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Abstract

www.cellulardynamics.com Madison, WI USA +1 (608) 310-5100

Human iPS Cell-derived Cardiomyocytes Carrying MYH7-R403Q Exhibit Aspects of Hypertrophic Cardiomyopathy In Vitro

Eugenia Jones, Natsuyo Aoyama, Jun Wang, Michael McLachlan, Randy Learish, Tom Burke, Coby Carlson, and Blake Anson

Hypertrophic cardiomyopathy (HCM) is a common genetic heart condition affecting approximately 1 in 500 individuals, where the heart muscle becomes thick and blood flow is restricted. The condition is characterized by a thickening of the ventricular wall as a result of enlarged cardiac myocytes, changes in blood pressure due to restricted blood flow, and arrhythmias. The most prevalent form of familial HCM arises from a missense mutation in the gene encoding the beta-myosin heavy chain protein, resulting in a change of amino acid 403, from Arg-to-Gln (MYH7-R403Q). The study of diseases affecting cardiomyocytes has been advanced by the advent of stem cell technology which has enabled the production of stem cell-derived cardiomyocytes in sufficient quantities to facilitate large scale in vitro research. Further advances in stem cell technology enabled the production of human induced pluripotent stem (iPS) cells from any individual, apparently healthy normal as well as affected individuals, prompting production of large collections of iPS cells. Cardiomyocytes (CM) can be produced from any iPS cell in a collection and used to gain a better understanding of mechanisms involved in complex heart disease. Here we describe the study of iPS cell-derived CM from normal and MYH7-R403Q.

Hypertrophy can be induced in normal human donor iPS cell-derived CM with exposure to Endothelin-1 (ET-1). HCM-induced CMs exhibit classic hallmarks of cardiac hypertrophy including up-regulation of fetal genes, cytoskeletal rearrangements, and an increase in cardiomyocyte size. We show that induced and inherited HCM in iPS cell-derived CM have common features. CMs differentiated from MYH7-R403Q iPS cells exhibit cardiac morphology, and showed autonomous contractile activity similar to the control iPS cell-derived CM. MYH7-R403Q CM and ET-1 induced HCM in normal CM have similar basal gene expression. ET-1 induction increases BNP expression in both control and MYH7-R403Q cardiomyocytes, but basal BNP levels are higher in MYH7-R403Q cardiomyocytes. These data show the progression of HCM characteristics in MYH7-R403Q cardiomyocytes and underscore the advantages of modeling cardiovascular disease with iPS cell technology.

Power of iPSC Technology

Cryopreserved iCell Neurons Human Donor Terminally

differentiated cell types

Induced Pluripotent Stem

(iPS) Cells

Innate Disease Modeling

MyCell R403Q iCell CM

98% 96%

NPPB 5ACTA1 4DUSP4 3ACTC1 2ACTN1 1CREB5 0MYH7 -1NPPA -2MYH6 -3

TRIM63 -4ADM -5

FBXO32PDCD4

Relative Expression

ET-1 induced iCell CM

MyCell MYH7

R403Q CM

Cell Type Viability Plating Efficiency

iCell CM 79% 56% MyCell R403Q 76% 45%

● Hundreds of genetic defects are linked to diseases of theheart muscle, however the pathways connected to the cellular phenotype remain largely unknown. MYH7 R403Q is linked to familial hypertrophic cardiomyopathy. We have produced cardiomyocytes from MYH7-R403Q iPS cells to study mechanisms involved in hypertrophy.

Functional Data Induced Disease Modeling

● Recapitulation of the disease phenotype can be accomplished by induction with endothelin (ET-1). Reactivation offetal genes, such as NPPB (dose-response curve in Panel A) or ACTA1, ACTA2, TAGLN (bar graph in Panel B), as well as changes in cell size (Panel C), or cytoskeletal actin rearrangements (F/G actin ratios; Panel D). Scale bar = 100 µm.

● Hypertrophy applicationsuite: qPCR assay for NPPB (Panel A above), ELISA to quantify levels of secreted BNP (E), Flow Cytometry to detect BNP in the cell (F), and High Content Analysis to image BNP expression (G).

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Somatic cells from human adult tissue are obtained via non-invasive methods and reprogrammed to generate iPS Cells using non-integrating vectors expressing a variety factors. The resulting iPS cells were cultured and expanded in defined media in feeder-free conditions. These cells can be grown indefinitely or cryopreserved. Truly pluripotent iPS cells can be differentiated into terminal cell types representing those in the human body derived from all three germ layers; mesoderm, endoderm, and ectoderm. Our reprogramming technology ensures the iPS cells we produce are truly pluripotent, enabling us to produce differentiated cells for diverse donor samples.

