs to the heart to form new blood vessels.

In conclusion, the authors write that “MSCs genetically-modified with human TK are a potential therapeutic for ischemic heart diseases.”

Getting FDA approval for genetically engineered stem cells will not be easy, but TK engineering seems much safer than some of the other modifications that have been used. Also the vascular and cardiac benefits of this gene seem clear in this rodent model. Pre-clinical trials with larger animals whose cardiac physiology is more similar to humans is definitely warranted and should be done before any talk of human clinical trials ensues.

Making New Neurons When You Need Them


Western societies are aging societies, and the incidence of dementias, Alzheimer’s disease, and other diseases of the aged are on the rise. Treatments for these conditions are largely supportive, but being able to make new neurons to replace the ones that have died is almost certainly where it’s at.

At INSERM and CEA in Marseille, France, researchers have shown that chemicals that block the activity of a growth factor called TGF-beta improves the generation of new neurons in aged mice. These findings have spurred new investigations into compounds that can enable new neuron production in order to mitigate the symptoms of neurodegenerative diseases. Such treatments could also restore the cognitive abilities of those who have suffered neuron loss as a result of radiation therapy or a stroke.

The brain forms new neurons regularly to maintain our cognitive abilities, but aging or radiation therapy to treat tumors can greatly perturb this function. Radiation therapy is the adjunctive therapy of choice for brain tumors in children and adults.

Various studies suggest that the reduction in our cache of neurons contributes to cognitive decline. For example, exposure of mice to 15 Grays of radiation is accompanied by disruption to the olfactory memory and reduction in neuron production. A similar event occurs as a result of aging, but in human patients undergoing radiation treatment, cognitive decline is accelerated and seems to result from the death of neurons.

How then, can we preserve the cache of neurons in our brains? The first step is to determine the factors responsible for the decline is neuron production. In contrast to contemporary theory, neither heavy doses of radiation nor aging causes completely destruction of the neural stem cells that can replenish neurons. Even after doses of radiation and aging, neuron stem cell activity remains highly localized in the subventricular zone (a paired brain structure located in the outer walls of the lateral ventricles), but they do not work properly.

Subventricular Zone
Subventricular Zone

Experiments at the INSERM and CEA strongly suggest that in response to aging and high doses of radiation, the brain makes high levels of a signaling molecule called TGF-beta, and this signaling molecule pushes neural stem cell populations into dormancy. This dormancy also increases the susceptibility of neural stem cells into apoptosis.

Marc-Andre Mouthon, one of the main authors of this research, explained his results in this manner: “Our study concluded that although neurogenesis is reduced in aging and after a high dose of radiation, many stem cells survive for several months, retaining their ‘stem’ characteristics.”

Part two of this project showed that blocking TGFbeta with drugs restored the production of new neurons in aging or irradiated mice.

Thus targeted therapies that block TGFbeta in the brains of older patients or cancer patients who have undergone high dose radiation for a brain tumor might reduce the impact of brain lesions caused by such events in elderly patients who show distinct signs of cognitive decline.

Mesenchymal Stem Cells from Diabetic Patients Show Impaired Abilities


When using a patient’s own stem cells to treat their diseases, there is a caveat to such a treatment. Things like great age, diabetes mellitus, or a heart attack can seriously compromise the quality of the patient’s stem cells.

To determine if a patient’s stem cells can potentially work under certain circumstances, it is necessary that we test them. With that in mind, Huishan Wang’s laboratory from Shenyang Northern Hospital in Liaoning, China, has extracted mesenchymal stem cells from the bone marrow of patients with type II diabetes. These bone marrow stem cells were used to treat rats that had suffered heart attacks. As a comparison, Wang and his associates used mesenchymal stem cells from the bone marrow of patients diagnosed with coronary artery disease, but not diabetes mellitus.

In these experiments, all patients were between the ages of 50 to 60 years of age, had type II diabetes for at least 10 years, previously suffered a heart attack, and had no signs of liver, kidney or infectious diseases, and no cancer. Bone marrow samples were taken from the breastbone (sternum) during coronary bypass surgery, and the mesenchymal stem cells (MSCs) were extracted from the bone marrow and grown in culture for up to three passages. Ten diabetic patients were selected and two non-diabetic patients were used as a control group.

Male rats (Sprague-Dawley rats for those who are interested) were given heart attacks and then the MSCs were injected into the heart tissue in the area of the heart scar and in the areas adjacent to the heart scar. One group of rats received injections of MSCs from the patients that had type II diabetes, the second group with MSCs from the non-diabetic patients, and a third group rats received only injections of culture medium. The rats were given shots on the drug cyclosporine to ensure that none of the mice rejected their grafted cells. Heart function was assessed with echocardiography, and the tissue was examined, post-mortem, with a “TUNEL” assay, to determine the number of dead cells in the heart, and protein expression was also determined with Western blots.

