Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Summary

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Abstract

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

Introduction

Human induced pluripotent stem cells (hiPSCs) are among the most promising agents for regenerative cell therapy because they can be differentiated into a potentially unlimited range and quantity of cells that are not rejected by the patient's immune system. However, their capacity for self-replication and differentiation can also lead to tumor formation and, consequently, hiPSCs need to be fully differentiated into specific cell types, such as cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs), before administration. One of the simplest and most common methods of cell administration is direct intramyocardial injection, but the number of transplanted cells that are engrafted by the native myocardial tissue is exceptionally low. Much of this attrition can be attributed to the cytotoxic environment of the ischemic tissue; however, when murine embryonic stem cells (ESCs) were injected directly into the myocardium of uninjured hearts, only ~40% of the 5 million cells delivered were retained for 3-5 hr¹, which suggests that a substantial proportion of the administered cells exited the administration site, perhaps because they were squeezed out through the needle track by the high pressures produced during myocardial contraction.

Here, we present novel and substantially more efficient methods for generating hiPSC-derived cardiomyocytes (hiPSC-CMs)², endothelial cells (hiPSC-ECs)³, and smooth muscle cells (SMCs)⁴. Notably, this hiPSC-SMC protocol is the first to mimic the wide range of morphological and functional characteristics observed in somatic SMCs⁵ by directing the cells toward a predominantly synthetic or contractile SMC phenotype. We also provide a method of cell delivery that improves the engraftment rate of injected cells by creating a cytokine-containing fibrin patch over the injection site. The patch appears to improve both cell retention, by sealing the needle track to prevent the cells from exiting the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over a period of at least three days.

Protocol

All experimental procedures are performed in accordance with the Animal Guidelines of the University of Alabama at Birmingham.

1. Differentiating hiPSCs into hiPSC-CMs

1. Coat the wells of a 6-well plate with pre-cooled growth-factor-reduced gelatinous protein mixture at 4 °C for overnight. Aspirate the gelatinous protein mixture before use. Seed the hiPSCs onto the pre-coated plates, and culture the cells (1 x 10⁵ cell per well) at 5% CO₂ and 37 °C in mTeSR1 medium supplement with 10 µM ROCK inhibitor.
2. Refresh the medium daily until the cells reach 90% confluence; then, add growth-factor-reduced gelatinous protein mixture to the medium (0.5 mg gelatinous protein mixture per 6-well plate, 2 ml medium per well), and culture the cells for 5% CO₂ and 37 °C two more days.
   1. To replace the medium, gently suck out the medium from the petri dish via vacuum without touching the cells, and add new medium using a transfer pipette.
3. Initiate differentiation by replacing the medium with RPMI1640 medium supplemented with growth-factor-reduced gelatinous protein mixture, B27 without insulin, and 100 ng/ml Activin A; culture the cells at 5% CO₂ and 37 °C for 24 hr.
4. Replace the medium with RPMI1640 medium that has been supplemented with B27 without insulin, 10 ng/ml bone morphogenic protein 4 (BMP-4), and 10 ng/ml basic fibroblast growth factor (bFGF); culture the cells at 5% CO₂ and 37 °C for 96 hr.
5. Replace the medium with RPMI1640 medium supplemented with B27 and continue culture the cells at 5% CO₂ and 37 °C; refresh the medium every 3 days.
6. Observe clusters of contracting cells under the microscope ~3 days after initiating differentiation. Collect the clusters ~7 days after first observing contractions and wash them in Hanks Balanced Salt Solution.
   1. Dissociate the clustered-cells in the plate by incubating them in Hanks buffer containing 100 U/ml collagenase IV for 10 min at 37 ºC with gentle shaking; then, add 0.25% trypsin in ethylenediaminetetraacetic acid (EDTA) for 5 min.
   2. Neutralize the enzyme solution with 10% fetal bovine serum in RPMI/B27 medium and then resuspend the cells in RPMI/B27 medium.
   3. Count the number of cells by hemocytometer. Culture the cells on 10 cm dishes at 5% CO₂ and 37 °C for at least 3 hr, which will allow the non-cardiomyocyte cells to adhere to the surface of the culture dish.
7. Collect the cell suspension, which contains the hiPSC-CMs, and maintain the cells (around 1 x 10⁶ cells per 10 cm dish) by culturing them on a gelatinous protein mixture -coated surface.