Large scale manufacturing capabilities, allows us to produce reproducibly terminal cells from any iPS cell line in quantities sufficient for screening experiments. Consistent production makes these terminally differentiated cells an ideal model system for studying biology in vitro. In addition, we have devised cryopreservation methods that enable researchers to store terminally differentiated materials, and enhanced workflows taking cells from the freeze to assay in less than five days.. Taken together these technologies are revolutionizing our ability to study mechanisms of induced, infectious, and inherited human diseases in a dish and to develop treatments for these disorders.

One powerful feature of iPSC technology is the ability to generate patient-derived stem cell lines from individuals affected by inherited disorders. This poster describes the production and characterization of cardiomyocytes from a patient with hypertrophic cardiomyopathy.

iCell CM MyCell MYH7 R403Q CM

● Cardiac hypertrophy is characterized by anincrease in cardiomyocyte cell size

● Reversion to a cardiac fetal gene profile is ahallmark trait of hypertrophy; B-type natriuretic peptide (BNP) expression is a common marker of the hypertrophic response

+ET-1 (10 nM)

Unstim + ET-1 (10 nM)

A qPCR B

C D

E F G

Cell Size Cytoskeletal Rearrangements

Measure R403Q Control Beat Rate (BR) 8.2 ± 0.5 27.6 ± 0.8

Amp 0.087 ± 0.008 0.088 ± 0.005

BRCV 0.12 ± 0.08 0.038 ± 0.024

● (A) The viability and plating efficiency ofiCell and R403Q Cardiomyocytes are nearly equivalent.

● (B) Cardiomyocytes produced from MYH7R403Q are high purity (>95%), similar to iCell Cardiomyocytes. Showing the mutation does not effect differentiation.

● (C) MYH7 R403Q Cardiomyocytes exhibitclassic iPSC-derived cardiac morphology.

● (D) Comparative analysis of cardiac beatrate via xCELLigence. Control and R403Q beats are similar amplitudes, but the BR of R403Q is about 30% of the control MyCell Cardiomyocytes.

● (E) Expression profiling for genes known tobe involved in cardiac hypertrophy were analyzed in ET-1-induced iCell CM and uninduced MYH7 R403Q cardiomyocytes were compared.

A

B C

D

Highly Pure CM Cardiac Morphology

Altered Beat Rate

High Viability at Thaw

E Gene Expression Profiling

● BNP expression: there is adramatic difference between cells that are unstimulated (0 nM) or induced with ET-1 (10 nM). This can be quantified by high content imaging and observed visually after fluorescence labeling (red = BNP; blue = DAPI).

● Baseline differences: iCell CM (left) and MYH7 R403Q (right)differ in the amount of BNP expressed in the uninduced state (red = BNP; blue = DAPI), suggesting innate signs of a cardiachypertrophy phenotype in the mutant CM. Additionally, thenumber of BNP-positive cells can be rescued by small molecules such as verapamil.

Conclusions The data presented here show human cardiomyocytes derived from iPS cell carrying the familial hypertrophic cardiomyopathy MYH7 R403Q mutation:

• shows increased expression of BNP in the unstimulated state, that is responsive todrug treatment.

• MYH7 R403Q cardiomyocytes differ from ET-1 induced-hypertrophy cardiomyocytes,in their gene expression profile, and beat rate.

• MYH7 R403Q cardiomyocytes can be induced to further express hypertrophy markerslike wild-type cardiomyocytes

Future Directions

Our next step is to differentiate genetically engineered iPS cells, harboring arrhythmogenic alleles in ion channels to determine if the phenotype is recapitulated in cardiomyocytes in vitro. In addition, we are producing cardiomyocytes from iPS cells derived from patients with polygenic cardiac hypertrophy, who are part of the Hypergen study cohort. These cells will be available to researchers as cardiomyocytes, later this year as part of the MyCell Disease and Diversity panel. The iPS cell lines will be available in an NHLBI bank.

Induced pluripotent stem cell technology enables disease research with the human cell type of interest. Using genetic engineering, isogenic backgrounds facilitate more robust studies of phenotype/genotype relationships. This poster demonstrates the use of genetically engineered iPSC-derived cardiomyocytes for the study of hypertrophy.