Inhibition of AKT Kinase Increases Umbilical Cord Blood Growth in Culture and Engraftment in Mice


Dr. Yan Liu from the Department of Pediatrics and the Herman B Wells Center for Pediatric Research at the Indiana University School of Medicine in Indianapolis, Indiana and his colleagues have increased the engraftment efficiency of umbilical cord hematopoietic (blood cell-making) stem cells in immunodeficient mice. The technique developed by Lui and his colleagues is simple and increases the proliferation of umbilical cord blood hematopoietic stem cells (UCB-HSCs) in culture, which potentially solves several long-standing problems with umbilical cord blood transplantation.

Umbilical cord blood has been used in the clinic for more than 40 years in hematopoietic stem cell transplantation therapies to treat patients with bone marrow diseases or to reconstitute the bone of those cancer patients who had to have theirs wiped out to cure their leukemia or lymphoma.

One of the problems with umbilical cord blood transplantations, however, is the small amount of material in a typical cord blood collection and, therefore, the small number of hematopoietic stem cells (HSCs) available for transplantation. To ameliorate these shortcomings, hematologists will transplant more than one lot of cord blood (a so-called “double umbilical cord blood transplantation”), which, unfortunately, also increases the risk of immunological rejection (so-called Graft Versus Host response).

A second strategy to get around the low numbers of UCB-HSCs is to expand them in culture, which has proven difficult. However, some experiments have given us more than enough hope to suspect this this is a feasible option (see Flores-Guzmán P, et al., Stem Cells Transl Med. 2013 Nov;2(11):830-8; Bari S., et al., Biol Blood Marrow Transplant. 2015 Jun;21(6):1008-1; Pineault N, Abu-Khader A. Exp Hematol. 2015 Jul;43(7):498-513).

Dr. Lui and his coworkers wanted to examine the role of the signaling protein AKT (also known and protein kinase B) in UCB-HSC expansion in culture. To this end, they used silencing RNAs to knock-down AKT gene expression in cultured UCB-HSCs. AKT knock-down enhanced UCB-HSC quiescence and growth in culture. In a separate experiment, Lui and others treated human UCB-HSCs (so-called CD34+ cells) with a chemical that specifically inhibits AKT activity. Then they subjected these cells to a battery of tests in culture and in laboratory mice.

The results were astounding.  Treatment of human UCB-HSCs did not affect the identity of the HSCs and enhanced their ability to form isolated colonies in cell culture growth tests known as “replating assays.”  Additionally, the short-term inhibition of AKT with drugs also enhanced the ability of UBC-HSCs to repopulate the bone marrow of immunodeficient mice.

ubc-hsc-engraftment-improved-with-akt-inhibition

In summary, inhibition of AKT increases human UCB-HSC quiescence, growth potential, and engraftment in laboratory mice.

These interesting pre-clinical results suggest that AKT inhibitor can increase the expansion of UCB-HSCs in culture and potential increase their tendency of these cells to engraft in patients.

Elabela, A New Human Embryonic Stem Cell Growth Factor


When embryonic stem cell lines are made, they are traditionally grown on a layer of “feeder cells” that secrete growth factors that keep the embryonic stem cells (ESCs) from differentiating and drive them to grow. These feeder cells are usually irradiated mouse fibroblasts that coat the culture dish, but do not divide. Mouse ESCs can be grown without feeder cells if the growth factor LIF is provided in the medium. LIF, however, is not the growth factor required by human ESCs, and therefore, designing culture media for human ESCs to help them grow without feeder cells has proven more difficult.

Having said that, several laboratories have designed media that can be used to derive human embryonic stem cells without feeder cells. Such a procedure is very important if such cells are to be used for therapeutic purposes, since animal cells can harbor difficult to detect viruses and unusual sugars on their cell surfaces that can also be transferred to human ESCs in culture. These unusual sugars can elicit a strong immune response against them, and for this reason, ESCs must be cultivated or derived under cell-free conditions. However, to design good cell-free culture media, we must know more about the growth factors required by ESCs.

To that end, Bruno Reversade from The Institute of Molecular and Cell Biology in Singapore and others have identified a new growth factor that human ESCs secrete themselves. This protein, ELABELA (ELA), was first identified as a signal for heart development. However, Reversade’s laboratory has discovered that ELA is also abundantly secreted by human ESCs and is required for human ESCs to maintain their ability to self-renew.

