Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

Our data demonstrates that transient inhibition of Rho-kinase activity enhances adhesion, survival, and engraftment of hiPSC-derived cardiomyocytes after being transplanted into the infarcted animal hearts. We provide a simple, low-cost, while highly efficient method to increase engraftment of transplanted hiPSC-derived cardiomyocytes in the injured animal hearts. As this technique can improve cell engraftment rates safely and efficiently in acute cardiac infarction and heart failure, it contributes for the resulting improvements in cardiac function, vascularities, and apoptosis.

Although we only tested our protocols in hiPSCs, these methods can be applied to other types of cells, such as embryonic stem cells, mesenchymal stem cells, et cetera. The dose and duration of Y-27632 treatment is critical. The visual demonstration can demonstrate in great detail all key steps in the protocol, which is beneficial for someone who has never performed this technique before.

On day 21 after hiPSC-CM differentiation, aspirate the medium and wash the cells with sterile PBS once. Incubate the cells with 0.5 milliliters of cell-dissociation enzymes per well at 37 degrees Celsius for five to seven minutes. After the incubation time, repeatedly pipette the cell suspension using a one-milliliter pipette in order to thoroughly dissociate the cells.

Then, add one milliliter of neutralizing solution to each well, collect the cell mixture in a 15-milliliter centrifuge tube, and centrifuge at 200 times g for three minutes. After the centrifugation, discard the supernatant and resuspend the cells in the neutralizing solution. Then, replant the cells onto gelatin-coated, six-well plates at a density of two million cells per well.

After 24 to 48 hours, replace the culture medium with the purification medium for three to five days. Prior to transplantation, culture the cells in the treatment group for 12 hours in RB-plus medium supplemented with 10-micromolar Y-27632. Similarly, perform verapamil treatment on the cells in the RB-plus medium with one micromole of verapamil for 12 hours.

After the treatment, wash the hiPSC-CMs with PBS once, and incubate the cells with 0.5 microliters of cell-dissociation enzymes per well for a maximum of two minutes. Repeatedly pipette the cell suspension using a one-milliliter pipette to thoroughly dissociate the cells. Next, neutralize the cells with neutralizing solution, collect the cell mixture in a 15-milliliter centrifuge tube, and centrifuge at 200 times g for three minutes.

After centrifugation, resuspend the cells in PBS at a concentration of 0.1 million cells per five microliters in preparation for injection. Immediately following myocardial infarction induction, inject five microliters of control hiPSC-CMs, Y-27632-pretreated hiPSC-CMs, verapamil-treated hiPSC-CMs, or an equal volume of PBS into the mouse's myocardium at each site, one in the infarct area and two in the areas around the infarct. 12 hours before performing calcium transient and contractility detection, add 10-micromolar Y-27632, 100-nanomolar RA, one-micromolar verapamil, or an equal volume of PBS into the culture medium of hiPSC-CMs.

Next, discard the medium, and wash the plate with PBS once. Change the medium to a phenol red-free DMEM containing 0.02 weight per volume percent of surfactant polyol, five-micromolar calcium indicator, and the corresponding Y-27632, RA, verapamil, or an equal volume of PBS, and incubate the plate at 37 degrees Celsius for 30 minutes. After the incubation, discard the supernatant, wash the cells with the dye-free DMEM three times, and let the medium rest for 30 minutes to de-esterify the mapping dye.

Place the cell-seeded coverslip in an open bath chamber, and insert the chamber into a microincubation system equilibrated to 37 degrees Celsius with an automatic temperature controller. Perfuse it with Tyrode's solution, and continuously add RI, RA, or PBS to the perfusion solution. Use an inverted fluorescence microscope and a laser scanning head to record spontaneous calcium transient by employing an x-t line scan.

Record the spontaneous contraction of hiPSC-CMs using the differential interference contrast functionality of the confocal microscope. The survival rate of the transplanted cells was confirmed by the increased luciferase activity. The increased human cardiac troponin T and human nuclear antigen expression showed that Y-27632 pretreatment could significantly improve the cell engraftment rate.

Y-27632 pretreated cells exhibited a larger and more defined rod-shaped cytoskeletal structure. Also, Y-27632 pretreatment decreased TUNEL-positive cells, indicating the reduced transplanted hiPSC-CM apoptosis in vivo. Western blot suggested that Y-27632 pretreatment increased expression of integrin beta-1 and N-cadherin and decreased the expression of phospho-myosin light chain 2.

This change was normalized 72 hours after Y-27632 withdrawal. In vitro mouse HL-1 cell attachment experiments indicated that Y-27632 pretreatment significantly increased hiPSC-CM adherence relative to HL-1. Y-27632 pretreatment reduced the contractile force, the peak calcium transient fluorescence, and the calcium transient duration.

Y-27632 pretreatment regulated cardiomyocyte contraction by significantly reducing the expression of cTnI and cTnT. Similar to Y-27632, verapamil pretreatment increased the luciferase activity and numbers of hcTnT/HNA double-positive cells. The most important step is to culture cells in the treatment group for 12 hours in Rb-plus medium supplemented with 10-micromolar Y-27632.

Based on this method, slow release of Y-27632 to further increase the cell engraftment rate is a promising strategy. This technique paves the way for researchers to study the physiology and function of hiPSC of CMs in vivo. The small molecule CHIR99021 used for cardiomyocytes differentiation is hazardous, which may cause respiratory irritation.

Please don't intake or inhale by any way.