A review of protocols for human iPSC culture, cardiac differentiation, subtype-specification, maturation, and direct reprogramming.

The methods for the culture and cardiomyocyte differentiation of human embryonic stem cells, and later human induced pluripotent stem cells (hiPSC), have moved from a complex and uncontrolled systems to simplified and relatively robust protocols, using the knowledge and cues gathered at each step. HiPSC-derived cardiomyocytes have proven to be a useful tool in human disease modelling, drug discovery, developmental biology, and regenerative medicine. In this protocol review, we will highlight the evolution of protocols associated with hPSC culture, cardiomyocyte differentiation, sub-type specification, and cardiomyocyte maturation. We also discuss protocols for somatic cell direct reprogramming to cardiomyocyte-like cells.

PI(18:1/18:1) is a SCD1-derived lipokine that limits stress signaling.

Cytotoxic stress activates stress-activated kinases, initiates adaptive mechanisms, including the unfolded protein response (UPR) and autophagy, and induces programmed cell death. Fatty acid unsaturation, controlled by stearoyl-CoA desaturase (SCD)1, prevents cytotoxic stress but the mechanisms are diffuse. Here, we show that 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) [PI(18:1/18:1)] is a SCD1-derived signaling lipid, which inhibits p38 mitogen-activated protein kinase activation, counteracts UPR, endoplasmic reticulum-associated protein degradation, and apoptosis, regulates autophagy, and maintains cell morphology and proliferation. SCD1 expression and the cellular PI(18:1/18:1) proportion decrease during the onset of cell death, thereby repressing protein phosphatase 2 A and enhancing stress signaling. This counter-regulation applies to mechanistically diverse death-inducing conditions and is found in multiple human and mouse cell lines and tissues of Scd1-defective mice. PI(18:1/18:1) ratios reflect stress tolerance in tumorigenesis, chemoresistance, infection, high-fat diet, and immune aging. Together, PI(18:1/18:1) is a lipokine that links fatty acid unsaturation with stress responses, and its depletion evokes stress signaling.

Regeneration in starved planarians depends on TRiC/CCT subunits modulating the unfolded protein response.

Planarians are able to stand long periods of starvation by maintaining adult stem cell pools and regenerative capacity. The molecular pathways that are needed for the maintenance of regeneration during starvation are not known. Here, we show that down-regulation of chaperonin TRiC/CCT subunits abrogates the regeneration capacity of planarians during starvation, but TRiC/CCT subunits are dispensable for regeneration in fed planarians. Under starvation, they are required to maintain mitotic fidelity and for blastema formation. We show that TRiC subunits modulate the unfolded protein response (UPR) and are required to maintain ATP levels in starved planarians. Regenerative defects in starved CCT-depleted planarians can be rescued by either chemical induction of mild endoplasmic reticulum stress, which leads to induction of the UPR, or by the supplementation of fatty acids. Together, these results indicate that CCT-dependent UPR induction promotes regeneration of planarians under food restriction.

Staphylococcus aureus-Derived α-Hemolysin Evokes Generation of Specialized Pro-resolving Mediators Promoting Inflammation Resolution.

Underlying mechanisms of how infectious inflammation is resolved by the host are incompletely understood. One hallmark of inflammation resolution is the activation of specialized pro-resolving mediators (SPMs) that enhance bacterial clearance and promote tissue repair. Here, we reveal α-hemolysin (Hla) from Staphylococcus aureus as a potent elicitor of SPM biosynthesis in human M2-like macrophages and in the mouse peritoneum through selective activation of host 15-lipoxygenase-1 (15-LOX-1). S. aureus-induced SPM formation in M2 is abolished upon Hla depletion or 15-LOX-1 knockdown. Isolated Hla elicits SPM formation in M2 that is reverted by inhibition of the Hla receptor ADAM10. Lipid mediators derived from Hla-treated M2 accelerate planarian tissue regeneration. Hla but not zymosan provokes substantial SPM formation in the mouse peritoneum, devoid of leukocyte infiltration and pro-inflammatory cytokine secretion. Besides harming the host, Hla may also exert beneficial functions by stimulating SPM production to promote the resolution of infectious inflammation.