Reversade and others deleted the ELA gene with the CRISPR/Cas9 system, and they also knocked the expression of this gene down in other cells with small interfering RNAs. Alternatively, they also incubated human ESCs with antibodies against ELA, which neutralized ELA and prevented it from binding to the cell surface. However Ela was inhibited, the results were the same; reduced ESC growth, increased amounts of cell death, and loss of pluripotency.

How does ELA signal to cells to grow? Global signaling studies of growing human ESCs showed that ELA activates the PI3K/AKT/mTORC1 signaling pathway, which has been show in other work to be required for cell survival. By activating this pathway, ELA drives human ESCs through the cell-cycle progression, activates protein synthesis, and inhibits stress-induced apoptosis.

fx1 (2)

Interestingly, INSULIN and ELA have partially overlapping functions in human ESC culture medium, but only ELA seems to prime human ESCs toward the endoderm lineage. In the heart, ELA binds to the Apelin receptor APLNR. This receptor, however, is not expressed in human ESCs, which suggests that another receptor, whose identity remains unknown at the moment, binds ELA in human ESCs.

Thus ELA seems to act through an alternate cell-surface receptor, is an endogenous secreted growth factor in human

This paper was published in the journal Cell Stem Cell.

Heart Cells Expressing Stem Cell Factor Show Less Cell Death After a Heart Attack


Stem Cell Factor is a cell surface protein that is expressed by several different cells, including tissue fibroblasts, heart cells, cells in the bone marrow, and blood vessel cells. Stem Cell Factor (SCF) plays important roles in the migration, proliferation, and adhesion of any cell that expresses the receptor for SCF, a molecule called c-kit. Cells that express c-kit include cardiac stem cells, endothelial progenitor cells, and hematopoietic stem cells. When c-kit binds to SCF, the SCF-containing cell activate their Akt /PI3K pathway, and this pathway prevents cells from dying and drives them to divide, differentiate, more, adhere, and even secrete new molecules.

pi3k-resized-600

Fu-Li Xang in the laboratory of Qingping Feng at the University of Western Ontario has done several experiments with SCF in the heart. His goal is to determine if heart cells that have SCF fare better after a heart attack than hearts that do not have quite so much SCF.

To that end, Feng and his team showed that SCF does help heal the heart after a heart attack in 2009 (Xiang et al, Circulation 120: 1065-74). The next step was to determine if SCF could attenuate cell death in the heart that results from a heart attack.

SCF-ckit

 

The strategy behind this experiments involved making genetically engineered mice that expressed lots of SCF in their heart muscle. The particular mouse strain that Feng and his crew made had the SCF gene activated by a heart muscle-specific promoter, but the expression of SCF could be shut off by giving the mice the drug doxycycline. These SCF transgenic mice and normal mice were given heart attacks and then some were treated with a doxycycline while others were given a drug called LY294002, which inhibits the Akt pathway. These animals were then analyzed three hours after the induced heart attack and the amount of cell death, the size of the infact, the number of stem cells that moved into the heart were all measured.

 LY294002
LY294002

The upshot of all this work is this: SCF decreased the amount of cell death by about 40%. Also the size of the infarct was also smaller. These benefits were abrogated by the co-administration of either doxycycline or LY294002. When a search for molecules that are indicative of cell death were examined, the results were completely unsurprising: the markers of cells death like fragmented DNA or caspase-3 were decreased in the SCF mice and this attenuation was abrogated by co-administration with doxycycline or LY294002.

Other experiments examined the activation of the Akt/PI3K pathway in the SCF-expressing animals, and it was quite clear that the SCF-expressing animals showed a robustly active Akt/PI3K pathway compared to the non-SCF-expressing mice.

A different experiment examined the presence of c-kit-expressing cells in the hearts of these mice. Remember that c-kit expressing cells are stem cells that have been recruited to the heart by the SCF. Once again, it was exceedingly clear that the SCF-expressing mice had hearts with a large excess of c-kit-expressing cells and this recruitment of stem cells was abrogated by neutralizing c-kit with an antibody against it. The incoming stem cells also tend to secrete a host of interesting molecules that help heal the heart, and one of these molecules, HGF (hepatic growth factor), which also goes up in concentration in the hearts of the SCF-expressing mice, is blocked by a drug called crizotinib. If SCF-expressing mice were pre-treated with crizotinib, the infarct size tended to be just as large as the non-SCF-expressing cells.

