Based on our proprietary induction reagents and protocol, Creative Biolabs now has the ability to provide high-quality functional cardiomyocytes derived from iPSCs. Moreover, multiple cell types such as neuronal cells and hepatocytes can be granted based on our advanced platform.
In recent decades, cardiovascular diseases were the major causes of morbidity and mortality in the world. As adult cardiomyocytes are terminally differentiated, and they are impossible to further proliferate in application of cell replacement therapy, the most promising cell types for cardiovascular regenerative medicine are human induced pluripotent stem cells (hiPSCs) which have the ability to differentiate into various cell types and proliferate in pluripotent state. The technique for differentiating iPSC into cardiomyocytes involves three stages:
These stages usually involve the manipulation of growth factors or small molecules that regulate important signaling pathways. However, there are also challenges to the differentiation process. These include heterogeneous differentiation, where not all iPSCs become cardiomyocytes, and the relative immaturity of the produced cardiomyocytes.
Now scientists at Creative Biolabs have developed three major approaches for differentiation of iPSCs to cardiomyocytes: Embryoid (EB), monolayer culture, and inductive coculture.
Embryoid bodies (EBs) are three-dimensional aggregates which consist of cells from ectoderm, endoderm, and mesoderm. The differentiation of EBs is the first and most common method to obtain cardiomyocytes. The first step, iPSCs are dispersed to small clumps of cells and removed from the pluripotency maintaining conditions to suspension culture where EBs were formed. The second step, the formation of EBs initiates and facilitates the differentiation process with gene expression for mesodermal， and early cardiac cell lineages is stimulated via cell to cell interactions. Finally, the formed EBs are re-plated onto a matrix-coated tissue culture plate for several days, and contracting outgrowth appears. In addition, the introduction of growth factors and small molecules can further enhance and direct the differentiation towards the cardiac lineage.
Fig.1 Current methods for cardiac differentiation of human iPSCs.
Another alternative approach for differentiation of hiPSCs is based on the monolayer. First of all, iPSCs are plated on Matrigel as a monolayer in the presence of embryonic fibroblast-conditioned media. Then cells are treated with activin A one day followed by BMP4 in serum-free RPMI medium plus a B27 supplement, to induce cardiac differentiation. Compared with the EBs, the absence of complex diffusional barriers makes growth factor regulation and other interventions more readily controllable and reproducible. Moreover, the monolayer protocol also does not need the complex replating steps thus reducing the procedural steps and saving time.
Inductive coculture is a differentiation strategy in co-culturing iPSCs with a visceral endoderm-like cell line (END-2). In this protocol, iPSCs are mechanically passaged as undifferentiated colonies onto mitotically inactivated END-2 culture platform. As endoderm plays a critical role of in the cardiac induction during the development of the heart, cardio-inductive signals of END-2 cells induce cardiomyocytes differentiation from iPSC.
Through our customized iPSC reprogramming and differentiating services, Creative Biolabs now can generate various iPSC derived cell types from patient samples. If you are interested in our services, please do not hesitate to contact us for more details.
Below are the findings presented in the article related to cardiomyocyte differentiation from iPSC.
Kyla Bourque, et al. conducted a comparative study to assess cell signaling in rat neonatal cardiomyocytes (RNCM) and human iPSC-derived cardiomyocytes. They used genetically encoded biosensors to explore GPCR-mediated nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/2 (ERK1/2) activity at the single-cell level in both cardiomyocyte populations.
As shown in the figure below, the biosensors were expressed at higher levels in hiPSC-CM compared to RNCM, despite the same transduction time. Subsequently, bulk RNA-seq also revealed key differences in the expression patterns of GPCR, G protein, and downstream effector expression levels. The results demonstrate that the hiPSC-CM model indeed offers significant advantages.
Fig. 2 Measuring protein kinase activity in RNCMs and hiPSC-CMs.3
References
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