Finding the Optimal Spot for Stem Cell Injections In Spinal Cord Injured Patients


A gaggle of laboratory animal experiments and clinical studies in human patients have established that stem cell injections into the spinal cord after spinal cord injury promote functional recovery (see Beattie, M. S., et al., Exp. Neurol. 148(2):453‐463; 1997; Bennett, D. L., et al., J. Neurosci. 20(1):427‐437; 2000; Kim HK, et al., PLos One 4(3): e4987 2009; Lu, P.; Tuszynski, M. H. Exp. Neurol. 209(2):313‐320; 2008; McTigue, D. M., et al., J. Neurosci. 18(14):5354-5365; 1998; Widenfalk, J.; Lundströmer, K. J. Neurosci. 21(10):3457‐3475; 2001; also see Salazar DL, et al., PLoS ONE, August 2010; Hooshmand M, et al., PLoS ONE, June 2009; Cummings BJ, et al., Neurological Research, July 2006; and Cummings BJ, et al., PNAS, September 19, 2005).  Stem Cell, Inc., for example, has conducted several tests with human patients using their HuCNS-SC human neural stem cell line, and transplantation of these stem cells promotes functional recovery in human patients who have suffered spinal cord injury.

However, one factor that has yet to be properly determined is the best site for stem cell injection. Previous work by scientists at the Keio University School of Medicine in Japan has shown that injection of neural stem cells and neural progenitor cells (NS/PCs) into non-injured sites by either intravenous or intrathecal (introduced directly into the space under the arachnoid membrane of the brain or spinal cord) administration failed to produce sufficient engraftment of stem cells at the site of injury.

Arachnoid space

Instead cells were trapped in the lungs and kidneys, and many mice even developed fatal lung conditions as a result of intravenous administration (see Takahashi Y., et al., Cell Transplant. 2011;20(5):727-39). These data convinced them that intralesional application of the stem cells (injections directly into the damaged site of the spinal cord) might be the most effective and reliable method for NS/PC tranplantations.

A new study by the Keio group has attempted to ascertain the efficacy of the intralesional injections. Mice with spinal cord injuries were injected with NS/PCs that had been derived from mice that expression glowing proteins. This allowed the injected cells to be tracked with bio-luminescence imaging (BLI).

The principal investigator of this research is Masaya Nakamura from the Department of Orthopedic Surgery at the Keio University School of Medicine. Dr. Nakamura and his team gave mice spinal contusions at the level of the tenth thoracic vertebra. Then some mice were given low doses and others high doses of NS/PCs that were derived from fetal mice (for those who are interested, low dose – 250,000 cells per mouse; high dose – 1 million cells per mouse) nine days after spinal cord injury. These mice were further divided into two groups: those injected at the lesion epicenter (E), those injected at sites at the front and back of the lesion (RC for “rostral/caudal”). Thus there were four groups total: High dose E, High dose RC, Low dose E, and Low dose RC.

All four groups showed better functional recovery than the control group, which was injected with phosphate buffered saline. BLI showed that the number of cells that survived in each of the four cell-transplanted groups was about the same across these groups.  Thus injecting more cells does not lead to greater numbers of surviving neural stem cells.  This makes sense, since the damaged spinal cord in  very inhospitable place for transplanted cells.

However, when the mice were examined for the expression of particular brain-derived neurotropic factors, the expression of such genes was higher in the RC-injected mice than in the E-injected mice. These results seems to explain why the transplanted NS/PCs differentiated more readily into neurons in the RC-injected mice rather than a type of glial cell known as an astrocyte, as was the case in the E-injected mice.

Human Astrocytes
Human Astrocytes

Nakamura and his team interpreted these results to mean that the environments of the E and RC sites can both support the survival of transplanted NS/PCs during the sub-acute phase of spinal cord injury. The authors conclude with a practical note: “Therefore, we conclude that it is optimal to graft a certain threshold number of NS/PCs into the epicenter lesion during the sub-acute phase of SCI, and thereby avoid causing further iatrogenic injury to the intact RC regions of the spinal cord.”

Hopefully Nakamura’s work will be translated into further human clinical trials. One feature of this study is that a particular threshold of stem cells survive when injected into the spinal cord and injecting larger numbers of cells does not increase the number of surviving cells. Injecting more cells might only contribute to the cell debris in the spinal cord. This is certainly a good thing to know when conducting clinical trials with neural stem cells in spinal cord-injured patients.

