Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Summary

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Introduction

Primary human cardiomyocytes are difficult to obtain because of the requirement for invasive cardiac biopsies, difficulty in dissociating to single cells, and because of poor long-term cell survival in culture. Given this lack of primary human cardiomyocytes, patient-specific human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology has been regarded as a powerful alternative cardiomyocyte source for basic research as well as clinical and translational applications such as disease modeling and drug discovery¹. Early efforts in differentiating pluripotent stem cells into cardiomyocytes employed differentiation protocols using embryoid bodies (EBs), but this method is inefficient in producing cardiomyocytes because often less than 25% of cells in an EB are beating cardiomyocytes^2,3. Comparatively, a monolayer-based differentiation protocol using the cytokines activin A and BMP4 displayed a higher efficiency than EBs, but this protocol is still relatively inefficient, requires expensive growth factors, and only functions in a limited number of human pluripotent stem cell lines⁴. Recently, a highly efficient, hiPSC monolayer-based cardiomyocyte differentiation protocol was developed by modulating Wnt/β-Catenin signaling⁵. These hiPSC-CMs express cardiac troponin T and alpha actinin, two sarcomeric proteins that are standard markers of cardiomyocytes⁶. The protocol describe here is an adaptation of this small molecule-based, feeder cell-free, monolayer differentiation method^5,7. We are able to obtain beating cardiomyocytes from hiPSCs after 7-10 days (Figure 1). However, following a cardiomyocyte differentiation resulting in 50% beating cells, immunostaining consistently shows the existence of a population of non-cardiomyocytes that are negative for cardiomyocyte-specific markers such as cardiac-specific troponin T and alpha-actinin. To further purify cardiomyocytes and eliminate non-cardiomyocytes, heterogeneous differentiated cell populations were subjected to glucose starvation by treating them with an extremely low glucose culture medium for multiple days (Figure 2). This treatment selectively eliminates non-cardiomyocytes due to the ability of cardiomyocytes, but not non-cardiomyocytes, to metabolize lactate as the primary energy source in order to survive in a low glucose environment⁸. After this purification step, a 40% increase in the ratio of cardiomyocytes to non-cardiomyocytes is observed, (Figure 3, Figure 4) and these cells can be used for downstream gene expression analysis, disease modeling, and drug screening assays.

Protocol

NOTE: Vendor information for all reagents used in this protocol has been listed in Table 1 and Materials List. All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly. Perform all culture incubations in a humidified 37 °C, 5% CO₂ incubator unless otherwise specified. In this protocol, all differentiations are performed in 6-well plates, in which the hiPSCs are seeded. Following differentiation and purification, cells can be dissociated and replated for downstream use.

1. Medium Preparation

1. For the E8 medium, make a stock solution for each medium component (NaHCO₃, L-ascorbic acid 2-phosphate, sodium selenite, transferrin, insulin, FGF2, TGFB1) and store the solutions at -20 °C. Add appropriate amount of stock solutions to the bottle of DMEM/F12 and filter to sterilize. The solution concentration and reaction setup for the stock components and E8 medium can be found in Table 1.
2. For the extracellular matrix solution, thaw the basement membrane matrix (commonly supplied at 10 mg/ml) at 4 °C O/N. Matrigel is commonly used for pluripotent stem cell culture because human pluripotent stem cells can adhere well to this material using the alpha-6-beta-1 integrin⁹. Once thawed, make 2.25 mg (250 µl) aliquots on ice using ice cold pipet tips and tubes and store the aliquots at −80 °C.
   1. When precoating plates with ECMS, thaw and aliquot the extracellular matrix solution (ECMS) on ice. Mix the ECMS and ice-cold DMEM/F12 medium in a ratio of 1:200 on ice. Keep cool to prevent premature ECMS solidification.
3. For the RPMI/B27 without insulin medium, add 10 ml of B27 Minus Insulin into 500 ml of RPMI media.
4. For the RPMI/B27 (with insulin) medium, add 10 ml of B27 Supplement (with insulin) and 5 ml of Pen-strep antibiotic into 500 ml of RPMI media.
5. For the low glucose medium, add 10 ml of B27 Supplement and 5 ml of Pen-strep antibiotic into 500 ml of glucose-free RPMI media.
6. For the freezing/cryopreservation medium, mix fetal bovine serum together with dimethylsulfoxide (DMSO) at a 9:1 ratio. The freezing medium can be stored up to 1 month at 4 °C.
   NOTE: All media should be filtered and stored in 4 °C and used within 2 weeks unless otherwise specified. All reagent/solution volumes used below are intended for a single well in a 6-well plate, unless otherwise specified.

2. Pre-coating 6-well Plates with ECMS

1. Make ECMS by mixing basement membrane matrix (e.g., Matrigel) with ice-cold DMEM/F12 media in a ratio of 1:200, then apply 2 ml of freshly made ECMS to each well for a 6-well plate. Each well in a 6-well plate will receive approximately 90 mg ECMS.
2. Incubate the ECMS coated plates for 1 hr at 37 °C. Immediately prior to plating cells, aspirate DMEM/F12 solution from each well. The plates will then be ready to receive cells.