Feng and his group also examined the resident stem cells in the heart, the cardiac stem cells population, which, by the way, also express c-kit. These cells also were induced to express HGF and IGF (insulin-like growth factor) as a result of SCF, and if the c-kit receptor was blocked with an antibody, then this effect was abrogated.

There is a lot of data in this paper, but the news is almost all good. Basically SCF will recruit stem cells to the heart after a heart attack and this recruitment happens quickly (within 3 hours) and does the heart a world of good. Translating this work into human patients will not be easy, but SCF is available. If it could be localized to the heart by some means soon after a heart attack, there is good reason to believe, based on these pre-clinical results that it would do the patient quite a bit of good. The next piece is figuring our how to go about doing just that.

Thymosin beta4-Overexpressing Cells Heal Heart After a Heart Attack


Thymosin beta4 is a very highly conserved 43-amino acid peptide that plays a very important role in cell proliferation, migration, and angiogenesis (blood vessel production). Experiments with thymosin beta4 in laboratory animals that have had a heart attack have shown that treatment with thymosin beta4 can reduce cell death in the heart and reduce the size of the infarct, while increasing heart function (see Hannappel E, et al., Arch Biochem Biophys 240 (1985): 236-241; Bock-Marquette, et al., Nature 432 (2007): 466-472; Srivastava D, et al., Ann NY Acad Sci 1112 (2007): 161-170; Grant DS et al., Angiogenesis 3 (1999): 125-135). Also, knocking down thymosin beta4 in endothelial progenitor cells (cells that make blood vessels) prevents these cells from healing the heart after a heart attack (Hinkel, et al., Circulation 117 (2008): 2232-2240).

Thymosin beta4
Thymosin beta4

Given the ability of thymosin beta4 to heal the heart, Dinender Singla and colleagues at the University of Central Florida have engineered embryonic stem cells to express thymosin beta4 and used them to treat laboratory animals that have suffered a heart attack. The results were truly tremendous.

Singla and his team genetically engineered mouse embryonic stem cells to express either red fluorescent protein or red fluorescent protein and thymosin beta4. In culture, those cells that expressed thymosin beta4 showed much more efficient differentiation into heart muscle cells (3-5 times greater).

Effect of Tβ4 Expression on ES Cell Differentiation. A. Fluorescent microscopy of EBs derived from RFP-ES and Tβ4-ES cells. At D12 EBs were stained with anti- sarcomeric α-actin (S-actin) (green) and counterstained with DAPI for nuclear visualization (blue). The lower panel shows S-actin staining in a beating area (square box) in the EBs derived from Tβ4-ES cells. Scale = 200µm. B. Percentage of beating EBs during cardiac myocyte differentiation. Spontaneously beating EBs were examined and counted under a light microscope at D9, 12 and 15. C. Real-time PCR analysis of gene expression of GATA-4, Mef2c and Tbx6 at D12. Data are represented as mean ± SEM, *p< 0.05; vs. RFP ESCs.
Effect of Tβ4 Expression on ES Cell Differentiation.
A. Fluorescent microscopy of EBs derived from RFP-ES and Tβ4-ES cells. At D12 EBs were stained with anti- sarcomeric α-actin (S-actin) (green) and counterstained with DAPI for nuclear visualization (blue). The lower panel shows S-actin staining in a beating area (square box) in the EBs derived from Tβ4-ES cells. Scale = 200µm. B. Percentage of beating EBs during cardiac myocyte differentiation. Spontaneously beating EBs were examined and counted under a light microscope at D9, 12 and 15. C. Real-time PCR analysis of gene expression of GATA-4, Mef2c and Tbx6 at D12. Data are represented as mean ± SEM, *p< 0.05; vs. RFP ESCs.

Next, they gave laboratory mice heart attacks and implanted these cells into the heart. Those mice that received no cells had bucket loads of cell death. Those mice who received embryonic stem cells that did not express thymosin beta4 showed a decrease in cell death 2 weeks after the heart attack. However those mice that received the embryonic stem cells that expressed thymosin beta4 showed a third of the cell death found in the control mice. The same applied to the amount of scarring in the hearts. Animals treated with embryonic stem cells (ESCs) that did not express thymosin beta4 had about half the scarring of the control mice that received no cells, but the hearts treated with thymosin beta4-expressing ESCs showed about a third of the scarring.

