Applications Development at CDI: Improving Workflows, Pushing Biology, and Enabling Screening

www.cellulardynamics.com Madison, WI USA (608) 310-5100

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Coby Carlson, Shannon Einhorn, Steve Fiene, Rachel Llanas,

Giorgia Salvagiotto, and Arne Thompson Cellular Dynamics International, Inc., Madison, WI USA

Cellular Dynamics International (CDI) is the world’s largest producer of fully functional,

terminally differentiated human cell types derived from induced pluripotent stem cells (iPSC).

The quality, quantity, and purity of iCell® products has been a driving force for adoption of this

technology in the scientific community. iCell products have been tested and evaluated by

investigators around the world to push the current limits of biology, expand the utility of iPSC-

derived cell models, and improve the predictive capability of new and existing assays.

CDI strives to enhance the customer experience with iCell products through educational

training and engaged technical support, with a keen focus on application development. The

Applications Support Team works collaboratively with platform providers and key opinion

leaders in the field to continuously develop novel assays and unique approaches to asking

specific biological questions. An output of this work is to then transfer this knowledge back to

the end-user to enable a smooth transition into their current and future workflows.

This poster highlights some of the on-going application development projects at CDI. These

include general workflow improvements, phenotypic modeling of cardiac hypertrophy by high

content screening (HCS) assay in 384-well format, modulating neuronal activity on multi-

electrode arrays (MEA), and investigation of the bioenergetics of hepatotoxicity.

Abstract

CDI’s core competencies are in the reprogramming, engineering, and differentiation aspects of human iPSC-technology.

However, in order to help bring the use of these iCell Products to routine laboratory workflows, the existence of the

Applications Support Team is critical. iCell Cardiomyocytes, iCell Neurons, and iCell Hepatocytes more fully recapitulate

native biology and the Application Support Team at CDI is developing the protocols to enable implementation of these

products into assays and instruments that effectively address the complex endogenous biological processes shown here.

Summary

Improving Workflows

Pushing Biology Enabling Screening

Patterned Cell Culture

Cellular Electrophysiology

One advantage to screening with iCell tissue cells is that they recapitulate native biology. CDI is enabling the drug

discovery community by developing culture conditions for each of the terminally-differentiated cell types into HTS-

compatible workflows in miniaturized formats; all without sacrificing the expected (and complex) biology. In this example,

iCell Cardiomyocytes represent an in vitro model for cardiac hypertrophy whereby induction with Endothelin 1 (ET-1) re-

activates the fetal gene program (represented by B-type natriuretic peptide (BNP) a well established marker for cellular

hypertrophy). Building of an HTS-compatible high content imaging assay for cardiac hypertrophy required a shortening

of the protocol to 5 days and a miniaturization of the assay to a 384-well format. Now, the ability to titrate agonists in a

dose-response or screen multiple compounds for inhibition is possible with this assay.

An important feature of iCells is their broad

utility across a diverse set of applications.

In order to better support these products,

the Applications Support Team is continually

looking for ways to improve cell handling

protocols and to identify potential “shortcuts”

that might help users run their assays more

quickly and in a more cost effective manner.

In this example, the typical procedure for plating iCell Neurons (ie. two-step plate coating

process with PLO and laminin prior to seeding cells) was updated to offer a simpler

workflow that features mixing of laminin directly with the neurons at the time of seeding

onto pre-coated Poly-D-Lysine (PDL) plates. The result is equivalent cell morphology

when cultured across different multi-well formats (see above). The efficiencies gained

from an improvement like this would most likely be realized in a fluorescence-based

high-throughput screening application.

Cellular electrophysiology is a complex process that can be acutely modulated by various stimuli. iCell

Neurons are mixed population of GABAergic and Glutamatergic subtypes that form intricate networks in

vitro. The Maestro Multi-Electrode Array (MEA) System from Axion BioSystems (Atlanta, GA) enables

label-free detection of neuronal activity. This representative image is a “heat map”-style depiction of the

impact of electrical modulation of iCell Neurons.

