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

Summary

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

Abstract

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs^+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

Introduction

Stem cell-based therapies have shown considerable potential as a treatment for cardiac damage caused by MI¹. The use of differentiated hiPSCs provides an inexhaustible source of hiPSC-CMs² and opens the door for the rapid development of breakthrough treatments. However, many limitations to therapeutic translation remain, including the challenge of the severely low engraftment rate of implanted cells.

Dissociating cells with trypsin initiates anoikis³, which is only accelerated once these cells are injected into harsh environments like the ischemic myocardium, where the hypoxic environment accelerates the course toward cell death. Of the remaining cells, a large proportion is washed out from the implantation site into the bloodstream and spread throughout the periphery. One of the key apoptotic pathways is the RhoA/ROCK pathway⁴. Based on previous research, the RhoA/ROCK pathway regulates the actin cytoskeletal organization^5,6, which is responsible for cell dysfunction^7,8. The ROCK inhibitor Y-27632 is widely used during somatic and stem cell dissociation and passaging, to increase cell adhesion and reduce cell apoptosis^9,10,11. In this study, Y-27632 is used to treat hiPSC-CMs prior to transplantation in an attempt to increase the cell engraftment rate.

Several methods aimed at improving the cell engraftment rate, such as heat shock and basement membrane matrix coating¹², have been established. Aside from these methods, genetic technology can also promote cardiomyocyte proliferation¹³ or reverse nonmyocardial cells into cardiomyocytes¹⁴. From the bioengineering perspective, cardiomyocytes are seeded onto a biomaterial scaffold to improve the transplantation efficiency¹⁵. Unfortunately, the majority of these methods are complicated and costly. On the contrary, the method proposed here is simple, cost-efficient, and effective, and it can be used as a basal treatment before transplantation, as well as in conjugation with other technologies.

Protocol

All animal procedures in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham and were based on the National Institutes of Health Laboratory Animal Care and Use Guidelines (NIH Publication No 85-23).

1. Preparation of Culture Media and Culture Plates

1. Medium preparation
   1. For hiPSC medium, mix 400 mL of human pluripotent stem cell (hPSC) basal medium (Table of Materials 1) and 100 mL of hPSC 5x supplement; store the mixture at 4 °C.
   2. For RPMI 1640/B27 minus insulin (RB-) medium, mix 500 mL of RPMI 1640 and 10 mL of B27 supplement minus insulin.
   3. For RPMI 1640/B27 (RB+) medium, mix 500 mL of RPMI 1640, 10 mL of B27 supplement, and 5 mL of penicillin-streptomycin (pen-strep) antibiotic.
   4. For purification medium¹⁶, mix 500 mL of no-glucose RPMI 1640, 10 mL of B27 supplement, 5 mL of pen-strep antibiotic, and 1,000 μL of sodium DL-lactate solution (at a final concentration of 4 mM).
   5. For neutralizing solution, mix 200 mL of RPMI 1640, 50 mL of fetal bovine serum (FBS), 2.5 mL of pen-strep antibiotic, and 50 μL of Y-27632 (at a final concentration of 10 μM).
   6. For freezing medium, mix 9 mL of FBS, 1 mL of dimethyl sulfoxide (DMSO), and 10 μL of Y-27632 (at a final concentration of 10 μM).
   7. For extracellular matrix solution (EMS), thaw concentrated extracellular matrix (Table of Materials 1) at 4 °C until it has reached an evenly consistent liquid state. Precool pipette tips and tubes prior to making matrix aliquots of 350 µL each on ice, and store the aliquots at -20 °C. Before coating the plates, dilute one thawed aliquot into 24 mL of ice-cold DMEM/F12 medium (EMS); keep the EMS in 4 °C for up to 2 weeks.
      NOTE: Perform all coating steps on ice to prevent basement membrane matrix solidification.
   8. For Tyrode’s solution, take 90% of the final required volume of tissue culture grade water, add the appropriate amount of the following reagents, and stir until dissolved. Then, use 1 N NaOH to adjust the pH to 7.4. Add extra water to a final concentration of 140 mmol/L sodium chloride, 1 mmol/L magnesium chloride, 10 mmol/L HEPES, 5 mmol/L potassium chloride, 10 mmol/L glucose, and 1.8 mmol/L calcium chloride. Sterilize by filtration, using a 0.22 µm membrane.
2. Cell culture plate coating
   1. For hiPSC culture plates, apply 1 mL of EMS to each well of a 6-well plate and incubate the plate for at least 1 h at 37 °C. Aspirate DMEM/F12 solution before seeding cells.
   2. For gelatin coating, dissolve 0.2 g of gelatin powder in tissue culture grade water to make a 0.2% (w/v) solution. Sterilize by autoclaving at 121 °C, 15 psi for 30 min. Coat each well of a 6-well plate with 2 mL of the solution and incubate for 1 h at 37 °C. Before passaging the cells, aspirate the solution and allow the plate to dry for at least 1 h at room temperature inside the tissue culture hood.

