Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension

Tendon injury is one of the most prevalent musculoskeletal problems in the US, affecting an estimated 33 million people annually. Our research focuses on developing a cell and tissue engineering approach for repairing tendons that can drastically improve patient outcomes. Current technologies used to advance tendon research include the use of stem cells from different sources, like induced pluripotent stem cells, in combination with the use of bioreactors, genetic engineering biomaterials, and other tissue engineering techniques to mimic the environmental cues needed for tendon.

Our understanding of how tendons develop from their origins is limited. There are several well-known genes that are very highly expressed during tendon maturation. However, they're unfortunately not unique to the tendon tissue.

This makes it difficult for researchers to standardly assess successful tendon differentiation. Our protocol takes advantage of remarkable IPC change potential and what we know so far on tendon development. Our goal is to develop an allogeneic cell source for tendon cell therapy applications.

Our laboratory's focused on elucidating the pathways involved with tendon cell fate to better understand tendon development. We want to implement this knowledge in combination with tissue engineering technologies and biomaterials to develop more effective alternatives for traditional tendon repair. After autoclaving two silicone plates, under sterile conditions remove the plates from the autoclave bag and place them in 150-millimeter plates.

Add 400 microliters of fibronectin solution to each well of the silicone plates. Next, re-suspend the scleraxis-positive induced mesenchymal stromal cells in MSC medium and count the viable cells using an automated cell counter. Calculate the volume of cell suspension required to obtain 1.25 times 10 of the fourth cells per square centimeter.

Remove this volume of MSC stretch media from the 15-milliliter conical tube and add the required volume of scleraxis-positive induced mesenchymal stromal cells suspension. Invert the tube for homogeneity of the new cell suspension. Add 400 microliters of cell suspension to each well of both silicone plates.

Put the lid back on the 150-millimeter plates and incubate the plates at 37 degrees Celsius for 24 hours. For uniaxial tension, rinse the stretching apparatus with double-distilled water, followed by 70%ethanol. Wipe down the apparatus, place it in the cell culture hood, and expose it to ultraviolet light for 15 minutes.

Next, place one plate in the incubator as static control. Place the second seated silicone plate in the stretching apparatus by aligning the screws with the holes in the silicone plate. Then, place the lid on the apparatus.

Attach the apparatus with the seated silicone plate to the electronic source and place it in the incubator at 37 degrees Celsius. Set the cyclic stretching program to 4%sinusoidal strain in 0.5 hertz for two hours per day. Initiate the stretching by pressing the start button.

During mechanical loading of scleraxis-positive induced mesenchymal stromal cells, excessively high seating densities led to premature cell contraction and early cell death. After several days of stretching, some degree of cell organization was observed in the scleraxis-positive induced mesenchymal stromal cells stretched group, compared to the random cell organization in the static group. Phalloidin staining of the acton filaments showed that cells grew perpendicular to the direction of stretch, in contrast to the random cell organization observed in the static plates.

Gene expression analysis revealed significant upregulation in tenogenic genes at both three and seven days, compared to just the iMSCs at day zero. The collagen deposition in the media after seven days of stretching was significantly higher in the scleraxis-positive induced mesenchymal stromal cells stretched group compared to all other groups.