Hepatocyte Bioenergetics

Compatibility of the iCell products with various assay kits and

instrument platforms is an important factor in their successful

implementation. Additionally, engineered surfaces that push the

boundaries of traditional cell culture are also of interest. This example

above illustrates the unique cell morphologies observed when iCell

Cardiomyocytes are grown on an aligned substrates from Nanofiber

Solutions (Columbus, OH). CDI is investigating how the use of

different materials and/or pattern surfaces might impact biology.

Understanding cellular bioenergetics is a crucial step

in drug discovery, development, and toxicity testing.

By analyzing iCells on instrumentation such as the

XF Extracellular Flux Analyzer from Seahorse

Biosciences (Billerica, MA), we can gain a better

understanding of the processes by which cells

produce and consume energy. CDI is building its

library of Application Notes and Protocols to highlight

the system advantages and provide easy to follow

step-by-step instructions for use.

The data to the right illustrate a metabolic profile of

iCell Hepatocytes. Similar data can be generated to

gain a better understanding of hepatotoxicity, as well

as provide insight as to how genetic differences in

patient-derived cell lines might influence human liver

metabolic diseases or drug metabolism.

Fluorescent images of iCell Cardiomyocytes either untreated (Left)

or fully stimulated with ET-1 (Right) using an anti-pro-BNP antibody

to detect BNP expression (red). Cell nuclei are stained with Hoechst

33342 (blue).

Cells were stimulated with ET-1 (Left) to induce hypertrophy, or

treated with Verapamil (Right) prior to activation with ET-1 to inhibit

the hypertrophic response. High Content Screening was performed

on an ImageXpress Micro System and the data was analyzed with

MetaXpress software, both from Molecular Devices (Sunnyvale, CA).

Modeling Cardiac Hypertrophy

Analysis of iCell Hepatocytes on this platform after 3 days in culture.

Above, the data show 1) basal respiration, 2) ATP production, 3) proton

leak, 4) maximal respiration, 5) spare respiratory capacity, and 6) non-

mitochondrial respiration.

iCell Neurons were cultured on an Axion

48-well Microplate for 4 days prior to

treatment with Gabazine (GABAA

antagonist). Cells were treated with

increasing concentrations of compound

from left to right.

iCell Cardiomyocytes (endogenously expressing RFP) were transiently transfected with GFP 12 days after thaw and

then imaged 48 hours later (Left). The high level of GFP expression indicates good transfection efficiency in this cell

type. Following transfection with pGL4.29[luc2P/CRE/Hygro] vector at Day 4, Isoproterenol was added either 24 hours

(Center) or 9 days (Right) post-transfection, demonstrating that reporter activity remained viable after prolonged culture.

Transfect (Day 12); Image (Day 14)

Gene Delivery

Monitoring gene delivery and cell signaling pathways in an in vitro environment allows for extensive cellular analysis and

increased understanding of drug toxicity. iCell Cardiomyocytes are amenable to DNA transfection and exhibit the

expected responses to activation of various pathways. In this example, a luciferase reporter-gene assay from Promega

(Madison, WI) driven by the cAMP response element (CRE) was “turned on” by stimulation with Isoproterenol. Assays

like this one performed with iCell Cardiomyocytes provide a means for isolating relevant targets in drug discovery as well

as early detection of toxicity.

(Left) Phase contrast images of iCell Neurons

plated in different multi-well plates. (Right)

Neurons cultured in 1536-well PDL-coated

plates for 10 days (with laminin mixed in the

plating medium). Live cell fluorescent staining

is compatible with the CDI Application

Protocol for Neurite Outgrowth and can be

acquired on an ImageXpress Micro from

Molecular Devices, (Sunnyvale, CA).

Fluorescent images of iCell Cardiomyocytes

cultured on (A) standard 24-well tissue culture plate

at 100X magnification, (C) 24-well NanoAligned™

PCL treated plate at 100X, (D) same as C but at

200X. Panel (D) shows a brightfield image of the

Nanofiber-coated plate at 100X total magnification.

Immunofluorescent staining for sarcomeric alpha

actinin (green), N-cadherin (red), and nuclei (blue).

A B C D

Transfect (Day 4); Assay (Day 5) Transfect (Day 4); Assay (Day 13)