During the last two decades, induced pluripotent stem cell-derived organoids have emerged as crucial tools in virtually all fields of biology. Depending on the differentiation protocols used, they can mimic in vitro key functions of a large variety of organs, including the intestine. Those so-called Human Intestinal Organoids (HIO) have proven useful at various degrees: studying developmental processes with human cells, exploring host-microbiota interactions, and testing drugs during preclinical trials.

However, HIO still fails to reproduce the in vivo complexity of mesenchyme derivatives and particularly visceral smooth muscle development, which make them not suitable to model mature contractile intestines. Our goal is to improve our knowledges of the mechanisms shaping human intestinal development via the prism of smooth muscle differentiation, using organoid model.

Methods

Knowing that physiological cues may be lacking in current differentiation protocols of HIO, we explored the impact of matrix composition on intestinal muscular development. As there is accumulating evidence highlighting the importance of stiffness and viscoelasticity for muscle differentiation, we studied mechanical properties of our matrixes. To that end, we studied custom-made matrixes with different compositions and stiffness, including Matrigel, collagen and alginate hydrogels. We intended to screen the impact on matrix composition on organoid development and maturation through the prism of smooth muscle development. We monitored the impact of matrixes at gene and protein level, and the architecture of organoids via confocal microscopy.

Results

We found that collagen-based matrix promoted muscular development in HIO independently of stiffness. We observed increased expression of SMC differentiation markers and modified organization of cells expressing those markers. In the contrary, alginate hydrogels prevented mesenchyme development and differentiation.

Conclusion

We developed a robust and simple protocol to achieve HIO bearing intestinal SMCs. Ultimately, our goal is to generate mature, functional smooth muscle cells to model and monitor the physiology of smooth muscle layers. Translating it to Visceral Myopathies by using pluripotent stem cells from patients will help us understand the pathophysiology of the disease and offer a platform to test therapeutics.