Paper 1 – Xenograft of Human Pluripotent Stem Cell-Derived Cardiac Lineage Cells on Zebrafish Embryo Heart
Overview:
In their 2023 paper, Takahi et al. (2023) describes the use of zebrafish as a model cardiomyocyte transplant100. They microinjected fluorescent-tagged cardiac cells into the zebrafish yolk one day post-fertilization (dpf). They then tracked their movement and localization throughout the formation of the chimera heart (chimera meaning the heart forms partially as a result of engrafted stem cells) (Figure 1). In most cases, the cells both survived and exhibited heartbeat-like movements following injection. The paper establishes a precedent for the use of zebrafish to model xenograft effectiveness.
Why Zebrafish?
This article is novel in that it establishes the use of zebrafish as a model of cardiomyocyte xenografts. Animal models are a well-established technique that is valuable in the analysis of many drugs, treatment, and disease mechanisms. Traditionally, cardiac models are performed using mammalian models, and more specifically mice. However, the average heart rate of mice as compared to humans is approximately four times faster, complicating any comparisons drawn between the models. In addition, the use of mammalian models tends to spark ethical controversy in the scientific field. Zebrafish have long been used as models for xenografting experiments of both human and cancerous cells. They have many different advantages as compared to other model organisms, including an immature immune system that does not tend to reject foreign cells, and a similar electromechanical cardiac and conduction system to humans. While xenografting in zebrafish is well-established, there is little data to indicate the compatibility of human cardiac lineage cells specifically. This paper therefore aims to construct a zebrafish-model for a xenograft of cardiac cells derived from human induced pluripotent stem cells (hiPSCs).
Cardiomyocyte differentiation and injection:
The hiPSCs were cultured following the precedent established in previous peer-reviewed literature. The cells are grown in pluripotent conditions with excess fibronectin (see fibrin glue and the cardiac patch). The differentiation to mesoderm cells was then induced via a 24-hour treatment with 6 μM CHIR99021 and 10 ng/mL activin A in DF7 medium with 10 μg/mL insulin. CHIR99021 is a GSK-3 inhibitor, meaning it activates the Wnt signaling pathway (see cardiomyocyte differentiation from stem cells). Activation of the Wnt pathway promotes a complex of transcription factors involved in expressing a wide variety of cardiac-specific genes. Ideally, upregulation of this pathway would cause the stem cells to pursue a cardiomyocyte fate, allowing for them to be transplanted into the heart as a xenograft.
Visualizing the injected cells:
The yolks were injected with the differentiated cardiomyocyte graft 1 dpf. Fluorescence activity was then visualized and quantified 2 hours and 1 day post injection (dpi) using an epifluorescence microscope and confocal microscopy (Figure 1). Epifluorescence microscopy uses a beam of light to illuminate a sample of interest and allows the corresponding excitation spectrum to reflect back through the emission filter and detector for visualization. Confocal microscopy similarly quantifies fluorescence by passing emission spectrums generated from irradiating the sample with a beam of light through a detector. These imaging techniques were used to visualize the fluorescent-tagged stem cells to determine both viability and localization during heart development.
At one dpi, approximately 80% of the cells survived and were localized to the embryonic heart as opposed to other developing organ systems. An example of how this data might be presented is included in Figure 2. This successfully illustrates that the cells were readily accepted by the zebrafish’s immune system, highlighting its use as a model organism. In addition, the xenograft was viable and integrated into the zebrafish’s developing cardiac system, illuminating the therapeutic potential of cardiac cell xenografts in treating myocardial infarction pending further research. The transplanted cells were also analyzed using a video analysis platform. Recording showed that at 1dpi, approximately 30% of the derived cardiomyocytes in the embryonic heart region exhibited periodic movement. We encourage you to take a look at these figures in the original article (Figures 3 and 4 in Takahi et al.) Taken together, these findings indicate that the transplanted cells were successfully implanted and incorporated into the zebrafish’s cardiac system. This major conclusion paints a bright future for xenograft animal models that could provide major insights into stem cell implantation in humans. Animal models can provide incredibly important information along the basis of both drugs and therapeutics, with an emphasis on investigating cellular and molecular mechanisms. As we’ve previously emphasized the importance of understanding the chemistry of the heart in developing therapeutics, we believe the zebrafish model holds incredible potential to expand the scope of available cardiovascular treatment options from a more ethical and compatible frame.
December 8, 2023 at 4:41 pm
The use of zebrafish, for me, is quite surprising, but the less robust immune system makes a lot sense. That being said, I would be interested to see if other human xenographs, or any variety, have been successful with mammals. I was also curious if, like for heart transplants, rejection risk for these grafts would also be an issue. Individuals with a heart transplant often require a lot of drugs to reduce their immune response, and I was curious what potential limitations the grafts may have.
December 8, 2023 at 7:53 pm
Human-human grafts do require an immunosuppressive regiment, like other transplants. I assume that zebra fish xenografts would require immunosuppressants too as risk of immune attack on the heart is too high a risk.
December 8, 2023 at 7:48 pm
I appreciated your detailed exploration of the advantages of using zebrafish as a model for cardiomyocyte xenografts. The rationale for choosing zebrafish over traditional mammalian models is well-articulated. I was left wondering about further limitations or challenges associated with using zebrafish as a model. Addressing factors like the scalability of findings to larger organisms or the specific differences in the cardiac systems between zebrafish and humans could offer a more well-rounded perspective, complementing your discussion of the similarities by offering a different perspective.
December 8, 2023 at 7:56 pm
One small change: The title of this general page is misspelled (Relavant -> relevant)
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Interesting paper, and certainly a deep dive into an organism I wasn’t familiar was used as a biological model! I think the infographics worked very well on this page to help illustrate the process (and to show the reader what a zebrafish looks like). I was wondering why the ethical considerations might be different for the zebrafish vs. mice? I thought that was an interesting point and I’m not very familiar with animal studies so would love to know more. Other than that, I would say that including a few sentences on where your group sees this development going in the future (opinion) and how it ties in with heart therapies would be nice to have.
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