Transplanted Tβ4-ES Cells Reduce Cardiac Fibrosis in the Infarcted Mouse Heart. A. Representative photomicrographs of tissue sections stained with Masson’s trichrome at D14 post MI surgery. Scale =100µm. B. Quantitative analysis of interstitial fibrosis for control and experimental groups. #p<0.05 vs. sham, *p<0.05 vs. MI, and $p<0.05 vs. RFP-ESCs. C. Histogram illustrates quantitative MMP-9 expression. #p<0.05 vs sham, *p<0.05 vs. MI. n = 5-7 animals per group.
Transplanted Tβ4-ES Cells Reduce Cardiac Fibrosis in the Infarcted Mouse Heart.
A. Representative photomicrographs of tissue sections stained with Masson’s trichrome at D14 post MI surgery. Scale =100µm. B. Quantitative analysis of interstitial fibrosis for control and experimental groups. #p

When it came to heart function, things were really remarkable. The ESC-treated hearts showed definite improvement over the control animals, but the ESC-thymosin beta4 cells restored heart function so that the hearts worked almost as well as the sham hearts that were never given a heart attack. The fractional shortening was not as high, nor was the end diastolic volume as low, but most of the other functional parameters were close to the sham hearts.

Transplanted Tβ4-ES Cells Improve Cardiac Function in the Infarcted Heart. Echocardiography was performed D14 following MI. A. Raw functional data. Histograms show average quantified measurements of B. left ventricular internal diameter during diastole (LVIDd) C. left ventricular internal diameter during systole (LVIDs) D. fractional shortening FS% E. end diastolic volume (EDV) F. end systolic volume (ESV) G. and ejection fraction EF% at 2 weeks after MI for all treatment groups. #p<0.05 vs. sham, *p<0.05 vs. MI, and $p<0.05 vs. RFP-ESCs. Data set are from n=6-8 animals/group.
Transplanted Tβ4-ES Cells Improve Cardiac Function in the Infarcted Heart.
Echocardiography was performed D14 following MI. A. Raw functional data. Histograms show average quantified measurements of B. left ventricular internal diameter during diastole (LVIDd) C. left ventricular internal diameter during systole (LVIDs) D. fractional shortening FS% E. end diastolic volume (EDV) F. end systolic volume (ESV) G. and ejection fraction EF% at 2 weeks after MI for all treatment groups. #p

Mechanistically, the thymosin beta4 appears to down-regulate PTEN and upregulated the AKT kinase. AKT kinase activation is associated with cell survival and growth. PTEN tends to slow down growth and prevent healing under some conditions.

Effects of Tβ4 Expression on Caspase-3, pAkt, and p-PTEN Activities. Heart homogenates from each group were prepared for ELISA analysis of caspase-3, Akt, and p-PTEN. A. Quantitative analysis of caspase-3, B. p-PTEN, and C. pAkt activity in the hearts following cell transplantation. Data were represented as Mean ± SEM; *p<0.01 vs. MI, #p<0.05 vs. sham. n = 4-5 animals per group.
Effects of Tβ4 Expression on Caspase-3, pAkt, and p-PTEN Activities.
Heart homogenates from each group were prepared for ELISA analysis of caspase-3, Akt, and p-PTEN. A. Quantitative analysis of caspase-3, B. p-PTEN, and C. pAkt activity in the hearts following cell transplantation. Data were represented as Mean ± SEM; *p

This suggests that thymosin beta4 expression seems to augment healing in the heart after a heart attack. Such a therapy could potentially be used to treat heart attack patients, however, more animal experiments will need to be done. What is the proper time frame for thymosin beta4 treatment? How many cells should be implanted in order to provide the maximum therapeutic effect. Can such a treatment be provided via intracoronary delivery? Can conditional expression provide a robust enough response to heal the heart? Can other cells, like mesenchymal stem cells to used to deliver the thymosin beta4? Can c-kit cardiac progenitor cells be used to deliver thymosin beta4?

Many questions remain, but hopefully, this remarkable treatment regime can be ramped up to eventually go to clinical trials.

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?

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.