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Making cardiomyocytes from Pluripotent Cells: Pure and Simple!

Review of “A Massive Suspension Culture System with Metabolic Purification for Human Pluripotent Stem Cell-Derived Cardiomyocytes” from Stem Cells TM by Stuart P. Atkinson

The generation and grafting of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) – the muscle cells which help to keep the heart beating – is a potential exciting treatment for cell loss brought around by ischaemic events such as heart attack. However, a large scale, cost effective production strategy to produce sufficient numbers of functional cells has still to be fully developed. Massive suspension culture systems (MSCSs) have been employed to generate seemingly bona fide CMs from human embryonic stem cells (hESCs) [1], although these studies did not assess potential mechanisms to purify CMs from potentially tumor forming cells [2]. The transfer of potentially pluripotent non-differentiated cells is one of the major concerns with stem cell based therapies such as this. Researchers from the laboratory of Jun Fujita (Keio University School of Medicine, Tokyo, Japan) have previously identified differences between CMs and other unwanted cell-types at the metabolomic level [3], and they have used this knowledge to outline a strategy to grow vast amounts of pure functional CMs from hPSCs by combining MSCS with metabolic selection [4].

The group first compared CM differentiation from PSC-embryoid bodies (EBs) using standard techniques (standard suspension culture in non-adherent culture dishes) and MCSCs (125 ml spinner flasks at 40–100 revolutions per minute). The differentiation technique used BMP4, activin A, and ascorbic acid [5, 6] to generate mesodermal lineage cells, followed by CM specification using the Wnt inhibitor, IWR-1 [7] (15 days total). While the proliferation rate was not different between the two platforms, the MCSC generated smaller, more homogenous EBs, which led to a significantly smaller amount of apoptotic cells. Looking specifically at cells generated by MCSC, pluripotency marker expression (OCT4, TRA-1-60) remained detectable at the two week stage, at which time cardiac marker gene expression (NKX2-5, ACTC1, and TNNT2) was readily detectable. These non-purified CMs readily generated teratomas containing components of all three germ layers in NOD-SCID mice.

The researchers then modified the protocol to include a metabolic purification step; the addition of glucose-depleted/lactate-supplemented medium (See Figure). CMs can effectively utilise lactate in place of glucose, giving them a growth advantage over other “contaminating” cell types. In fact, the researchers detected no TRA-1-60 cells after the inclusion of this step, OCT4 expression was significantly reduced, up to 99% of cells were positive for the cardiac marker gene ACTC1, and other cardiac differentiation markers were significantly induced. Importantly, teratoma formation assays demonstrated a complete lack of teratoma growth using metabolically-purified CMs. The purified CMs demonstrated all three types of action potentials (nodal-, atrial-, and ventricular-like) in whole-cell patch-clamp assays, a typical electrocardiograph which responded appropriately to stimulants and blockers, and spontaneous and synchronized Ca2+ oscillations, all of which are observed in normal CMs.

This all suggests that combining large scale suspension culture with simple metabolic purification is sufficient to generate a population of functional CMs capable of being safely transplanted into recipients. The large scale nature of this differentiation will produce the millions upon millions of cells required, and will entail a significant cost and time saving, a common theme appearing in stem cell publications at this present time. This represents a significant step towards the clinical use of CMs in human patients, although further safety assessments still lie ahead.

References

  1. Niebruegge S, Nehring A, Bar H, et al. Cardiomyocyte production in mass suspension culture: embryonic stem cells as a source for great amounts of functional cardiomyocytes. Tissue engineering. Part A 2008;14:1591-1601.
  2. Miura K, Okada Y, Aoi T, et al. Variation in the safety of induced pluripotent stem cell lines. Nature biotechnology 2009;27:743-745.
  3. Tohyama S, Hattori F, Sano M, et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 2013;12:127-137.
  4. Hemmi N, Tohyama S, Nakajima K, et al. A Massive Suspension Culture System With Metabolic Purification for Human Pluripotent Stem Cell-Derived Cardiomyocytes. Stem Cells Translational Medicine 2014;
  5. 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.
  6. Takahashi T, Lord B, Schulze PC, et al. Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 2003;107:1912-1916.
  7. Willems E, Spiering S, Davidovics H, et al. Small-molecule inhibitors of the Wnt pathway potently promote cardiomyocytes from human embryonic stem cell-derived mesoderm. Circulation research 2011;109:360-364.