Improving Cardiac Cell Production through Enhancing Early Mesoderm Formation

Review of “IGF promotes cardiac lineage induction in vitro by selective expansion of early mesoderm” from Stem Cells, reviewed by Stuart P. Atkinson.

While cell based therapies, especially those based on the use of induced pluripotent stem cells (iPSCs), hold great promise for the treatment of a multitude of diseases and disorders, the differentiation of sufficient specific somatic cell types remains a significant problem. Cardiomyocyte production from iPSCs to replace cells lost during myocardial infarction is modest and highly variable [1, 2] and requires further “fine tuning” which may be informed through the study of normal early development [3]. A group led by Sean M. Wu from the Stanford University School of Medicine, CA, USA have now evaluated signaling pathway activation during early cardiac lineage induction [4] to search for factors which can enhance the formation of multipotent cardiac progenitor cells (CPCs) in order to give rise to cardiomyocytes, smooth muscle cells and endothelial cells [5, 6].

Engels et al first assessed 44 factors in differentiating Nkx2.5‐eGFP murine ESCs (day 3) for their ability to enhance Nkx2.5+ CPC formation (see Figure). Of these 44 factors, IGF1, IGF2, insulin and Wnt3a all significantly increased CPC formation, while Activin A, BMP2 or BMP4 decreased CPC formation. Positive factors led to a dose‐dependent increase in the number of eGFP cells, although the positive effect was only seen at day 3 and not after sorting at day 6. The CPC boosting effect was associated with increases in the expression of cardiac mesoderm‐ and CPC‐specific genes (Mesp1, Isl‐1, Nkx2.5, GATA4 and MLC2a), and the production of cardiomyocyte‐like cells with proper sarcomeric architecture.

Focusing on how IGFs and Insulin treatment boosts CPC formation, the group observed a significant increase in the expression levels of mesoderm‐ and endoderm‐specific genes (Eomes, Bra, Gata4, Goosecoid, Afp, Hnf1b, Hnf3b and Sox17), whilst ectoderm‐specific genes (Nestin, Gbx2, Fgf5 and Pax6) were unchanged or slightly decreased. Further research found that Bra+ mesodermal cells, but not Bra- cells, were highly proliferative following IGF/Insulin treatment, suggesting that these factors may selectively induce mesodermal cell proliferation to boost CPC formation. AKT phosphorylation, a mediator of IGF signaling [27], was also selectively induced in Bra+ cells following IGF/insulin treatment. Selective inhibition of Akt, as well as PI3K and mTOR, signalling during differentiation blocked IGF/insulin‐induced formation of eGFP+ CPCs, while Akt inhibition also reduced the frequency of Brachyury/Ki‐67‐double‐positive cells suggesting that IGF signaling through PI3K, Akt and mTOR can enhance mesodermal cell proliferation which allows increased CPC formation.

The authors note that this is the first study to show a direct role for IGF/Insulin in the mesodermal differentiation of ESCs, via PI3K, Akt and mTOR signalling-mediated expansion of Bra+ cells during differentiation. This will not only inform us on early developmental processes which may occur in vivo, but will also aid to improve cardiac differentiation strategies towards possible clinical use. Hopefully this research can be taken forward into human pluripotent stem cell types in order to amplify the production of much needed cells which will be required for effective cell therapy.

Discussion
• Does this method lead to enhanced cardiomyocyte production?
• Are they more or less functional than for previous strategies?
• Can we apply such strategic thinking to boost other differentiation protocols?
References

1. Kattman SJ, Witty AD, Gagliardi M, et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 2011;8:228-240.
2. Burridge PW, Thompson S, Millrod MA, et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One 2011;6:e18293.
3. Van Vliet P, Wu SM, Zaffran S, et al. Early cardiac development: a view from stem cells to embryos. Cardiovasc Res 2012;96:352-362.
4. Engels MC, Rajarajan K, Feistritzer R, et al. IGF promotes cardiac lineage induction in vitro by selective expansion of early mesoderm. Stem Cells 2014
5. Wu SM, Fujiwara Y, Cibulsky SM, et al. Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart. Cell 2006;127:1137-1150.
6. Moretti A, Caron L, Nakano A, et al. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell 2006;127:1151-1165.