January 15, 2015 (Vol. 35, No. 2)

iPSC-Derived Tissue Cells Are a Powerful Addition to the Biologist’s Tool Box

Human induced pluripotent stem cell (iPSC) technology has significant potential to revolutionize biomedical research. Derived from skin or blood of any individual, iPSCs provide unprecedented access to native cellular biology exhibiting healthy and disease phenotypes for basic research, drug development, and regenerative medicine.

Cellular Dynamics International has industrialized this technology and routinely creates iPSC-derived tissue cells (e.g., cardiomyocytes, neurons, hepatocytes) in bulk quantities at high purity and high quality.  CDI’s iCell® and MyCell® products have advanced the discovery of new therapeutic approaches for hepatitis infection, Alzheimer’s disease,1-2 latent effects of Varicella Zoster Virus (VZV) infection,3 cardiac hypertrophy,4 and diabetic cardiomyopathy,5 to name a few.

In addition, toxicity testing paradigms utilizing iCell Cardiomyocytes have demonstrated higher predictivity for detecting adverse drug-induced effects in cost-efficient assays.6-7 These data have contributed to the launch of the Comprehensive In Vitro Proarrhythmia Assay (CIPA) initiative by the U.S. FDA, HESI, CSRC, and other regulatory partners, with the goal of integrating human stem cell-derived cardiomyocyte cells into routine cardiotoxicity assessment.8

Genetic Manipulation Technologies

Advanced genetic and molecular tools are needed to fully interrogate native human cellular biology. Human iPSC-cardiomyocytes exhibit the three primary functional characteristics of native cardiomyocytes: (1) electrical, (2) biochemical, and (3) mechanical activities. Electrical signals in the form of action potentials at the sarcolemmal membrane are transduced through biochemical and calcium signaling via the sarcoplasmic reticulum into mechanical (contractile) activity of sarcomeric myosin and actin proteins.

While the basic flow of information travels from the myocyte membrane to the sarcomeric proteins, feedback in the other direction and interactions with other cellular processes significantly affect activity. Therefore the ability to dissect and manipulate these diverse pathways is critical to understanding the mechanistic underpinnings of cardiac biology and drug-induced effects on cardiac function.

Cellular processes are typically interrogated through reporter constructs that convey fluorescent or luminescent signals in response to transcriptional activity, protein modulation, or protein-protein interactions. Transient transfection of DNA encoding reporter constructs is commonly employed for such experiments. Primary tissue cells, which prior to the advent of iPSC technology provided the most relevant biological background, have traditionally been very difficult to transfect and they remain so despite advances in transfection technologies.

However, next-generation transfection reagents such as ViaFect™ Transfection Reagent (Promega) work quite well with human iPSC-tissue cells showing increased transfection efficiencies with lower toxicity. Using green fluorescent protein (GFP) as a marker, Viafect achieved transfection efficiencies of ~60, 50, and 40% in iCell Cardiomyocytes, iCell DopaNeurons, and iCell Hepatocytes, respectively (Figures 1A and 1B). Importantly, the improved transfection workflow (no washout) had minimal impact on cell viability as shown using iCell Cardiomyocytes (Figure 1C).

Figure 1. Transfecting human iPSC-derived tissue cells at high efficiency with minimal toxicity. Images at 4X, 20X, and 4X in (A) for iCell Cardiomyocytes, iCell DopaNeurons, and iCell Hepatocytes transfected with GFP, respectively. Efficiency (B) was assessed via flow cytometry and toxicity (C) was assessed with CellTiterGlo®.

Interrogation of Native Human Biology

Target-based screens interrogate a single receptor or pathway while phenotypic screens provide a holistic assessment of a condition. Both screens have utilized transfected reporters and engineered heterologous cell systems and are often limited by the biology of the engineered cell system. Human iPSC-derived tissue cells do not have such constraints as they recapitulate native biology and therefore provide contextually relevant results.

This paradigm is illustrated in Figure 2, which shows activation of cAMP, anti-oxidant, and hypoxic response pathways in iCell Cardiomyocytes via Viafect transfected CRE-, ARE-, and HRE-luciferase reporters (Promega), respectively. The inset in the middle panel of Figure 2A shows potential screening use by illustrating the alleviating effect of ascorbic acid on oxidative stress. iCell Cardiomyocytes and luciferase reporter constructs can be used together in phenotypic screens to leverage endogenous human cardiac biology and identify therapeutic agents without preconceived notions.9

Transfecting iPSC derived-tissue cells has other advantages as well. Heterologous cell lines are mitotic and transient transfections must be completed before the reporter construct is “kicked out” or diluted through cell division. In contrast, many differentiated iPSC derived-tissue cells, including cardiomyocytes, neurons, and hepatocytes, are post-mitotic and experiments are not under such temporal constraints.
For example, reporter constructs can be transfected into iCell Cardiomyocytes at variable times post-thaw followed by induction from anywhere between one and nine days post transfection (Figure 2B). The flexible transfection and induction window enables novel acute and sub-acute treatments and experimental paradigms.

