Nonhematopoietic Stem Cells from Umbilical Cord Blood Improve Heart Function After a Heart Attack


Xin Yu, who has dual appointments at Case Western Reserve University, in Cleveland, Ohio and the University of Minnesota Medical School in Minneapolis, Minnesota, has published a remarkable paper in the journal Cell Transplantation that describes the use of a stem cell population from umbilical cord blood to treat mice that had suffered heart attacks. The non-invasive way in which these cells were administered and the tremendous healing qualities of these cells makes paper unique.

In 2005, Water Low at the University of Minnesota Medical School described the isolation and characterization of a unique stem cell population from umbilical cord blood that he called nonhematopoietic umbilical cord blood stem cells or nh-UCBSCs. These cells were used to treat animals with strokes and they induced the growth of new brain cells in the brains of treated mice (see J Xiao, Z Nan, Y Motooka, and WC Low, Stem Cells Dev. 2005 Dec;14(6):722-33).

Yu used these cells to treat male Lewis rats that had suffered heart attacks. In all cases, the rats were subjected to open-heart surgery and the left anterior descending artery was tied off to induce a heart attack. One group of rats were operated on but no heart attacks were induced. A second group was given heart attacks, and then two days later were given intravenous saline infusions. The third group was given a heart attack and then two days later, were injected with one million nonhematopoietic umbilical cord stem cells into their tail veins.

Ten months after the surgery, the heart structure and function of animals from all three groups was assessed with tensor diffusion magnetic imaging, and a pressure‐volume conductance catheter. The hearts were also extirpated from the animals and structurally assessed by means of staining and 3-D imaging.

The stem cell-treated animals were compared with the sham-operated animals and the saline-treated animals. In almost all categories, the stem cell-treated animals had better function. Also, the overall structure of the heart was preserved and looked more like the normal heart than the saline-treated hearts. For example, in the saline-treated group, the heart wall thickness in the infarct zone was reduced by 50% compared to the control rats, and wall thickness at the border zone was also significantly
decreased. However, there were no statistical difference in wall thickness between the stem cell-treated group and the control group.

Additional finds were that the stem cell-treated group had significantly smaller areas of dead cells, more blood vessels, and better heart muscle fiber structure that contracted better.

These data show that the long-term effects of nh-UCBSC administration was to preserve the structure, and, consequently, the function of the heart after a heart attack.

However, the added bonus to this work is that the animals were injected with these cells into the tail vein. The animals did not have to have their chests cracked, or have over-the-wire stent technology to implant these cells; they merely introduced them intravenously. Apparently, the nh-UCBSCs homed to the damaged heart and mediated its healing. If such healing can be translated to human patients, this could truly be a revolutionary find.

Umbilical Cord Blood Stem Cells in a Biodegradable Scaffold Regenerate Full-Thickness Skin Defects


In a new study published in the ASAIO Journal by Reza Zeinali and others in the laboratory of Kamal Asadipour, specific stem cell from umbilical cord blood called unrestricted somatic stem cells (USSCs) have been grown on a biodegradable scaffold to promote skin regeneration and wound healing.

USSCs are considered by many stem cell scientists to be a type of mesenchymal stem cell, but USSCs can be grown in the laboratory and have the ability to differentiate into a wide variety of adult cell types.

Asadipour and others used a material called PHBV or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) to make a skin-like scaffold upon which the USSCs were grown. They discovered that attaching a molecule called “chitosan” to the PHBV made it quite resilient and a very good substrate for growing cells. When grown on these scaffolds, the USSCs adhered nicely to them and grew robustly.

Then Zeinali and his colleagues used these cell-impregnated scaffolds to treat open surgical wounds in laboratory rodents. After three weeks, the group treated with the cell grown on the scaffolds healed significantly better than those animals treated with just cells, just scaffolds, or neither.

Thus it seems likely that tissue-engineered skin made from modified PHBV scaffolds and embedded umbilical cord blood-based stem cells might be a potent treatment for wound patients with large injuries that do heal slowly.  In the words of the abstract of this paper, “These data suggest that chitosan-modified PHBV scaffold loaded with CB-derived USSCs could significantly contribute to wound repair and be potentially used in the tissue engineering.”

Some larger animal studies should further test this protocol and if it can augment the healing of large animal wounds, then human clinical trials should be considered.

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.