Primed Fat-Based Stem Cells Enhance Heart Muscle Proliferation


A Dutch group from the University of Groningen has shown that fat-based stem cells can enhance the proliferation of cultured heart muscle cells. The stem cells used in these experiments were preconditioned and this pretreatment greatly enhanced their ability to activate heart muscle cells.

This paper, by Ewa Przybyt, Guido Krenning, Marja Brinker, and Martin Harmsen was published in the Journal of Translational Medicine. To begin, Przybyt and others extracted human adipose derived stromal cells (ADSC) from fat tissue extracted from human liposuction surgeries. To do this, they digested the fat with enzymes, centrifuged and washed it, and then grew the remaining cells in culture.

Then they used rat neonatal heart muscle cells and infected them with viruses that causes them to glow when certain types of light was shined on them. Then Przybyt and others co-cultured these rat heart cells with human ADSCs.

In the first experiment, the ADSCs were treated with drugs to prevent them from dividing and then they were cultured with rat heart cells in a one-to-one ratio. The heart muscle cells grew faster with the ADSCs than they did without them. To determine if cell-cell contact was required for this stimulation, they used the culture medium from ADSCs and grew the heart cell on this culture medium. Once again, the heart cells grew faster with the ADSC culture medium than without it. These results suggest that the ADSCs stimulate heart cell proliferation by secreting factors that activate heart cell division.

Another experiment subjected the cultured heart cells to the types of conditions they might experience inside the heart after a heart attack. For example, heart cells were subjected to low oxygen tensions (2% oxygen), and inflammation – two conditions found within the heart after a heart attack. These treatments slowed heart cell growth, but this heart cell growth was restored by adding the growth medium of ADSCs. Even more remarkably, when ADSCs were grown in low-oxygen conditions or treated with inflammatory molecules (tumor necrosis factor-alpha or interleukin-1beta), the culture medium increased the fractions of cells that grew. Therefore, ADSCs secrete molecules that increase heart muscle cell proliferation, and increase proliferation even more after the ADSCs are preconditioned by either low oxygen tensions or inflammation.

In the next experiment, Przybyt and others examined the molecules secreted by ADSCs under normal or low-oxygen tensions to ascertain what secreted molecules stimulated heart cell growth. It was clear that the production of a small protein called interleukin-6 was greatly upregulated.

Could interleukin-6 account for the increased proliferation of heart cells? Another experiment showed that the answer was yes. Cultured heart cells treated with interleukin-6 showed increased proliferation, and when antibodies against interleukin-6 were used to prevent interleukin-6 from binding to the heart cells, these antibodies abrogated the effects of interleukin-6.

Przybyt and others then took these results one step further. Since the signaling pathways used by interleukin-6 are well-known, they examined these pathways. Now interleukin-6 signals through pathways, once of which enhances cell survival, and another pathway that stimulated cell proliferation. The cell proliferation pathway uses a protein called “STAT3” and the survival function uses a protein called “Akt.” Both pathways were activated by interleukin-6. Also, the culture medium of ADSCs that were treated with interleukin-6 induced the interleukin-6 receptor proteins (gp80 and gp130) in cultured heart muscle cells. This gives heart muscle cells a greater capacity to respond secreted interleukin-6.

This paper shows that stromal stem cells from fat has the capacity, in culture, to activate the growth of cultured heart muscle cells. Also, if these cells were preconditioned with low oxygen tensions or pro-inflammatory molecules, those fat-based stem cells secreted interleukin-6, which enhanced heart muscle cell survival, and proliferation, even if those heart muscle cells are exposed to low-oxygen tensions or inflammatory molecules.

This suggests that preconditioned stem cells from fat might be able to protect heart muscle cells and augment heart healing after a heart attack. Alternatively, cardiac administration of interleukin-6 after a heart attack might prove even more effective to protect heart muscle cells and stimulate heart muscle cell proliferation. Human trials anyone?

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.

Treating the Heart with Mesenchymal Stem Cells: Timing and Dosage


Stephen Worthley from the Cardiovascular Investigation Unit at the Royal Adelaide Hospital in Adelaide, Australia and his colleagues have conducted a timely experiment with rodents that examines the effects of dosage and timing on stem cell treatments in the heart after a heart attack.