3. Thawing the Frozen hiPSCs

NOTE: Closely follow this procedure as it may lead to optimal culturing and downstream differentiation of hiPSCs into hiPSC-CMs following the freeze/thaw cycle.

1. Thaw one vial of hiPSCs for each well of a 6-well plate. Prepare a 15 ml conical tube with 9 ml of cold E8 medium with ROCK inhibitor (10 μM) for each vial. ROCK inhibitor improves hiPSCs survival following cryopreservation¹⁰. Keep the vials in 37 °C water bath to thaw the cells until an approximately 5 mm diameter ice crystal is left.
2. Spray the exterior of the vials with 70% ethanol. Wipe the vials with tissue paper before moving them to the sterile laminar hood.
3. Transfer the cells into the prepared conical tube. Rinse the vial once with 500 μl of E8 medium and transfer the medium containing the thawed cells to the same conical tube. Centrifuge at RT for 4 min at 200 x g.
4. Aspirate the supernatant after centrifugation and gently resuspend the cell pellet with 2 ml of E8 medium with ROCK inhibitor (10 μM). Transfer the resuspended cells to one well of a 6-well plate pre-coated with ECMS.
5. Replace the medium with E8 medium without ROCK inhibitor after 24 hr of culturing. Cells should have adhered by this time.

4. Passaging of hiPSCs

1. Typically, passage hiPSCs after reaching 75%-80% confluency in a 6-well plate. Aspirate the culture medium. Quickly wash wells with 1 ml of sterile, RT PBS. Aspirate PBS.
   1. Add 500 μl of 0.5 mM EDTA or 1x cell detachment solution (e.g., Accutase) and incubate for 1-7 min at RT. Appropriate time for EDTA incubation varies with the hiPSC line. Expect to see visible curling or thickening of colonies around the edges, as this will indicate that EDTA or cell detachment solution is ready to be removed.
2. Aspirate the EDTA or the cell detachment solution. Add 1 ml of E8 medium supplemented with 10 μM ROCK inhibitor. Displace hiPSC colonies in the well by repeatedly pipetting up and down and spraying colonies off with a P1000 pipette. Colonies should be broken into 5-10 cell clumps after completion.
3. Transfer the colonies that are suspended in 1 ml of E8 medium with ROCK inhibitor into a 15 ml conical tube.
4. Disrupt the large cell clumps into small clumps in the E8 medium. Add an appropriate volume of E8 media with inhibitor to the cell suspension to dilute the cells. The media volume to add for dilution depends on the cell density and the number of wells to be seeded. Ideally, a single, 80% confluent well of hiPSCs should be diluted 1:12.
   1. Add 11 ml E8 medium with ROCK inhibitor to the existing 1 ml of cell solution in aforementioned 15 ml conical tube to obtain a 1:12 dilution.
5. Further triturate the cell clumps into small cell fragments (around 50-200 cells in each fragment is best). Avoid over-triturating as this reduces cell survival.
6. Aspirate ECMS from the pre-coated plates and add 2 ml of resuspended cells into each well. Approximately 100,000 cells per well of a 6-well plate is ideal. Aim to dispense the cells evenly around the well to avoid clustering of cells in the center of the well.
7. After 24 hr of culturing, replace the medium with E8 medium without ROCK inhibitor. Change the culture medium every 24 hr until cells become 80% confluent. Then, the cells are ready to passage again. It usually takes 3-6 days between passages to reach 80% confluency.

5. Freezing hiPSCs

NOTE: Closely follow this procedure as it may lead to optimal culturing and downstream differentiation of hiPSCs into hiPSC-CMs following the freeze/thaw cycle.

1. Label cryogenic tubes with the cell line name, cell type, passage number, and freezing date. As a general guideline, use one vial for each well of a 6-well plate. Prepare a 15 ml conical tube filled with 9 ml of E8 medium with ROCK inhibitor (10 μM). Prepare freezing medium with 90% FBS and 10% DMSO, and keep the medium at 4 °C until ready to use.
2. Aspirate the culture media, add 500 μl of 0.5 mM EDTA or 1x cell detachment solution and incubate for 2-7 min at RT. The duration of EDTA or cell detachment solution exposure varies from cell line to cell line.
3. Aspirate EDTA or cell detachment solution. Add 1 ml of E8 medium.
4. Displace hiPSC colonies in the well by pipetting up and down and spraying colonies off with a P1000 pipette. Colonies should not be broken into smaller than 100 cell clumps. Transfer the suspended cells to the prepared conical tube containing E8. Centrifuge at RT for 4 min at 200 x g.
5. Aspirate the supernatant and add 500 μl of cold freezing medium to the pellet. Resuspend the pellet by pipetting one to two times. Cell survival is improved by keeping cells in large clumps.
6. Transfer the resuspended cells into a labeled cryogenic tube. Quickly move the vials into a freezing container containing isopropanol, which will allow for gradual cooling. Keep the container with cells at -80 °C for 24 hr, then transfer the cells to liquid nitrogen for long-term storage.