Figure 2. iCell cardiomyocytes transfection enables experimental flexibility. (A) iCell Cardiomyocytes were transfected with CRE-, ARE-, and HRE-luciferase reporter constructs and treated with isoproterenol (ISO), tert-Butylhydroquinone (tBHQ), and phenantholine, CoCl2, ML-228, and IOX-2 (COMPD). (B) iCell Cardiomyocytes were transfected with CRE-luciferase reporter construct and treated with isoproterenol (ISO) at the indicated concentrations and time points.

Richer Data

Combining iPSC-derived tissue cells with advanced genetic manipulation techniques also allows multiplexing biochemical and functional endpoints within the same experiment. This process is illustrated by examining multiple effects of beta-adrenergic GPCR activation with isoproterenol in iCell Cardiomyocytes transfected with CRE-luciferase (Figure 3), specifically examining increases in CRE activity and cardiomyocyte contractility with the latter endpoint measured using the xCelligence RTCA-Cardio system from ACEA Biosciences.

The RTCA-Cardio system detects myocyte movement (i.e., beating) and thus provides a phenotypic output of cardiomyocyte electrical and contractile activity. In this example, iCell Cardiomyocytes were transfected 6 days post-thaw, transferred to the xCelligence assay plate on day 8, and challenged with isoproterenol on day 14.

Contractile activity was measured noninvasively on the RTCA-Cardio system, and  followed assessment of CRE activity through cell lysis, addition of the Promega luciferase reagent, and CRE-luminescence analysis on a Tecan plate reader. Transfection using the Viafect Transfection Reagent was highly efficient and as expected, isoproterenol caused dose-dependent increases in spontaneous beating activity and CRE-luciferase reporter activity. This study demonstrated the ability to simultaneously measure cardiomyocyte movement and cAMP signaling and the ease with which biochemical and functional endpoints can be linked through multiplexing in iPSC-derived tissue cells.

In summary, human iPSC-derived tissue cells bring relevant human biology and demonstrated advantages to the basic and applied biomedical research laboratory. Incorporating biochemical and signaling pathway readouts using highly efficient and low-toxicity transfection techniques provides novel tools for developing therapeutic interventions in human tissue cells. Together, multiplexing biomolecular readouts with functional endpoints takes full advantage of the native biology of human iPSC-derived tissue cells and enables cost-efficient and highly effective experimental paradigms. Ultimately these research tools will increase our knowledge base and speed drug discovery efforts.

Figure 3. Multiplexing biochemical and functional assays provide deeper data. Label-free functional assays coupled with high efficiency, low-toxicity transfection allows multiplexing across biochemical and functional endpoints. Experimental timeline with; (A) Qualitative transfection efficiency with GFP and CRE-Luc (0.5 ug DNA each), (B) Qualitative (upper) and quantitative (lower) functional increases in cardiomyocyte beat rate in response to isoproterenol across controls (GFP, Mock, Media) and reporter (GFP-CRE Luc) transfected cardiomyocytes, (C) CRE transcriptional response from the same cardiomyocytes following induction with isoproterenol.

Blake Anson, Ph.D. ([email protected]), is a product manager at Cellular Dynamics.

1 Chai X, Dage JL, et al. (2012) Constitutive Secretion of Tau Protein by an Unconventional Mechanism. Neurobiol Dis 48:356-366
2 Xu X, Lei Y, et al. (2013) Prevention of ß-amyloid Induced Toxicity in Human iPS Cell-derived Neurons by Inhibition of Cyclin-dependent Kinases and Associated Cell Cycle Events. Stem Cell Res 10:213-227
3 Yu X, Sietz S, et al. (2013) Varicella Zoster Virus Infection of Highly Pure Terminally Differentiated Human Neurons. J Neurovirol 19:75-81
4 Aggarwal P, Turner A, et al. (2014) RNA Expression Profiling of Human iPSC-Derived Cardiomyocytes in a Cardiac Hypertrophy Model. PlosOne 9:e108051
5 Drawnel FM, Boccardo S, et al. (2014) Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells. Cell Reports
6 Guo L, Coyle l, et al. (2013) Refining the Human iPSC-cardiomyocyte Arrhythmic Risk Assessment Model. Toxicol Sci 136(2):581-94
7 Harris K, Aylott M, et al. (2013) Comparison of Electrophysiological Data from Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (hiPSC-CMs) to Functional Preclinical Safety Assays. Toxicol Sci 134(2):412-26
8 Sager PT, Gintant G (2014) Rechanneling the cardiac Proarrhythmia safety paradigm: a meeting report from the Cardiac Safety Research Consortium. Am Heart J 167(3):292-300
9 Swinney DC, Anthony J (2011) How Were New Medicines Discovered? Nat Rev Drug Disc 10:507-519

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