2. hiPSC maintenance and Cardiomyocyte Ddifferentiation

1. Culture the hiPSCs in hiPSC medium (see step 1.1.1) with a daily medium change until the cells reach 80%–90% confluence per well.
   NOTE: For maintenance purposes, hiPSCs are generally ready for passage every 4 days.
2. To passage hiPSCs, aspirate the medium and wash the well 1x with 1x phosphate-buffered saline (PBS). Add 0.5 mL of stem cell detachment solution (Table of Materials 1) to each well and incubate at 37 °C for 5–7 min.
   NOTE: The incubation time depends on the density of the cells and the rate of dissociation, which can be observed under a microscope.
3. Neutralize the stem cell detachment solution with 1 mL of hiPSC medium supplemented with 5 μM Y-27632. Collect the mixture in a 15 mL centrifuge tube. Centrifuge the tube for 5 min at 200 x g, aspirate the supernatant without disturbing the cell pellet, and then, resuspend the cells in 1 mL of hiPSC medium containing 5 μM Y-27632.
4. Seed the hiPSCs onto a new EMS-coated 6-well plate at a ratio between 1:6 to 1:18. Change the medium to hiPSC medium without Y27632 after 24 h.
5. To freeze the cells, resuspend the dissociated hiPSCs in freezing medium at a concentration of 0.5–1 million/500 μL, place the suspension in cryovials, and store them in a -80 °C freezer. Transfer the frozen vials to liquid nitrogen after 24 h.
6. To differentiate, replace the hiPSC medium with 2 mL of RB- medium supplemented with 10 μM GSK-3β inhibitor CHIR99021 (CHIR) at 80%–90% cell-confluence. Replace the medium with 3 mL of RB- medium after 24 h and incubate for an additional 48 h (day 3).
7. On day 3, change the medium to 3 mL of RB- medium with 10 μM Wnt inhibitor IWR1 for 48 h (day 5), and then replace the medium with 3 mL of RB- medium and maintain for 48 h (day 7).
8. On day 7, replace the medium with 3 mL of RB medium and change the medium every 3 days going forward. Spontaneous beating of hiPSC-CMs should be observed between days 7–10.

3. hiPSC-CMs Purification and Small Molecule Pre-treatment

NOTE: Highly purified, recombinant cell-dissociation enzymes (Table of Materials 1) were used to dissociate hiPSC-CMs.