The MSCs were tested for growth characteristics in culture and gene expression patterns were assayed with microarray studies.

Wang and others found that the MSCs from diabetic patients grew noticeably slower in culture than MSCs from non-diabetic patients. Also, the gene expression profiles a few significant examples; levels of the anti-cell death protein Bcl-2 were significantly lower in MSCs from diabetics.

When it came to the heart function of rats that had received MSC injections after a heart attack, those rats that had received MSCs from diabetic patients fared far worse than those that had received the MSCs from non-diabetics. Also, hearts that had received MSCs from diabetic patients had great amounts of cell death, and expressed significantly lower amounts of growth factors, and the anti-cell death protein Bcl-2.

These data show that MSCs from diabetic patients are impaired in the proliferative ability, and in their survival. This poor survival is due to lower levels of the anti-cell death protein Bcl-2.

Bcl2 activity
Bcl2 activity

A consequence of these experiments is that autologous or self stem cell transplantations in type I diabetics will probably be unsuccessful. This means that allogeneic transplantations or transplants that use stem cells from donors who are not diabetics are a better strategy for treating diabetics.

Mesenchymal Stem Cell Transplantation Improves Heart Remodeling After a Heart Attack


Stem cell scientists from the University of Maryland, Baltimore have used bone marrow mesenchymal stem cells (MSCs) to treat sheep that had suffered a heart attack. They found that the injected stem cells prevented the heart from deteriorating.

This work was a collaboration between the laboratories of Mark Pittenger, ZhonGjun Wu and Bartley Griffith from the Department of Surgery and the Artificial Organ Laboratory.

After a heart attack, the region of the heart that was deprived of oxygen undergoes cell death and is replaced by a heart scar. However, the region next to the dead cells also undergo problematic changes. The cells in these regions adjacent to dead region must contract more forcibly in order to compensate for the noncontracting dead region. These cells enlarge, but some undergo cell death due to inadequate blood supply. There are other changes that can occur, such as abnormalities in Calcium ion handling and poor contractability.

Thus, the problems that result from a heart attack can spread throughout the heart and cause heart failure. In this experiment, the U of Maryland scientists injected MSCs into the sheep hearts four hours after a heart attack to determine if the stem cells could prevent the region adjacent to the dead heart cells from deteriorating.

In this experiment, bone marrow MSCs were isolated from sheep bone marrow and put through a battery of tests to ensure that they could differentiate into bone, cartilage, and fat. Once the researchers were satisfied that the MSCs were proper MSCs, they induced heart attacks in the sheep, and then injected ~200 million MSCs into the area right next to the region of the heart that died.

After 12 weeks, tissue biopsies from these sheep hearts were taken and examined. Also, the sheep hearts were measured for their heart function and structure.

The sheep that did not receive any MSC injections continued to deteriorate and showed signs of stress. The cells adjacent to the dead region expressed a cadre of genes associated with increased cell stress. Furthermore, there was increased cell death and evidence of scarring in the region adjacent to the death region. There was also evidence of Calcium ion-handling problems in the adjacent tissue and increased cell death.

On the other hand, the hearts of the sheep that had received injections of MSCs into the area adjacent to the dead region showed a reduced expression of those genes associated with increased cell stress. Also, these hearts contracted better than those that had not received stem cell injections. There was also less cell death, less scarring, and no evidence of Calcium ion-handling problems.

Changes that occur in the heart after a heart attack are collectively referred to as “remodeling.” Remodeling begins regionally, in those areas near the dead heart cells, but these deleterious changes spread to the rest of the heart, resulting in heart failure. The injections of MSCs into the area next to the dead region clearly prevented remodeling from occurring.

This pre-clinical study is a remarkable study for another reason: the MSCs used in this study were allogeneic. Allogeneic is a fancy way of saying that they did not come from the same animal that suffered the heart attack, but from some other healthy animal. Therefore, the delivery of a donor’s MSCs into the heart of a heart attack patient could potentially prevent heart remodeling.

The main problem with this experiment is that the MSCs were injected directly into the heart muscle. In humans, such a procedure requires special equipment and carries potential risks that include perforation of the heart wall, rupture of the heart wall, or further damaging the heart muscle. Therefore, if such a technology could be adapted to a more practical delivery system in humans, then certainly human clinical trials should be forthcoming.

See Yunshan Zhao, et al., “Mesenchymal stem cell transplantation improves regional cardiac remodeling following ovine infarction.” Stem Cells Translational Medicine 2012;1:685-95.