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    Tissue Engineering

    Altered development of PKD2-/-kidney organoids and cysts in response to substrate stiffness

    (I-323) Altered development of PKD2-/- kidney organoids and cysts in response to substrate stiffness

    Thursday, October 12, 2023
    2:00 PM – 3:00 PM PDT
    Location: Exhibit Hall - Row I - Poster # 323

      Presenting Author(s)

    • Isabella R. Jennings (she/her/hers)

      Bioengineering PhD Student
      University of Washington
      Seattle, Washington, United States

      • Primary Investigator(s)

    • BF

      Benjamin S. Freedman

      Assistant Professor in the Division of Nephrology, Department of Medicine
      Institute for Stem Cell and Regenerative Medicine, University of Washington, United States

    Introduction::

    Affecting approximately 1 in 500 to 1 in 1000 adults, autosomal dominant polycystic kidney disease (ADPKD) is one of the most prevalent genetic disorders. ADPKD is known for its development of renal cysts that enlarge the kidneys and disrupt renal function. Stem cell-derived human kidney organoid differentiation protocols are able to recreate this cystic phenotype in vitro using gene edited ADPKD cell lines. Utilizing this model, it has been reported that cystogenesis occurs more frequently after organoids are removed from adherent culture conditions and suspended in media without any modification of the biochemical environment. This suggests that mechanical changes in the organoids’ environment is key to inducing cysts. It is known that the mechanical environment plays a significant role in stem cell differentiation and cell phenotype modulation.


    To study the effects of the mechanical environment on the development of ADPKD cysts, polydimethylsiloxane (PDMS) was used to generate a range of stiffnesses from 5-13.3 kPa that mimic the natural stiffness of kidney tissue (10 kPa). These conditions were benchmarked against traditional differentiations conducted on tissue culture plastic (1 GPa). We hypothesized that lower stiffness substrates would promote cyst development similar to the effects of suspension culture. Control over cyst development using the mechanical environment would reveal a new avenue of study of ADPKD mechanotransduction that current suspension cultures cannot support.

    Sub-track: Stem Cells In Tissue Engineering and Disease Modeling



    Materials and Methods:: Mixtures of Sylgard 184 (2200 kPa) and Sylgard 527 (5 kPa) were combined to create a series of stiffnesses. Both types of PDMS were premixed according to their normal base to curing ratios before being combined to produce 5, 7.7, and 13.5 kPa. An isogenic pair of wild-type (WT) and PKD2-/- human induced pluripotent stem cells were used to generate kidney organoids on varying stiffnesses over 18 days. Organoid number and size on day 18 and cyst counts on day 25 of differentiation were collected. Immunofluorescent staining of day 18 organoids for podocalyxin, lotus tetragonolobus lectin, and E-cadherin, which indicate major nephron segments, was used to evaluate organoid health and structural development.

    Results, Conclusions, and Discussions::

    Neither WT or PKD2-/- organoid maturation was impacted by substrate stiffness, as all organoids stained positive for all three nephron segments. WT organoid yield on PDMS was inversely proportional to stiffness, with 5 kPa producing the greatest number of organoids at similar quantities as tissue culture plastic.  In contrast, PKD2-/- organoid yield was not affected by substrate stiffness. WT organoids were significantly larger on 5 kPa compared to 7 and 13.5 kPa and tissue culture plastic, indicating strong preference for 5 kPa. PKD2-/- organoids were also largest on 5 kPa but were only significantly larger than 13.5 kPa organoids, indicating reduced preference for softer substrates compared to WT organoids. It has been reported that PKD2-/- cells have altered adhesion and matrix remodeling abilities, and the observations here suggest that PKD2-/- cells may also incorrectly sense or respond to their mechanical environment. Dysregulation of mechanical homeostasis may contribute to behaviors such as cyst hyperproliferation.


    PKD2-/- cyst development appears to be sensitive to substrate stiffness. It was observed that 5 kPa reduced cyst yield compared to stiffer substrates, which was not expected. This observation may help reveal the sequence of events in cyst development, where fibrosis is observed surrounding renal cysts. These results suggest that stiffer surfaces may promote cyst development.


    Future work will examine the expression and activation states of mechanosensory molecules such as those in the focal adhesion complex to determine if there is faulty mechanotransduction in PKD2-/-. Additionally, observation of the time course of matrix remodeling genes will resolve whether fibrosis begins before or after cyst development. By focusing on the mechanical homeostasis functions of PKD2-/- the drivers of cyst development may be further revealed.



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