1. On day 21 after hiPSC-CM differentiation, aspirate the medium and wash the cells 1x with sterile PBS. Incubate the cells with 0.5 mL of cell-dissociation enzymes per well for 5–7 min at 37 °C. Repeatedly pipette the cell suspension using a 1 mL pipette in order to thoroughly dissociate the cells.
2. After the cells are dissociated, add 1 mL of neutralizing solution to each well, collect the cell mixture into a 15 mL centrifuge tube and centrifuge at 200 x g for 3 min. Discard the supernatant and resuspend the cells in neutralizing solution.
3. Replant the cells onto gelatin-coated 6-well plates at a density of 2 million cells per well.
4. After 24–48 h, replace the culture medium with purification medium for 3–5 days.
   NOTE: Purification is complete when more than 90% of the cells in each microscope view field are beating. To prevent further damage to the cardiomyocytes, do not extend the purification duration beyond 5 days. If the first round does not yield the necessary purity, replace the purification medium with RB+ medium for 1 day, and then complete a second round of purification.
5. Prior to transplantation, culture the cells in the treatment group for 12 h in RB+ medium supplemented with 10 μM Y-27632. Similarly, perform verapamil treatment on the cells in the RB+ medium with 1 μM verapamil for 12 h (hiPSC-CMs^+VER).
6. After treatment, wash the hiPSC-CMs 1x with PBS and incubate the cells with 0.5 mL of cell-dissociation enzymes per well for a maximum of 2 min. Repeatedly pipette the cell suspension, using a 1 mL pipette, to thoroughly dissociate the cells. Neutralize the cells with neutralizing solution, collect the cell mixture in a 15 mL centrifuge tube, and centrifuge at 200 x g for 3 min. Resuspend the cells in PBS at a concentration of 0.1 million cells/5 μL in preparation for injection.

4. Myocardial Infarction and Cell Transplantation

NOTE: All surgical instruments are presterilized with autoclave and are maintained in aseptic condition during multiple surgeries via a hot bead sterilizer (Table of Materials 2).

1. Anesthetize NOD/scid mice (Table of Materials 2) with inhaled isoflurane (1.5%–2%). Monitor the anesthesia levels by the mice’s responses to a toe pinch. Place vet optical ointment onto the eyes to prevent them from drying during surgery.
2. Supply 0.1 mg/kg buprenorphine subcutaneously for pain management prior to surgery.
3. Position and secure the mouse in a supine position on a heated operating table, remove the hair from the ventral neck region and the left thorax using a depilatory cream, expose the skin, and disinfect it with 70% alcohol.
4. Cut a 0.5 cm incision at the center of the neck using surgical scissors, separate the subcutaneous fat with sterile forceps, and expose the trachea. Introduce orally the intubation cannula into the trachea, connect the cannula to a small animal ventilator, and adjust the ventilation settings (set the tidal volume at 100–150 μL and the respiration rate at 100x–150x per min).
5. After the mouse stabilizes, cut a 0.5 cm incision in the middle of the left chest skin. Use forceps to bluntly separate the muscle layer and make a small incision in the fifth intercostal space to expose the chest cavity. Place the retractor in the incision to open the thoracic cavity and locate the left descending coronary artery (LAD). Under a dissecting surgical microscope, ligate the LAD with an 8-0 nonabsorbable suture.
6. Immediately following MI induction, inject 5 μL of hiPSC-CMs^-RI, hiPSC-CMs^+RI, hiPSC-CMs^-VER, hiPSC-CMs^+VER, or an equal volume of PBS into the mouse’s myocardium at each site (3 x 10⁵ cells/animal, 1 x 10⁵ cells/site), one in the infarct area and two in the areas around the infarct.
   NOTE: Since 5 μL is very small volume, it is not easy to accurately control the volume by directly sucking it with a syringe. The lid of a sterile Petri dish can be turned over, and 5 μL of the cells can be first deposited on the lid with a pipette. Due to the surface tension, it will not spread but will condense into a small liquid mass. This can be easily absorbed and injected into the mouse’s myocardium.
7. Eliminate residual air in the thoracic cavity by filling it up with warm isotonic saline solution. Stitch the ribs, muscles, and skin in sequence with a 6-0 absorbable suture. Turn off the isoflurane anesthesia injection. Keep the surgery animals on the heating pad and closely observe them while they regain full consciousness. Then, place the animals in a clean cage. Do not return the animals to the company of other animals until they are fully recovered.
8. Following surgery, inject the mice intraperitoneally with buprenorphine (0.1 mg/kg) every 12 h for 3 consecutive days and inject them with ibuprofen (5 mg/kg) every 12 h for one day. Perform subsequent analysis studies at specified time points.
   NOTE: Follow your local animal care committee guidelines for analgesia.

