Scientists report the development of a virus-free method for triggering blood stem cells to differentiate into beating heart cells. The approach uses plasmids to carry the seven genes necessary to promote differentiation and a culture medium which the team claims is just a 10th the cost of standard stem cell media.
The Johns Hopkins Medical Institutions researchers who have developed the culture system say the highly efficient method can be applied to both human embryonic stem cells (hESCs) and induced pluripotent stem cell (iPSC) lines. The approach also eliminates the variability in cardiac differentiation capacity of different human pluripotent stem cells including hiPSC generated from CD34+ cord blood.
While they admit that the cells aren’t yet ready for testing in humans, the researchers suggest that virus-free, iPSC-derived cardiac cells could in the nearer-term be used to test new drug candidates. The team describes its approach in PLoS One in a paper titled “A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability.”
The most commonly used basic protocol for hESC cardiac differentiation has a low efficiency of 8–22%, and takes up to 21 days to produce contracting areas, according to the Johns Hopkins team, led by Elias Zambidis, M.D., professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center. This protocol performs even less efficiently for human iPSCs (1–25%) and can take up to 30 days to generate contracting human embryoid bodies (hEBs).
The researchers’ work to develop a more efficient and cheaper system for differentiating stem cells into cardiomyocytes hinged on initial resaerch by Johns Hopkins colleague Paul Burridge, Ph.D., at the Johns Hopkins Institute for Cell Engineering. Dr Burridge assembled a catalogue of the buffers, enzymes, growth factors, timings, and other variables reported in 30 papers detailing the production of cardiac cells. He then tested out hundreds of combinations before homing in on 4–9 essential ingredients at each of the three stages of cardiac cell development.
The resulting growth medium, which the team has called the universal cardiac differentiation system, was initially tested with iPSCs derived from cord blood cells. The cells were first given an electric pulse to enable the delivery of plasmids carrying the genes necessary to reprogram them into a pluripotent state. The resulting iPSCs were then cultured in the simplified growth medium and incubated under low-oxygen conditions. “The idea is to recreate conditions experienced by an embryon when these primitive cells are developing into different cell types,” Dr Burridge explains.
In summary, the differentiation method employed forced aggregation hEB formation in the chemically defined medium along with staged exposure to physiological (5%) oxygen and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin.
The contracting hEB derived using these methods were composed of high percentages of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles that were responsive to cardioactive drugs. “Importantly we demonstrate that the contracting cells produced using this system expressed normal cardiomyocyte markers, were capable of electrically coupling, and displayed highly reproducible electrophysiological profiles,” the authors stress.
For each of 11 cell lines tested, the efficiency of differentiation appeared to be about 94.5%. “Most scientists get 10% efficiency for iPSC lines if they are lucky,” Dr. Zambidis notes.
“Many scientists previously thought that a nonviral method of inducing blood cells to turn into highly functioning cardiac cells was not within reach,” he points out. “”We’ve found a way to do it very efficiently and we want other scientists to test the method in their own lab.”
The authors conclude, “The development of a universal cardiac differentiation protocol that can translate across multiple pluripotent stem cell lines allows immediate application to genetically diverse human iPSC lines created from patients with cardiac related diseases. The uniformity of the electrophysiological profiles of these cells highlights the potential for translation of this methodology to future high-throughput cardiotoxicity testing and novel drug discovery assays that can be used at various stages of drug development."
The researchers also stress that for future clinical applications, the generation of cardiomyocytes from clinically safe cord blood-derived human iPSCs is especially attractive because this cell source is widely available, carries relatively few somatic mutations, and could ultimately be used to create an HLA-defined stem cell bank for human iPSC generation via worldwide networks of existing cord blood banks.
“Overall, the development of this efficient cardiac differentiation system should greatly facilitate the utility of human iPSC-derived cardiomyocytes in drug development and cardiotoxicity screening, cardiac developmental biology and disease modelling, and contribute to the future generation of clinically safe human cardiac cells for regenerative medicine”.
The team is still refining the culture conditions and recently managed to completely remove fetal bovine serum from one step in the procedure. Meanwhile, they claim to have also developed similar techniques for differentiating blood-derived iPSC lines into retinal, neural, and vascular cells.