Downregulation of mTOR Signaling Increases Stem Cell Population Telomere Length during Starvation of Immortal Planarians.

Reduction of caloric intake delays and prevents age-associated diseases and extends the life span in many organisms. It may be that these benefits are due to positive effects of caloric restriction on stem cell function. We use the planarian model Schmidtea mediterranea, an immortal animal that adapts to long periods of starvation by shrinking in size, to investigate the effects of starvation on telomere length. We show that the longest telomeres are a general signature of planarian adult stem cells. We also observe that starvation leads to an enrichment of stem cells with the longest telomeres and that this enrichment is dependent on mTOR signaling. We propose that one important effect of starvation for the rejuvenation of the adult stem cell pool is through increasing the median telomere length in somatic stem cells. Such a mechanism has broad implications for how dietary effects on aging are mediated at the whole-organism level.

It is not all about regeneration: Planarians striking power to stand starvation.

All living forms, prokaryotes as eukaryotes, have some means of adaptation to food scarcity, which extends the survival chances under extreme environmental conditions. Nowadays we know that dietary interventions, including fasting, extends lifespan of many organisms and can also protect against age-related diseases including in humans. Therefore, the capacity of adapting to periods of food scarcity may have evolved billions of years ago not only to allow immediate organismal survival but also to be able to extend organismal lifespan or at least to lead to a healthier remaining lifespan. Planarians have been the center of attention since more than two centuries because of their astonishing power of full body regeneration that relies on a large amount of adult stem cells or neoblasts. However, they also present an often-overlooked characteristic. They are able to stand long time starvation. Planarians have adapted to periods of fasting by shrinking or degrowing. Here we will review the published data about starvation in planarians and conclude with the possibility of starvation being one of the processes that rejuvenate the planarian, thus explaining the historical notion of non-ageing planarians.

Postnatal telomere dysfunction induces cardiomyocyte cell-cycle arrest through p21 activation.

The molecular mechanisms that drive mammalian cardiomyocytes out of the cell cycle soon after birth remain largely unknown. Here, we identify telomere dysfunction as a critical physiological signal for cardiomyocyte cell-cycle arrest. We show that telomerase activity and cardiomyocyte telomere length decrease sharply in wild-type mouse hearts after birth, resulting in cardiomyocytes with dysfunctional telomeres and anaphase bridges and positive for the cell-cycle arrest protein p21. We further show that premature telomere dysfunction pushes cardiomyocytes out of the cell cycle. Cardiomyocytes from telomerase-deficient mice with dysfunctional telomeres (G3 Terc(-/-)) show precocious development of anaphase-bridge formation, p21 up-regulation, and binucleation. In line with these findings, the cardiomyocyte proliferative response after cardiac injury was lost in G3 Terc(-/-) newborns but rescued in G3 Terc(-/-)/p21(-/-) mice. These results reveal telomere dysfunction as a crucial signal for cardiomyocyte cell-cycle arrest after birth and suggest interventions to augment the regeneration capacity of mammalian hearts.

Telomerase Is Essential for Zebrafish Heart Regeneration.

After myocardial infarction in humans, lost cardiomyocytes are replaced by an irreversible fibrotic scar. In contrast, zebrafish hearts efficiently regenerate after injury. Complete regeneration of the zebrafish heart is driven by the strong proliferation response of its cardiomyocytes to injury. Here we show that, after cardiac injury in zebrafish, telomerase becomes hyperactivated, and telomeres elongate transiently, preceding a peak of cardiomyocyte proliferation and full organ recovery. Using a telomerase-mutant zebrafish model, we found that telomerase loss drastically decreases cardiomyocyte proliferation and fibrotic tissue regression after cryoinjury and that cardiac function does not recover. The impaired cardiomyocyte proliferation response is accompanied by the absence of cardiomyocytes with long telomeres and an increased proportion of cardiomyocytes showing DNA damage and senescence characteristics. These findings demonstrate the importance of telomerase function in heart regeneration and highlight the potential of telomerase therapy as a means of stimulating cell proliferation upon myocardial infarction.