5. Calcium Transient and Contractility Recording

1. Autoclave 15 mm-diameter coverslips (Table of Materials 2) and tweezers in a small glass beaker at 121 °C, 15 psi for 15 min. Precool the coverslips and tweezers at 4 °C.
2. Using the tweezers, place the coverslip into a 12-well plate and pipette 300 μL of extracellular matrix onto each coverslip.
   NOTE: Do not let the matrix exceed the edge of the coverslip to prevent reducing the matrix coating amount. Incubate the 12-well plate in a 37 °C cell culture incubator for 1 h.
3. Aspirate the medium in the gelatin-coated 6-well plate with purified hiPSC-CMs, wash it 1x with PBS, and then digest it with 0.5 mL of cell-dissociation enzymes for 1.5 min. Add 1 mL of neutralizing solution, pipette repeatedly for no more than 5x–10x with a 1 mL pipette, and centrifuge the mixture at 200 x g for 3 min.
4. Aspirate the coated extracellular matrix from the coverslips. Count the cell number and resuspend the cells in the neutralizing solution to a concentration of 40,000/300 μL, and then add 300 μL of the cell suspension onto each coverslip. Culture the plate in a 37 °C incubator.
5. After 24 h, gently aspirate the neutralized solution, add 500 μL of RB+ medium to each well of the plate, and continue to culture for 2 days.
6. Add Y-27632 (10 μM), Rho kinase activator (RA, 100 nM), verapamil (1 μM), or an equal volume of PBS into the culture medium of hiPSC-CMs, 12 h before calcium transient and contractility detection.
   NOTE: In addition to the detection of the cells’ calcium transient and contractility after 12 h of chemical treatment, the detection of these parameters was also tested at 12, 24, 48, and 72 h after chemical withdrawal.
7. Take out the plate, discard the medium, and wash the plate 1x with PBS. Change the medium to a phenol-red-free DMEM containing 0.02% (w/v) of surfactant polyol (Table of Materials 1), 5 μM calcium indicator (Table of Materials 1), and the corresponding Y-27632 (10 μM), RA (100 nM), verapamil (1 μM), or an equal volume of PBS, and incubate the plate for 30 min at 37 °C.
8. Discard the supernatant, wash the cells 3x with dye-free DMEM medium, and let the medium rest for 30 min to de-esterify the mapping dye.
9. Place the cell-seeded coverslip in an open bath chamber and insert the chamber into a microincubation system equilibrated to 37 °C with an automatic temperature controller (Table of Materials 2), perfuse it with Tyrode’s solution, and continuously add Rho kinase inhibitor (RI), RA, or PBS to the perfusion solution.
10. Use an inverted fluorescence microscope (Table of Materials 2) and a laser scanning head (Table of Materials 2) to record spontaneous calcium transient by employing an x-t line scan, using a 488 nm argon laser for dye excitation and a 515 ± 10 nm barrier filter to collect emission light. Record the spontaneous contraction of hiPSC-CMs using the Differential Interference Contrast functionality of the the confocal microscope (Table of Materials 2).  
    NOTE: Calcium transient recordings were processed and analyzed using MATLAB R2016A software (Table of Materials 4). Cell contraction change assays were performed using ImageJ software (Table of Materials 4).

Results

The hiPSC-CMs used in this study were derived from human origin with luciferase reporter gene; therefore, the survival rate of the transplanted cells in vivo was detected by bioluminescence imaging (BLI)¹⁷ (Figure 1A,B). For histological heart sections, human-specific cardiac troponin T (hcTnT) and human nuclear antigen (HNA) double-positive cells were classified as engrafted hiPSC-CMs (Figure 1C

Discussion

The key steps of this study include obtaining pure hiPSC-CMs, improving the activity of hiPSC-CMs through Y-27632 pretreatment, and finally, transplanting a precise amount of hiPSC-CMs into a mouse MI model.

