Adult Directly Reprogrammed With Proteins into Cardiac Progenitor Cells Heal Heart After a Heart Attack and Make New Heart Muscle


Jianjun Wang from Wayne State School of Medicine in Detroit, Michigan and Xi-Yong Yu from Guangzhou Medical University and a host of graduate students and postdoctoral research fellows in their two laboratories have teamed up to make human cardiac progenitor cells (CPCs) from human skin fibroblasts through direct reprogramming. Direct reprogramming does not go through a pluripotent intermediate, and, therefore, produces cells that have a low chance of generating tumors.

To begin their study, Wang, and Yu and their colleagues isolated fibroblasts from the lower regions of the skin (dermis) and grew them in culture. Then they reprogrammed these cells in a relatively novel manner. This is a little complicated, but I will try to keep it simple.

Reprogramming cells usually requires scientists to infect cells with recombinant viruses that have been genetically engineered to express particular genes in cells or force cells to take up large foreign DNA. Both of these techniques can work relatively well in the laboratory, but you are left with cells that are filled with foreign DNA or recombinant viruses. It turns out that directly reprogramming cells only requires transient expression of specific genes, and once the cells have recommitted to a different cell fate, the expression of the genes used to get them there can be diminished.

To that end, some enterprising scientists have discovered that inducing cells to up modified proteins can also reprogram cells. Recently a new reagent called the QQ-reagent system can escort proteins across the cell membrane. The QQ-reagent has been patented and can sweep proteins into mammalian cells with high-efficiency and low toxicity (see Li Q, et al (2008) Methods Cell Biol 90:287–325).

Wang and Yu and their coworkers used genetically engineered bacteria to overexpress large quantities of four different proteins: Gata4, Hand2, Mef2c, and Tbx5. Then they mixed these proteins with their cultured human fibroblasts in the presence of the QQ reagent. This reagent drew the proteins into the cells and the fibroblasts were reprogrammed into cardiac progenitor cells (CPCs). Appropriate control experiments showed that cells that were treated with QQ reagent without these proteins were not reprogrammed. Wang and Yu and they research groups also exposed the cells to three growth factors, BMP4 and activin A, to drive the cells to become heart-specific cells, and basic fibroblast growth factor to turn the cells towards a progenitor cell fate.

The next set of experiment was intended to show that their newly reprogrammed were of a cardiac nature. First, the cells clearly expressed heart-specific genes. Flk-1 and Isl-1 are genes that earmark cardiac progenitor cells, and by the eighth day of induction, the vast majority of cells expressed both these genes.

 

Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.
Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.

Second, cardiac cells can differentiate into three different cell types: heart muscle cells, blood vessels cells, and smooth muscle cells that surround the blood vessels. In mesoderm progenitors made from embryonic stem cells, inhibition of the Wnt signaling pathway can drive such cells to become heart muscle cells (see Chen, et al Nat Chem Biol 5:100–107; Willems E, et al Circ Res 109:360–364; Hudson J, et al Stem Cells Dev 21:1513–1523). However, Wang, Yu and company showed that treating the cells with a small molecule called IWR-1 that inhibits Wnt signaling drove their cells to differentiate into, not only heart muscle cells, but also endothelial (blood vessel) cells and smooth muscle cells when the cells were grown on gelatin coated dishes. When left to differentiate in culture, the cells beat synchronously and released calcium in a wave-like fashion that spread from one cell to another, suggesting that some cells were acting as pacemakers and setting the beat.

 

Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.
Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.

Then these cells were transplanted into the heart of mice that had suffered heart attacks. When compared to control hearts that received fluid, but no cells, the hearts of the animals that received protein-induced CPCs showed decreased scarring by 4 weeks after the transplantations. They also showed the growth of new heart muscle. A variety of staining experiments established that the engrafted protein-induced CPCs positive for heart muscle- and endothelial-specific cell markers. These experiments showed that transplantation of cardiac progenitor cells can not only help attenuate remodeling of the left ventricular after a heart attack, but that the protein-induced CPCs (piCPCs) can develop into cells of the cardiac lineage.

In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.
In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.

These are exciting results. It shows that direct reprogramming can occur without introducing genes into cells by means that can complicate the safety of the implanted cells. Also, because the cells are differentiated into progenitor cells, they still have the ability to proliferate and expand their numbers, which is essential for proper regeneration of a damaged tissue.

After a heart attack, the ventricle wall scars over and can become thin. However, piCPCs that have been directly reprogrammed from mature, adult cells can be used to replace dead heart muscle in a living animal.

Despite these exciting advances, further questions remain. For example, are the physiological properties of cells made from piCPCs similar enough to match the functional parameters of the heart into which they are inserting themselves? More work is necessary to answer that question. Functional equivalence is important, since a heart that does not function similarly from one end to the other can become arrhythmic, which is clinically dangerous. Further work is also required to precisely determine how well cells derived from piCPCs mature and coupling with neighboring cells. Therefore, larger animal studies and further studies in culture dishes will be necessary before this technique can come to the clinic. Nevertheless, this is a tremendous start to what will hopefully be a powerful and fruitful technique for healing damaged hearts.

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Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).