2. Differentiating hiPSCs into hiPSC-ECs

1. Dissociate the hiPSC population into single cells by incubating them with chelating agent at 5% CO₂ and 37 °C for 5 min. Transfer the cells to a 15 ml centrifuge tube containing fresh mTeSR1 medium.
2. Spin down the cells at 200 x g for 5 min. Resuspend the cells in mTeSR1 medium supplemented with 10 µM ROCK inhibitor. Determine the cell density with a hemocytometer.
3. Add 250 µl of 20 NIH units/ml thrombin solution to one well of a 24-well plate.
4. Add 1 x 10⁶ hiPSCs to 250 µl of a 12.5 mg/ml fibrinogen solution. And add the 250 µl of cell-containing fibrinogen solution to the thrombin-loaded well. Observe the mixture solidify to form a hiPSC-containing fibrin scaffold within min.
5. Transfer the solidified, cell-containing scaffolds into the wells of a 6-well plate with the cover glass forceps, and initiate differentiation by culturing the scaffolds in 2 ml EBM2 medium supplemented with 2% B27 without insulin, activin-A (50 ng/ml), and BMP-4 (25 ng/ml) for 24 hr at 5% CO₂ and 37 °C.
   1. Replace the medium by gently sucking out the old medium via vacuum without touching the cells. Add new EBM2 medium supplemented with B27 without insulin, vascular endothelial growth factor (VEGF, 50 ng/ml), erythropoietin (EPO, 100 ng/ml), and transforming growth factor β1 (TGFβ1, 10 ng/ml), and culture the scaffolds at 5% CO₂ and 37 °C for 48 hr.
   2. Refresh the medium and culture the scaffolds at 5% CO₂ and 37 °C for another 48 hr; then, release the cells from the scaffold by adding 200 U collagenase IV (100 U/ml) to the medium.
   3. Spin down the cells after they are released. Replace the medium with 2 ml EGM2-MV medium supplemented with 2% of B27, VEGF-A (50 ng/ml), and SB-431542 (10 µM); refresh the medium every two days.
   4. Approximately 10 days later (i.e., on ~Day 14 after differentiation was initiated), dissociate the cells with 100 U/ml collagenase IV, isolate hiPSC-ECs from the population of differentiated cells via flow-cytometry analyses for the expression of EC-specific marker proteins (e.g., CD31, CD144)³.
   5. Expand the isolated hiPSC-ECs via an established protocol³.

3. Differentiating hiPSCs into hiPSC-SMCs

1. Seed the hiPSCs onto plates that have been coated with gelatinous protein mixture, and culture the cells in mTeSR1 medium at 5% CO₂ and 37 °C, with daily medium changes, until confluent (~2 days).
2. Initiate differentiation into mesodermal-lineage cells by culturing the cells with CHIR99021 (5 µM) and BMP-4 (10 ng/ml) in RPMI1640 medium and 2% B27 plus insulin for 3 days.
3. To produce hiPSC-SMCs with a predominantly synthetic phenotype:
   1. Culture the cells with VEGF-A (25 ng/ml), fibroblast growth factor β (FGFβ, 5 ng/ml) in RPMI1640 medium, and 2% B27 minus insulin at 5% CO₂ and 37 °C for 4 days.
   2. Culture the cells in VEGF-A (25 ng/ml), FGFβ (5 ng/ml) in RPMI1640 medium, and 2% B27 plus insulin at 5% CO₂ and 37 °C for 2 days.
   3. Culture the cells in platelet-derived growth factor β (PDGFβ, 10 ng/ml), TGFβ (3 ng/ml) in RPMI1640, and 2% B27 plus insulin at 5% CO₂ and 37 °C for 4 days.
4. To produce hiPSC-SMCs with a predominantly contractile phenotype:
   1. Culture the cells for 4 days with VEGF-A (25 ng/ml) and FGFβ (5 ng/ml) in RPMI1640 and 2% B27 minus insulin.
   2. Culture the cells for 7 days with PDGFβ (5 ng/ml) and TGFβ (2.5 ng/ml) in RPMI1640 and 2% B27 plus insulin.
5. Purify the final hiPSC-SMC populations by culturing the cells in lactate (4 mM) containing RPMI1640 metabolic medium for ~6 days.

4. Creating the IGF-containing Microspheres

1. Heat olive oil to 45 °C in a water bath.
2. Heat 5 ml of 10% gelatin solution to 50 °C.
3. Add the gelatin to the olive oil, stir, and then rapidly cool to 5 °C by adding ice to the water bath.
4. Twenty-five minutes later, add chilled (4 °C) acetone to the olive oil to induce microsphere formation.
5. Maintain the temperature at 5 °C for 1 hr; then, collect the microspheres, wash them 5 times with pre-cooled acetone to remove the olive oil, and allow them to air-dry at 4 °C.
6. Resuspend the microspheres in a solution of 70% ethanol and 0.25% glutaraldehyde overnight at 4 °C to induce cross-linking, and then neutralize the mixture with 100 mM glycine.
7. Load IGF into the microspheres by mixing 5 mg microspheres with 15 µl distilled H₂O containing 5 µg IGF and 0.1% bovine serum albumin for 30 min.

