Roth-Gibson Professor of Bioengineering University of Notre Dame, United States
Introduction: A primary goal of tissue engineering is the generation of functional organs from patient-derived stem cells. There are many obstacles preventing the generation of organs with sufficient structure and function. One of these is the generation of cells with adequate functional maturity. This is especially relevant to the heart, where stem cell-derived cardiomyocytes (CMs) lack metabolic and functional characteristics of in vivo CMs. This is a barrier to both whole organ engineering and drug screening, where the lack of functional maturity of CMs impairs our ability to detect cardiotoxic drugs. Early in human development, macrophages migrate from the liver into the heart where they take residence and persist throughout life. In mice, ablation of cardiac-resident macrophages leads to dysregulation of the energetics of CMs, which impairs heart function. It is unknown how the residence of macrophages in human development aids the maturation of CMs. Further, this has not been explored as an avenue to the generation of more mature iPSC-derived CMs for tissue engineering applications. We hypothesized that addition of macrophages to iCMs, particularly during differentiation, would improve the metabolism and function of human iCMs.
Materials and
Methods: Cardiomyocytes and macrophages were derived from the same iPSC line as per previously established protocols. Briefly, CMs were generated through a modified GiWi protocol, and macrophages were collected from embryoid body-based differentiation of hematopoietic precursors. During our modified CM differentiation protocol, cells are supplied with glucose-free media containing B27 and fatty acids on day 9, and the same media without fatty acids on day 17. On the 20th day of differentiation, cells are supplied media with low glucose in addition to B27 and a cocktail of other molecules previously demonstrated to improve CM metabolic function. On each of these days (9, 17, 20), macrophages were added to independent differentiations. Samples were collected 2 days after addition of macrophages to assess changes in mitochondria dynamics and cellular development, and others were allowed to continue differentiation to day 30. Cell proliferation, mitochondria network organization, and off-target cell amplification were assessed through immunostaining in samples taken 2 days post addition of macrophages. After day 30, metabolism was assessed through seahorse analysis. Further quantification of functional changes in CMs were assessed through video beating analysis over the course of the differentiation and immunostaining after day 30. In addition, macrophages were added to mature (post day 30) CM cultures and changes in beating characteristics and electrical handling were assessed through video (brightfield and calcium-reactive dye) and microelectrode array analyses. Mitochondria dynamics were interrogated through mitotracker staining and JC-1 fluorescence.
Results, Conclusions, and Discussions: In mature CM cultures, macrophages were observed to engulf CM-derived mitochondria. CM cultures with macrophages exhibited lower beating frequency than those without coculture treatment. During our differentiation protocol, CMs are subjected to low glucose conditions at specified timepoints to induce a switch to fatty acid oxidation-based metabolism. We hypothesized that this metabolic stress would promote CMs to export mitochondria to macrophages in the culture as observed in murine studies. Addition of macrophages to CM differentiation at day 9, one of these points, significantly increased measures of mitochondrial respiration at day 35. Additions at days 17 and 20 also appeared to increase respiration, but these changes were not significant. This appeared alongside changes in sarcomere structure and mitochondria network morphology. Addition of macrophages to iCM cultures during differentiation seems to improve functional and respiratory characteristics of these cells. This reflects human embryonic development when macrophages migrate to the heart and take residence. Further, we show that transfer of mitochondria material from CMs to macrophages is reflected in human cultures, suggesting conservation of this metabolic regulatory behavior in humans. These studies suggest that the crosstalk between macrophages and cardiomyocytes is important to the development of functionally mature CMs, which is an obstacle for the generation of patient-specific organs. By leveraging this interaction, future work will be able to more accurately model responses of mature CMs to stimuli such as ischemia and cardiotoxic drugs.