Mesenchymal stem cells from bone marrow and other sources have been used to treat the heart of laboratory animals and humans after a heart attack. The optimal timing for such a treatment remains uncertain despite a respectable amount of work on this topic. Early intervention (one week) seems offer the best hope for preserving cardiac function, but the heart at this stage is highly inflamed and cell survival is poor. If treatment is delayed (2-3 weeks after the heart attack), the prospects for cell survival are better, but the heart at this time is undergoing remodeling and scar formation. Therefore, stem cell therapy at this time seems unlikely to work. Human clinical trials seem to suggest that mesenchymal stem cell treatment 2-3 weeks after a heart attack does no good (see Traverse JH, et al JAMA 2011;306:2110-9). The efficacy of the delivering mesenchymal stem cells to the heart at these different times has also not been compared.

If that degree of uncertainty is not enough, dosage is also a mystery. Rodent studies have used doses of one million cells, but studies have not established a linear relationship between efficacy and dose, and higher dosages seem to plateau in effectiveness (see Dixon JA, et al Circulation 2009;120(11 Suppl):S220-9). High doses might even be deleterious.

So what is the best time to administer after a heart attack, and how much should be administered? These are not trivial questions. Therefore a systematic study is required and laboratory animals such as rodents are required.

In this study, five groups of rats were given heart attacks by ligation of the left anterior descending artery, and two groups of rats received bone marrow-derived mesenchymal stem cells immediately after the heart attack. The first group received a low dose (one million cells) and the second group received twice as many cells. The three other groups received their treatments one week after the heart attack. The third group received the low dose of stem cells received the low dose of cells (one million cells), and the fourth group received the higher dose (two million cells). The fifth group received no such cell treatment.

All mesenchymal stem cells were conditioned before injection by growing them under low oxygen conditions. Such pretreatments increase the viability of the stem cells in the heart.

The results were interesting to say the least. when assayed four weeks after the heart attacks, the hearts of the control animals showed a left ventricular function that tanked. The ejection fraction fell to 1/3rd the original ejection fraction (~60% to ~20%) and stayed there. The early high dose animals showed the lowest decrease in ejection fraction (-8%). The early low dose group showed a greater decrease in ejection fraction. Clearly dose made a difference in the early-treated animals with a higher dose working better than a lower dose.

In the later-treated animals, dose made little difference and the recovery was better than the early low dose animals. when ejection fraction alone was considered. However, when other measures were considered, the picture becomes much more complex. End diastolic and end systolic volumes were all least increased in the early high dose animals, but all four groups show significantly lower increases than the controls. The mass of the heart, however, was highest in the late high-dose animals as was ventricular wall thickness.

When the movement of the heart walls were considered, the early-treated animals showed the best repair of those territories of the heart near the site of injection, but the later-treated animals showed better repair at a distance from the site of injection. The same held for blood vessel density: higher density in the injected area in the early-treated animals, and higher blood vessel density in those areas further from the site of injection in the later-treated animals.

The size of the heart scar clearly favored the early injected animals, which the lower amount of scarring in the early high dose animals. Finally when migration of the mesenchymal stem cells throughout the heart was determined by using green fluorescent protein-labeled mesenchymal stem cells, the later injected mesenchymal stem cells were much more numerous at remote locations from the site of injection, and the early treated animals only had mesenchymal stem cells at the site of injection and close to it.

These results show that the later doses of mesenchymal stem cells improve the myocardium further from the site of the infarction and the early treatment improve the myocardium at the site of the infraction. Cell dosage is important in the early treatments favoring a higher dose, but not nearly as important in the later treatments, where, if anything, the data favors a lower dose of cells.

Mesenchymal stem cells affect the heart muscle by secreting growth factors and other molecules that aids and abets healing and decreases inflammation. However, research on these cells pretty clearly shows that they modulate their secretions under different environmental conditions (see for example, Thangarajah H et al Stem Cells 2009;27:266-74). Therefore, the cells almost certainly secrete different molecules under these conditions.

In order to confirm these results, similar experiments in larger animals are warranted, since the rodent heart is a relatively poor model for the human heart as it beats much faster than human hearts.

See James Richardson, et al Journal of Cardiac Failure 2013;19(5):342-53.