Serum Free hiPSC-Derived Cardiomyocytes

A series of experimental procedures were carried out to investigate the characteristics and possible applications of human iPSC-derived cardiomyocytes (iPSC-CMs) for cardiovascular research.

Introduction

  • Induced pluripotent stem cells (iPSCs) can be generated through reprogramming adult cells using defined factors OCT3/4, KLF4, SOX2 and c-MYC1-3
  • iPSCs, including cardiomyocytes, iPSC-CMs, can be separated into a variety of cell types
  • The expression of several cardiomyocyte-selective markers and electrical functioning is shown in iPSC-CMs
  • To demonstrate the application of these cells in drug toxicity, testing a simultaneous optical control/calcium imaging approach was developed

Methods

Cell Culture Human iPSC-CMs (Axol Bioscience, ax2505) were cultured in Cardiomyocyte Maintenance Medium (Axol Bioscience, ax2530-500) with 10% FBS, Pen/Strep for the initial 24hrs, and serum-free at 37  ̊C/5% CO2 thereafter.

Immunohistochemistry Cells were fixed at 3% PFA, permeabilized with 0.2% Triton X-100 and blocked with BSA. The primary antibody incubation was carried out overnight at 4 ̊C before a secondary antibody coupled to Alexa Fluor® dyes (Invitrogen) for 2hrs.

Western Blot A 30 μg protein ran on 10% SDS-PAGE gel for 70min at 130 V before being transferred to PVDF membrane. Membranes were then incubated with primary antibody overnight at 4  ̊C before being washed and incubated with secondary antibody for 1hr. Finally, chemiluminescent imaging was carried out.

Manual Patch Clamp Once thawed, cells were cultured for 7-14 days and syncytial monolayers were patched via perforated patch-clamp (100 μg/ml gramicidin).

Optical Control/Calcium Imaging Cells were cultured in iPSC  Cardiomyocyte Maintenance Media (Axol Bioscience, ax2530-500). Adenovirus expressing a light-sensitive optical control tool (Channel Rhodopsin 2) and a spectrally compatible genetically encoded calcium indicator (R-GECO) (Fig. 1). Cells were stimulated with 10 msec pulses of 405 nm light to induce depolarization and contraction. This was then followed with 568 nm light to visualize the evoked calcium transient.

Optical control/calcium imaging vector R_GECO.

Figure 1: Optical control/calcium imaging vector R_GECO. Image Credit: Axol Bioscience

Spontaneously BeatingiPSC-Derived Ventricular Cardiomyocytes

Data from Prof Matthew Daniels

Data from Prof Matthew Daniels

iPSC-Derived Ventricular Cardiomyocyte Marker Expression

Data from Dr Christian Zuppinger

Data from Dr Christian Zuppinger

A) 93.5% of iPSC-CMs are positive for cardiac tropinin-I (cTnI), B) Human iPSC-CMs (hiPSC-CMs) express more cardiac troponin-T (cTnT) & α-Actinin than human skin fibroblasts (hSFs). Data from Abigail Robertson, C) On average there were 15.3% ± 2.9 SEM (n=5) binucleate iPSC-CMs showing cardiomyocyte maturity (cTnI). Data from Prof Matthew Daniels.

A) 93.5% of iPSC-CMs are positive for cardiac tropinin-I (cTnI), B) Human iPSC-CMs (hiPSC-CMs) express more cardiac troponin-T (cTnT) & α-Actinin than human skin fibroblasts (hSFs). Data from Abigail Robertson, C) On average there were 15.3% ± 2.9 SEM (n=5) binucleate iPSC-CMs showing cardiomyocyte maturity (cTnI). Data from Prof Matthew Daniels.

Atrial vs Ventricular Cells

A) MLC2a & MLC2v staining to distinguish atrial and ventricular Cardiomyocyte populations, respectively (n=1). Data from Dr Christian Zuppinger, B) Negligible effect of atrial-selective agent on AP parameters in iPSC-CM cells (n=8) compared to positive effect of Carbachol observed in atrial-derived HL-1 cells. This suggests the majority of cells do not display an atrial phenotype Data from Metrion Biosciences.

A) MLC2a & MLC2v staining to distinguish atrial and ventricular Cardiomyocyte populations, respectively (n=1). Data from Dr Christian Zuppinger, B) Negligible effect of atrial-selective agent on AP parameters in iPSC-CM cells (n=8) compared to positive effect of Carbachol observed in atrial-derived HL-1 cells. This suggests the majority of cells do not display an atrial phenotype Data from Metrion Biosciences.

Dofetilide Treatment Prolongs the Calcium Transient

Data from Prof Matthew Daniels

Data from Prof Matthew Daniels

A) AP parameters for untreated control cells (n=32). Recorded from cell syncytium using perforated patch clamp (100 μg/mL gramicidin), B) Treatment with TTX & Mexiletine prolonged time to peak, C) Treatment with Nifedipine reduced peak voltage & shortened APD20 ,APD50 & APD90, D) Treatment with Dofetilide causes APD90 prolongation as observed with the calcium imaging above. (n=8). Data from Metrion Biosciences

A) AP parameters for untreated control cells (n=32). Recorded from cell syncytium using perforated patch clamp (100 μg/mL gramicidin), B) Treatment with TTX & Mexiletine prolonged time to peak, C) Treatment with Nifedipine reduced peak voltage & shortened APD20 ,APD50 & APD90, D) Treatment with Dofetilide causes APD90 prolongation as observed with the calcium imaging above. (n=8). Data from Metrion Biosciences

Conclusions

  • A range of characteristics was identified in the human iPSC-CMs, confirming their ability to function as a highly-pure population of single-beating human cardiomyocytes in vitro.
  • A technically simple and scalable platform was presented for cardiotoxicity screening assays. In the future, this could be incorporated into the cells directly via genome editing.
  • Under serum-free conditions, human iPSC-CMs can be cultured. This allows for examination of the effect of growth factors, cytokines and drugs on the development and functionality of human cardiomyocytes in vitro.

References and Further Reading

  1. iPS cell technologies: significance and applications to CNS regeneration and disease. Okano & Yamanaka. Mol Brain, 2014
  2. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Takahashi & Yamanaka. Cell, 2006
  3. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Takahashi et al., Cell, 2007

Acknowledgments

Produced from materials originally authored by Gibbons, G1; Sutton, KG2; Rogers, M2; Robertson, A3; El Haou, S2; Williams, S2; Ridley, JM2; Webdale, L2; Chang, Y4; Zuppinger, C5; and Daniels, M4 from Axol Bioscience Ltd.

1 Axol Bioscience Ltd., UK
2 Metrion Biosciences, UK
3 University of Manchester, UK
4 University of Oxford, UK
5 Bern University Hospital, Switzerland

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Feb 18, 2020 at 11:31 AM

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