6. Cardiac Differentiation of Human iPSC

NOTE: All media should be at least at RT when added.

1. After dissociation with EDTA or 1x cell detachment solution, seed approximately 100,000 human iPSCs on ECMS coated 6-well culture plates for differentiation (same steps as cell passaging procedures). When the cells reach 85% confluency, change the medium to RPMI/B27 without insulin medium with 6 µM GSK3-beta inhibitor CHIR99021 (CHIR) and maintain for 48 hr.
2. After 48 hr, replace the CHIR-containing culture medium with RPMI/B27 without insulin medium and leave alone for 24 hr (until day 3).
3. At day 3, change the media to RPMI/B27 without insulin with 5 µM Wnt inhibitor IWR1 and maintain for 48 hr (until day 5).
   NOTE: Wnt inhibition can also be attempted using other small molecule compounds, as described in earlier studies¹¹. IWR1 was selected over other small molecule Wnt inhibitors due to the increased range in which it has been shown to be effective in inhibiting Wnt signaling¹¹.
4. At day 5, change the medium back to RPMI/B27 without insulin medium and leave for 48 hr (until day 7).
5. At day 7, replace the medium with RPMI/B27 medium (with insulin) and replace medium every 3 days thereafter with the same medium. Spontaneous beating of cardiomyocytes should first be visible at approximately day 8 to day 10.

7. Purification of Human Cardiomyocytes through Glucose Starvation

1. At day 10 post-differentiation, change the medium in each well of the 6-well plate to 2 ml low glucose medium and maintain the cells in this medium for 3 days (until day 13).
2. At day 13, return cells to RPMI/B27 medium (with insulin).
3. Optionally, replate cardiomyocytes prior to the second round of glucose starvation to help loosen non-cardiomyocytes from the culture plate, allowing for easier dissociation of non-cardiomyocytes during glucose starvation.
   1. At day 13, aspirate medium, wash once with PBS, and dissociate the cells into single cells using 500 μl of cell disassociation enzyme for 5 min at 37 °C. Specifically, after 5 min of enzyme treatment, use a 1,000 µl pipette to manually dissociate cardiomyocytes from the 6-well plate by repeatedly pulling up the cell disassociation enzyme and spraying it against the cardiomyocyte monolayer. Up to 30 pipetting repetitions may be required to dissociate the cardiomyocytes into single cells.
   2. After cells are dissociated and are in single-cell form, collect all cells into a 15 ml conical tube filled with 5 ml of RPMI/B27 medium with insulin to dilute out the cell disassociation enzyme and centrifuge for 4 min at 200 x g. Aspirate and discard the supernatant.
   3. Re-suspend the cells with 2 ml RPMI/B27 medium and plate onto a new ECMS-coated 6-well plate. Typically, higher confluency of cardiomyocytes helps with cell survival during replating. Aim to replate 2 million cells per new 6-well dish for optimal survival during replating.
4. At day 14, change the medium back to 2 ml of low glucose medium for a second glucose deprivation cycle. Culture the cells in this low glucose state for 3 more days. Most of the non-cardiomyocytes will die in this low-glucose culture condition.
5. At day 17, change the medium to 2 ml of RPMI/B27 medium with insulin. The remaining cells will be highly purified cardiomyocytes. These cardiomyocytes can be used for gene expression analysis, drug screening, metabolic analysis, and various other downstream assays.

Results

The morphological changes during hiPSC differentiation.

The hiPSC cultured in feeder-free plates grew as flat, two-dimensional colonies. Upon reaching around 85% confluency, hiPSCs were treated with 6 µM CHIR for differentiation (Figure 1A). Substantial amounts of cell death, a normal and common phenomenon, were observed after 24 hr of CHIR treatment. After two days of CHIR treatment, the hiPSCs continued to differentiate towards a mesodermal fate. In comparis...

Discussion

Obtaining a large amount of highly purified hiPSC-derived cardiomyocytes is critical for basic cardiac research as well as clinical and translational applications. Cardiac differentiation protocols have undergone tremendous improvements in recent years, transitioning from embryoid body-based methods utilizing cardiogenic growth factors², to matrix sandwich methods¹², and finally to small molecule-modulated and monolayer-based methods⁵. Of the aforementioned protocols, the protocol describ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Matrigel (9-12 mg/ml)	BD Biosciences	354277
RPMI media	Invitrogen	11835055
Glucose free RPMI media	Invitrogen	11879-020
B27 Minus Insulin	Invitrogen	A1895601
B27 Supplement (w/ insulin)	Invitrogen	17504-044
Pen-strep antibiotic	Invitrogen	15140122
Fetal bovine serum	BenchMark	100-106
DMSO	Sigma	D-2650
ROCK inhibitor Y-27632	EMD Millipore	688000
CHIR99021	Thermo Fisher	508306
IWR1	Sigma	I0161
EDTA	Invitrogen	15575-020
Accutase	Millipore	SCR005
Cell lifter	Fisher	08-100-240
Cryovial	Fisher (NUNC tubes)	375418
TrypLE Select Enzyme	Invitrogen	12563-011