Preconditioning Your Way to Better Stem Cells


When stem cells are implanted into injured tissues, they often face a hostile environment that is inimical to their survival. A stroke, for example, can produce brain tissue without ample blood flow, low oxygen levels, and lots of cell debris and inflammation. The same can be said for the heart after a heart attack. If stem cells are going to help anyone we have to find a way for them to survive.

The first hints came in the form of genetically-engineered stem cells that expressed a host of genes that can help cells survive in low oxygen, high stress environments. However, the FDA is unlikely to approve genetically engineered cells for therapeutic purposes. Therefore, a more “user-friendly” way to precondition cells was sought, and found. Instead of loading cells up with extra genes, all you had to do was grow the cells under low oxygen, high stress conditions, and they would adapt and survive when implanted into damaged tissue. This, however, has a drawback: if you want to treat a patient, you do not always have the time it takes for extract and isolate their cells, grow them in culture over a week or two, and then implant them. Is there a better way?

The answer turns out to be yes. Treating cells with particular compounds or growth factors can induce resistance to low-oxygen, high-stress conditions, and two papers show us how it’s done.

The first paper is from the laboratory of Ling Wei at Emory University School of Medicine in Atlanta who has shown in the past that low-oxygen adaptation of mesenchymal stem cells from bone marrow made them better able to treat acute heart attacks in laboratory animals. In this paper, Wei and her colleagues exploited a biochemical pathway known to induce resistance of low-oxygen conditions known as the HIF-1 pathway. The HIF-1 pathway consists of two proteins that work as a pair; HIF1alpha and HIF1beta. HIF1beta is made all the time and HIF1alpha is oxygen sensitive. In the presence of oxygen, enzymes called prolyl hydroxylases modify HIF1alpha, marking it for destruction. In the absence of oxygen, the prolyl hydroxylases do not have enough oxygen to modify HIF1alpha and the HIF1alpha/beta complex activates the expression of a host of genes necessary for increased tolerance to low oxygen levels. Therefore, to make cells more tolerant to low oxygen levels, we need to turn on the HIF1 pathway and to do that we need to inhibit the prolyl hydroxylases.

This turns out to be pretty straight forward. A small molecule called dimethyloxalyglycine or DMOG can effectively inhibit prolyl hydroxylase and induce survival in low-oxygen, high-stress environments. Therefore Wei and her group used DMOG to treat cells and test them out.

In culture, the DMOG-treated cells made proteins known to be important for the establishment of new blood vessels and for survival. When they were compared to cultured stem cells that had not been treated with DMOG, the DMOG-treated cells expressed significantly more of VEGF, Glut-1 and HIF1alpha, all of which are important for surviving in low-oxygen environments. In a Matrigel assay, the DMOG-treated cells also made more blood vessels that were longer than their non-DMOG-treated counterparts.

When used in laboratory animals that had suffered heart attacks, the DMOG-treated cells distinguished themselves once again. They survived better than the control cells and hearts that had received the DMOG-treated cells had much smaller heart scars after heart attacks. Functional assays of heart function illustrated that the DMOG-treated cells helped their heart perform above and beyond what was shown observed in the animals implanted with stem cells that had not bee treated with DMOG.

Thus it is possible to precondition cells without long culture periods or genetic engineering. One compound can accomplish it and the cells only needed to be exposed to DMOG for 24 hours.

In a similar vein, Genshan Ma and others from Zhongda Hospital in Nanjing, China used a small peptide called bradykinin to precondition human umbilical cord endothelial progenitor cells (EPCs); the cells that form blood vessels. In this paper, Ma and colleagues used bradykinin-treated EPCs to treat heart attacks in mice. One nice aspect of this paper is the large number of controls they ran with their experimental runs.

The bradykinin-treated cells outperformed their untreated counterparts when it came to the size of the heart scar, the number of dead cells in the heart, and heart performance parameters. Cell culture experiments established that the bradykinin-treated cells expressed the Akt kinase at high levels, and expressed higher levels of VEGF, the blood vessel-inducing growth factor. Bradykinn-treated cells also were more resistant to being starved for oxygen, and survived better under unusual culture conditions. All of these benefits could be abrogated by inhibiting the activity of the Akt kinase by treating cells with LY294002, a compound that specifically inhibits the activator of Akt.

In this case, cells were treated with bradykinin for 10 minutes to 12 hours.

Two papers, two success stories. Stem cell preconditioning certainly works in laboratory animals. Since stem cell trials have been completed in human patients, it might be time to try preconditioned stem cells in human patients.

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.