The key issues addressed here were that, first, we optimized the glucose-free purification methods¹⁹ and established a novel efficient purification system. The system procedure included applying cell-dissociation enzymes, replanting cells in gelatin-coated plates, c...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
Accutase (stem cell detachment solution)	STEMCELL Technologies	#07920
B27 minus insulin	Fisher Scientific	A1895601
B27 Supplement	Fisher Scientific	17-504-044
CHIR99021	Stem Cell Technologies	72054
DMEM (1x), high glucose, HEPES, no phenol red	Thermofisher	20163029
Fetal bovine serum	Atlanta Biologicals	S11150
Fluo-4 AM (calcium indicator)	Invitrogen/Thermofisher	F14201
Glucose-free RPMI 1640	Fisher Scientific	11879020
IWR1	Stem Cell Technologies	72562
Matrigel (extracellular matrix )	Fisher Scientific	CB-40230C
mTeSR (human pluripotent stem cells medium)	STEMCELL Technologies	85850
Pen-strep antibiotic	Fisher Scientific	15-140-122
Pluronic F-127 (surfactant polyol)	Sigma-Aldrich	P2443
Rho activator II	Cytoskeleton	CN03
RPMI1640	Fisher Scientific	11875119
Sodium DL-lactate	Sigma-Aldrich	L4263
TrypLE (cell-dissociation enzymes)	Fisher Scientific	12-605-010
Verapamil	Sigma-Aldrich	V4629
Y-27632	STEMCELL Technologies	72304
Name	Company	Catalog Number	Comments
Equipment and Supplies
IVIS Lumina III Bioluminescence Instruments	PerkinElmer	CLS136334
15 mm Coverslips	Warner	CS-15R15
Centrifuge	Eppendorf	5415R
Confocal Microscope	Olympus	IX81
Cryostat	Thermo Scientific	NX50
Dual Automatic Temperature Controller	Warner Instruments	TC-344B
Electrophoresis Power Supply	BIO-RAD	1645050
Fluoresence Microscope	Olympus	IX83
High Speed Camera	pco	1200 s
Laser Scan Head	Olympus	FV-1000
Low Profile Open Bath Chamber (mounts into above microincubation system)	Warner Instruments	RC-42LP
Microincubation System	Warner Instruments	DH-40iL
Minivent Mouse Ventilator	Harvard Apparatus	845
NOD/SCID mice	Jackson Laboratory	001303
Precast Protein Gels	BIO-RAD	4561033
PVDF Transfer Packs	BIO-RAD	1704156
Trans-Blot System	BIO-RAD	Trans-Blot Turbo
Hot bead sterilizer	Fine Science Tools	18000-45
Name	Company	Catalog Number	Comments
Antibody
Anti-human Nucleolin (Alexa Fluor 647)	Abcam	ab198580
Cardiac Troponin T	R&D Systems	MAB1874
Cardiac Troponin C	Abcam	ab137130
Cardiac Troponin I	Abcam	ab47003
Cy5-donkey anti-mouse	Jackson ImmunoResearch Laboratory	715-175-150
Cy3-donkey anti-rabbit	Jackson ImmunoResearch Laboratory	711-165-152
Fitc-donkey anti-mouse	Jackson ImmunoResearch Laboratory	715-095-150
GAPDH	Abcam	ab22555
Human Cardiac Troponin T	Abcam	ab91605
Integrin β1	Abcam	ab24693
Ki67	EMD Millipore	ab9260
N-cadherin	Abcam	ab18203
Phospho-Myosin Light Chain 2	Cell Signaling Technology	3671s
Name	Company	Catalog Number	Comments
Software
Matlab	MathWorks	R2016A
ImageJ	NIH	1.52g