Harness the power of Hepatic Stellate Cells in Your Models

Written By: Nicholas Trompeter, Ph.D.

Technical Sales Specialist

Hepatic Stellate Cells in culture stained for αSMA at 20x magnification. Cell nuclei were stained with DAPI (blue).

Want to investigate the role of hepatic stellate cells in models of healthy or diseased livers?

You will need to account for culture-induced activation of hepatic stellate cells!

Residing within the space of Disse live the mesenchymal hepatic stellate cells (HSCs), named for their star-shaped morphology. HSCs play an integral role in human physiology by storing the majority of our bodies’ vitamin A. In addition to their ability to store vitamin A as lipid droplets, HSCs coordinate a complex network of signaling between the cells of the liver sinusoid (hepatocytes, liver sinusoidal endothelial cells, and Kupffer Cells) during liver homeostasis, disease, and regeneration. HSCs elicit their function within the liver through a few different mechanisms, including direct cell-cell interactions with hepatocytes and liver sinusoidal endothelial cells (LSECs), paracrine factors, autocrine signaling, and extracellular matrix production.  Under normal conditions, HSCs maintain a quiescent state, with minimal proliferation and production of extracellular matrix. However, during liver injury, infection, or disease, activation of hepatic stellate cells results in the production of extracellular matrix proteins (predominantly collagens), inflammatory mediators, and proliferation which can cause liver fibrosis.

When HSCs are isolated from human livers and are then cultured on plastic microplates they undergo a similar activation to HSCs in vivo during liver injury. Culture-induced activation of HSCs occurs rapidly (within 1 day), changing the quiescent star-shaped morphology to a more spindle like form. Concomitant with the alteration to morphology is the production of type I collagen and the expression of the myofibroblast marker α-smooth muscle actin, and more, by HSCs. Both biomechanical and biochemical strategies have been leveraged to avoid culture-induced activation of HSCs.

A contributing factor for the transdifferentiation of HSCs to a myofibroblastic phenotype during cultures stems from the use of plastics and/or coated glass. These substrates with Young’s moduli orders of magnitude greater than healthy livers can activate HSCs without the addition of transforming growth factor-β. Culturing fresh HSCs on biomaterials with mechanical properties mimicking the native environment can inhibit the activated phenotype, with the caveat that proliferation of HSCS is severely hampered.(1) As most researchers need to expand their HSCs to accommodate their in vitro models, additional biochemical methods have been devised to maintain HSC quiescence during culture.

One strategy to maintain quiescent HSCs utilizes paracrine signals by co-culturing HSCs with freshly isolated LSECs. Differentiated LSECs, with normal fenestrations, can reverse HSC activation, returning these mesenchymal cells to a quiescent state. Other mechanisms that attenuate HSC activation include the incorporation of insulin, glucose, and vitamin A within the culture media, while excluding fetal bovine serum. (2)  Furthermore, inhibition of s Rho-associated protein kinase (ROCK), a protein integral for stress fiber formation and myofibroblast formation, stems the production of type I collagen and α-smooth muscle actin in culture-induced HSCs.(3)

While activation of HSCs can influence your in vitro models, especially as monocultures, research from Viscient Biosciences, highlights that HSCs from MASH/MASLD donors elicit a diseased phenotype in a 3D bioprinted liver model, which was not observed when using HSCs from healthy individuals.(4) While both healthy and MASH/MASLD HSCs were expanded in culture, only the diseased HSCs could replicate the production of collagen in the bioprinted liver model. This suggests that the plasticity of HSCs, which allows for tissue regeneration after liver injury, may allow for the recreation of normal liver models within complex in vitro systems.

Do you need to empirically test the difference between healthy and MASH/MASLD HSCs for your in vitro model systems? Contact Nick Trompeter (ntrompeter@mosaiccellsci.com) to schedule a meeting.

Don’t forget to keep your eyes peeled for our upcoming gut-liver axis review, which will contain additional details on the role of HSC activation in the etiopathology of MASH/MASLD.

References:

1.          Olsen, A.L., Bloomer, S.A., Chan, E.P., Gaça, M.D.A., Georges, P.C., Sackey, B., Uemura, M., Janmey, P.A., and Wells, R.G. (2011). Hepatic stellate cells require a stiff environment for myofibroblastic differentiation. https://doi.org/10.1152/ajpgi.00412.2010.-The.

2.          Dang, T.M., van Le, T., Do, H.Q., Nguyen, V.T., Le Holterman, A.X., Thi Dang, L.T., Lu Phan, N.C., van Pham, P., Hoang, S.N., Le, L.T., et al. (2021). Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse. Biosci Rep 41. https://doi.org/10.1042/BSR20202514.

3.          Fukushima, M., Nakamuta, M., Kohjima, M., Kotoh, K., Enjoji, M., Kobayashi, N., and Nawata, H. (2005). Fasudil hydrochloride hydrate, a Rho-kinase (ROCK) inhibitor, suppresses collagen production and enhances collagenase activity in hepatic stellate cells. Liver International 25, 829–838. https://doi.org/10.1111/J.1478-3231.2005.01142.X.

4.          Tan, P.K., Ostertag, T., Rosenthal, S.B., Chilin-Fuentes, D., Aidnik, H., Linker, S., Murphy, K., Miner, J.N., and Brenner, D.A. (2024). Role of Hepatic Stellate and Liver Sinusoidal Endothelial Cells in a Human Primary Cell Three-Dimensional Model of Nonalcoholic Steatohepatitis. American Journal of Pathology 194, 353–368. https://doi.org/10.1016/j.ajpath.2023.12.005.