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.

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.