x

Module 1

Disease modeling

Familial HCM is an autosomal dominant disease of the cardiac sarcomere, mainly due to mutations in the MYH-7 protein, which results in abnormal thickening of the left ventricular myocardium and is associated with elevated circulating levels of the biomarker NT-proBNP in patients. HCM was induced by Endothelin-1 (ET-1, a potent pro-hypertrophic peptide produced by endothelial and smooth muscle cells) in bioreactor-derived cardiomyocytes. Overnight exposure of bioreactor-derived iPSC-CMs to ET-1 results in production of BNP as measured by immunofluorescence microscopy.

(A) Control or ET-1 treated cardiomyocytes were stained for BNP (green) and nuclei (blue). (B) Quantification of high content images showing total nuclear area (left) or BNP expression (right) in the presence or absence of ET-1. (C) ET-1 induced NT-proBNP secretion, measured by AlphaLISA, was rescued by treatment with Verapamil.

Module 2

Manufacturing

Cardiomyocytes were produced at large scale using controlled stirred tank bioreactors and Fluent® workstation (Tecan) allowed full automation of cell culturing in 384-well plates. We operate enough bioreactors for full factorial design = 2n , where n = the number of factors having impact on the process.

  • String speed
  • pH
  • Inoculum size
  • Chemical concentrations
  • Media refreshment pattern
  • More

Consecutive iPSC-CM lots were manufactured in an ISO 9001:2015-certified facility. Automated bioprocessing and integrated quality control help to reduce batch variability as evidenced by consistent cTNT expression and batch sizes. (A) Bioreactor derived cardiomyocytes exhibit consistent Troponin expression within a population and (B) across numerous production runs.

Download the Whitepaper on Bioprocessing

"Controlled manufacturing of hiPSC-derived cardiomyocytes in stirred-tank bioreactors enabling high-throughput phenotypic screening", a collaboration between Ncardia and Eppendorf.

Request a PDF version using the form below:

Module 3

Assay Development

Hypertrophy was assessed by measuring secretion of NT-proBNP using the AlphaLISA assay. The developed assay resulted in robust and reproducible assay quality. An increase in fluorescence units (AlphaLISA counts) in response to 5 nM ET-1 was observed. Verapamil, an inhibitor of L-type calcium channels that prevents (here, pathological) calcium-induced calcium release, was used as a positive control in this and subsequent assays. Verapamil was able to fully rescue ET-1 induced NT-proBNP release/BNP expression and was the positive comparator on every plate. S/B = Signal:Background; AW = Assay Window

(A) Pathological hypertrophy is characterized by asymmetric hypertrophy (or thickening) of the left ventricular wall, and is typically accompanied by contractile functional deficit, fibrosis, and cardiomyocyte disarray1 (B) Endothelin-induced secretion of NT-proBNP, a clinical marker of cardiac injury/hypertrophy, was rescued by verapamil treatment in iPSC-derived cardiomyocytes (C) Functional activity was assessed by calcium transient screening in 384 well format.

1Maron BJ et al. (2003)J Am Coll Cardiol, vol. 42; 1687-1713

Module 4

High Throughput Screening

Validation screening from duplicate plates as well as in a patient derived MYH7 mutated line confirmed the reproducibility of the assays (Pearson Correlation > 0.6). After successful validation of the assays we screened ~5000 data points including >1800 approved drugs, and a thousand further compounds with known mechanism of action. Compounds with percent inhibition (PIN) > 40% were confirmed as hits. Hits were confirmed via three distinct assays; NT-proBNP AlphaLISA assay (+re-test), AlphaLISATruHits assay (deselect false positives), and intracellular expression assessed by a high content imaging secondary assay. Finally, to determine compound efficacy, potency analyses with 8 point dose response curves were generated, and hits with various potencies identified (i.e. low potency EC50 > 10 uM, moderate potency EC50 > 1 uM and high at nM to sub-nM potency). Many confirmed hits have lead properties (sub-nM potency, favorable DMPK properties, good safety profile, etc.)

(A) Assay performance from 15 x 384 well plates screened in succession. (B) Hits grouped by % effect (rescue of hypertrophy) from >3000 compound screened. (C) Dose response curve for Bosentan, one of the validated hits identified from the primary screen.

INQUIRIES

Personal consultation

If you are interested in discussing the possibilities available with our DiscoverHIT Platform, please contact us to arrange a further conversation.

Johan te Koppele, PhD
Chief Services Officer, Ncardia

Get in contact