5. Creating the Patch over the Site of Injury and Injecting the Cells

1. Suspend the hiPSC-derived CMs (2 million), SMCs (1 million), and ECs (1 million) together in 1 ml Minimum Essential Medium (MEM).
2. Suspend 5 mg of microspheres in 1 ml fibrinogen solution (25 mg/ml).
3. Fill one syringe with the cell-containing solution, another syringe with the fibrinogen/microsphere solution, and a third syringe with 1 ml of thrombin solution (80 NIH units/ml, supplemented with 2 µl 400 mM CaCl₂ and 200 mM ε-aminocaproic acid).
4. Surgically induce myocardial infarction in swine heart via ligation of left anterior descending coronary artery as described before².
   NOTE: In brief, female Yorkshire swine (~13 kg, 45 days of age) are sedated with intramuscular injection of Telazol/Xylazine (4.4 mg/kg), and intubated under general anesthesia with isoflurane (0.5-5%). A 4^th intercostal thoracotomy is performed and left anterior descending coronary artery is exposed. The coronary artery is occluded by a ligature for 60 min and then the ligature is removed. Immediate electrical defibrillation is performed in the case of ventricular fibrillation.
5. Position a sterile plastic ring (~2.5 cm) on the epicardium of the infarcted region of swine hearts, and then simultaneously inject the microsphere/fibrinogen and thrombin solutions into the ring.
6. Allow the mixture to solidify (~30 sec), and then insert the needle of the cell-containing syringe through the solidified mixture into the infarcted myocardium and inject the cells. Close the muscle layers, subcutaneous tissue, and the chest wall with 3-0 or 2-0 Monocryl suture depending on animal size. Buprenorphine 0.01-0.02 mg/kg intramuscular injection every 8 hours will be used to control pain for the first 3 days after surgery.
7. Evaluate cardiac function using a cardiac magnetic resonance imaging (MRI) before surgical procedure, one week and four weeks after the surgical procedure^2,3,4. After the final MRI studies, sacrifice the animal and process their hearts for histology and molecular analysis^2,3,4.

Results

Characterization of Differentiated hiPSC-CMs, -ECs, and -SMCs

The differential capacity of hiPSCs were evaluated^2,3,4. Flow cytometry analyses of cardiac troponin T (cTnT) expression suggest that the purity of the final hiPSC-CM population can exceed 90% (Figure 1A, 1B, panel B1). Nearly all of the cells e...

Discussion

Improved Yield/Purity of hiPSC-CMs

Conventional protocols for differentiating human stem cells into CMs are often limited by low yield and purity; for example, just 35-66% of hESC-CMs obtained via Percoll separation and cardiac body formation expressed slow myosin heavy chain or cTnT⁶. The purity of differentiated hiPSC-CM populations can be substantially increased by selecting for the expression of a reporter gene that has been linked to the promoter of a CM-specific prot...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Protocol Section 1
mTeSR1 medium	Stem cell technologies	5850
Growth-factor-reduced matrigel	Corning lifescience	356231
Y-27632	Stem cell technologies	72304
B27 supplement, serum free	Fisher Scientific	17504044
RPMI1640	Fisher Scientific	11875-119
Activin A	R&D	338-AC-010
BMP-4	R&D	314-BP-010
bFGF	R&D	232-FA-025
Collagenase IV	Fisher Scientific	NC0217889
Hanks Balanced Salt Solution (Dextrose, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4 anhydrous)	Fisher Scientific	14175079
Fetal Bovine Serum	Fisher Scientific	10438018
6-well plate	Corning Lifescience	356721
10 cm dish	Corning Lifescience	354732
Cell incubator	Panasonic	MCO-18AC
Protocol Section 2
Versene	Fisher Scientific	15040066
Fibrinogen	Sigma-Aldrich	F8630-5g
Thrombin	Sigma-Aldrich	T7009-1KU
EMB2 medium	Lonza	CC-3156
VEGF	ProSpec-Tany	CYT-241
EPO	Life Technologies	PHC9431
TGF-β	Peprotech	100-21C
EGM2-MV medium	Lonza	CC-4147
SB-431542	Selleckchem	S1067
CD31	BD Bioscience	BDB555445
CD144	BD Bioscience	560411
15 ml centrifuge tube	Fisher Scientific	12565269
Eppendorff Centrifuge	Eppendorf	5702R
Protocol Section 3
CHIR99021	Stem cell technologies	720542
PDGF-β	Prospec	CYT-501-10ug
Protocol Section 4
Olive oil	Sigma-Aldrich	O1514
Gelatin	Sigma-Aldrich	G9391
Acetone	Sigma-Aldrich	179124
Ethanol	Fisher Scientific	BP2818100
Glutaraldehyde	Sigma-Aldrich	G5882
Glycine	Sigma-Aldrich	G8898
IGF	R&D	291-G1-01M
Bovine serum albumin	Fisher Scientific	15561020
Heating plate	Fisher Scientific	SP88850200
Water bath	Fisher Scientific	15-462-10Q
Protocol Section 5
CaCl2	Sigma-Aldrich	223506
ε-aminocaproic acid	Sigma-Aldrich	A0420000
MEM medium	Fisher Scientific	12561-056
Syringe	Fisher Scientific	1482748
Anesthesia ventilator	Datex-Ohmeda	47810
Anesthesia ventilator	Ohio Medical	V5A
Defibrillator	Physiol Control	LIFEPAK 15
1.5 T MRI	General Electric	Signa Horizon LX
7 T MRI	Siemens	10018532
Gadolinium Contrast Medium (Magnevist)	Berlex	50419-188-02
2-0 silk suture	Ethilon	685H
3-0 silk suture	Ethilon	622H
3-0 monofilament suture	Ethilon	627H