Introduction:: Insufficient differentiation of nephron progenitor (NP) cells during kidney formation can result in lower nephron number and glomerular density, which is a risk factor for progression to end-stage renal disease later in life. The amount of nephron differentiation in the developing kidney is correlated with the number of ureteric tubule tips present at the kidney surface. We predict that packing of ureteric tubule tips at the kidney surface translates into cyclical changes in mechanical micro-environments of adjacent NP populations. We ultimately seek to develop an in vitro method of inducing cyclic mechanical stress on NPs in order to quantify differentiation responses of cells toward early nephron lineages through the use of optogenetics. We can manipulate the mechanical stresses exerted on HEK cells through genomic insertion and activation of an optogenetic construct (BcLOV4-mCherry-RhoA) with exposure to blue light (488nm). RhoA induces cell contraction through stress fiber formation, and BcLOV4 encodes for a blue-light photoreceptor. In this work, we develop a three-dimensional spheroid HEK cell culture to examine how activation of the optogenetic construct leads to spheroid contraction and thus affects the mechanical forces seen by cells. Imaging spheroids using confocal microscopy before and after activation allows for quantification of the effect of blue-light exposure on spheroid area and perimeter using a MATLAB and ImageJ workflow. We find that each imaged spheroid exhibits decreased area following stimulation. These efforts provide proof of principle for controlling the interplay between morphogen and mechanotransduction signaling in fundamental and regenerative medicine applications of in vitro organogenesis.
Materials and Methods:: HEK Cell Spheroid Generation and Imaging The optogenetic construct was inserted into HEK cell genomes using a lentiviral vector. The transduced cells were sorted according to mCherry fluorescence expression so as to increase purity of the culture. After establishment of a two-dimensional culture of HEK cells transduced with BcLOV4-mCherry-RhoA, HEK cell spheroids were generated with varying cell counts (5000, 10000, 20000, 40000, 60000, and 100000 cells per spheroid) on a round-bottom ultra-low adhesion 96-well plate. Imaging of the spheroids was conducted using a confocal microscope. A laser was used to optically stimulate the cells with blue light (488 nm) and was operated at varying intensities. Four sets of laser excitation and imaging parameters were implemented for spheroids of each cell count. Three spheroids of each cell count were imaged using the parameters described by Settings 1-3. One spheroid of each cell count was imaged using the parameters of Setting 4 which is distinguished from Settings 1-3 by the longer imaging interval and increased frequency of images captured.
Quantification of Spheroid Contraction Following imaging of the HEK cell spheroids, a MATLAB and ImageJ workflow was implemented to measure the area and perimeter of the spheroids over the time of the imaging session. ImageJ was used to standardize the brightness and contrast values of each acquired image. MATLAB was used to generate quantitative values of the area and perimeter of each spheroid in each image through a pixel-counting algorithm.
Results, Conclusions, and Discussions:: Image analysis of the HEK cell spheroids presented a clear trend of decreasing spheroid area (um^2) over time (min) after activation with blue light. This trend was evident in spheroids of all tested cell counts. The average net decrease in area for eight spheroids of 60,000 cells from t=0 to t=60 minutes was 2.149e3 square microns (Fig. 1 b). A linear fit of the measured spheroid area over time for spheroids of 60,000 cells suggests that the spheroids will continue to contract and decrease in area as time increases past 60 minutes (Fig. 1 b). The displayed contraction of spheroids which consist of a non-homogeneous mixture of transduced HEK cells (Fig. 1 a,c,d) presents a method for inducing mechanical stress on developing kidney cells by altering the genomes of neighboring cells to include the optogenetic construct.
There was little difference in spheroid area over time due to changes in laser intensity and excitation parameters (Table 1). This is likely due to the fact that the two laser intensities tested (6mW and 12mW) both allowed for full saturation of the spheroids with blue light (488 nm), and as such there was no discernable difference between the contractile effects of the two laser intensities. There was no noticeable trend in perimeter change over time for the HEK cell spheroids. This can be attributed to generation of cell membrane blebs and to spheroid shape changes at the three-dimensional level which are not visible at the selected z-plane.
With the insertion of BcLOV4-mCherry-RhoA into HEK cells, we can control the contraction of HEK cells spheroids through activation of the optogenetic construct by exposure to blue light. These observations in cell spheroids provide a basic model for the induction of mechanical stress in tissues to control nephron differentiation rate in developing kidney tissues. Future work should be done to evaluate and refine this approach for inducing mechanical stress in IPSCs and nephron progenitors. These efforts will integrate biochemical and mechanical perspectives of nephrogenesis, which would have significant impacts on the more than third of all birth defects that are associated with kidney and urinary tract.