1. Volume 24, Issue 6, Pages 895-907.e6 (June 2019)
Drug Screening in Human PSC-Cardiac Organoids Identifies Pro-proliferative Compounds Acting via the Mevalonate Pathway 
Richard J. Mills, Benjamin L. Parker, Gregory A. Quaife-Ryan, Holly K. Voges, Elise J. Needham, Aurelie Bornot, Mei Ding, Henrik Andersson, Magnus Polla, David A. Elliott, Lauren Drowley, Maryam Clausen, Alleyn T. Plowright, Ian P. Barrett, Qing-Dong Wang, David E. James, Enzo R. Porrello, James E. Hudson 
Cell Stem Cell 
Volume 24, Issue 6, Pages e6 (June 2019)
DOI: /j.stem
Copyright © 2019 Elsevier Inc. Terms and Conditions

2. Cell Stem Cell 2019 24, 895-907.e6DOI: (10.1016/j.stem.2019.03.009)
Copyright © 2019 Elsevier Inc. Terms and Conditions

3. Figure 1 Schematic Outline of the Drug Development Strategy
A 5,000-compound library was screened for pro-proliferative effects in induced pluripotent stem cell (iPSC)-derived cardiomyocytes in 2D using EdU. The hits were eliminated if they also induced proliferation in fibroblasts. Hit compounds were then screened in immature hCOs using Ki-67. The hits were eliminated if they decreased force or increased relaxation time. Hit compounds were then screened in mature cell-cycle-arrested hCOs using Ki-67. Proliferation was confirmed by quantifying cardiomyocyte-specific Ki-67 and pH3 immunostaining, counting cardiomyocyte number, and analyzing hCO size following treatment.
Cell Stem Cell  , e6DOI: ( /j.stem )
Copyright © 2019 Elsevier Inc. Terms and Conditions

4. Figure 2
Screening for Proliferative Activators that Do Not Impact Contractile Force or Relaxation Time
(A) Screening protocol in immature hCOs.
(B) Ki-67 intensity in hCOs treated with positive control CHIR99021 for 2 days. n = 9 experiments.
(C) Force of contraction in hCOs treated with positive control CHIR99021 for 2 days. n = 8 experiments.
(D) Heatmap of hCO Ki-67 intensity after treatment with small molecules for 2 days. Molecules screened at 3 different concentrations with n = 2–6 per concentration.
(E) Heatmap of hCO contractile function after treatment with small molecules for 2 days. Molecules screened at 3 different concentrations with n = 2–6 per concentration.
(F) Correlation of maximum effect on proliferation in 3D hCOs versus 2D screening.
(G) Correlation of maximum Ki-67 effect versus force of contraction in hCOs.
(H) Correlation of maximum Ki-67 effect versus 50% relaxation time (Tr) in hCOs.
Triangles indicate hit compounds that reduce function, green triangles indicate compounds with GSK3 inhibition activity, and purple triangles indicate compounds activating adenosine receptor 2A. Data are presented as mean ± SEM. ∗∗ and ∗∗∗∗, denote p < 0.01 and p < , respectively, using the Mann-Whitney test (B and C).
Cell Stem Cell  , e6DOI: ( /j.stem )
Copyright © 2019 Elsevier Inc. Terms and Conditions

5. Figure 3 Follow-up of Hits in the Secondary Screen in Mature hCOs
(A) Schematic overview of protocol for hit validation.
(B) Force of contraction following treatment of compounds for 2 days. n = 6–10, 2 experiments.
(C) Ki-67+ cardiomyocytes (α-actinin) following treatment with hits for 2 days. n = 6–10, 2 experiments.
(D) Mitosis (pH3) of cardiomyocytes (α-actinin) following 2 days of treatment of hits at optimal concentrations. n = 17–19, 4 experiments for DMSO, compound 3, and compound 65 and n = 6, 2 experiments for compound 63.
(E) Cardiomyocyte number in the mature hCOs following treatment with 3 μM compound 3 or 1 μM compound 65. n = 11–13 from 2 experiments.
(F) The size of the cardiomyocyte (α-actinin) area increases in hCOs culture for 7 days with 3 μM compound 3 or 1 μM compound 65. n = 12–16 from 3 experiments.
(G) Structure of compound 3.
(H) Structure of compound 65.
Scale bars, 20 μm. Data are presented as mean ± SEM. ∗, ∗∗, ∗∗∗, and ∗∗∗∗ denote p < 0.05, p < 0.01, p < 0.001, and p < , respectively, using one-way ANOVA with Dunnett’s post-test relative to DMSO in (B)–(D) and (F) or using two-way ANOVA (E).
Cell Stem Cell  , e6DOI: ( /j.stem )
Copyright © 2019 Elsevier Inc. Terms and Conditions

6. Figure 4
Proliferation Is Activated by Distinct Processes for Compounds 3, 63, and 65
(A) Protocol. Mature hCOs were stimulated with DMSO or compounds 3, 63, or 65 (at concentrations of 3 μM, 1 μM, or 0.3 μM, respectively) for 2 days.
(B) Principal coordinate analysis of batch-corrected RNA-seq data. n = 10–20 hCOs per condition from 3–4 experiments (number in brackets).
(C) RNA-seq gene ontologies for compound 3.
(D) RNA-seq gene ontologies compound 65.
(E) Overlap of upregulated proteins in RNA-seq and proteomics.
(F) Key cell-cycle genes changed in response to stimulation with the different compounds to activate proliferation leading to changes in the proteome. All hit compounds upregulated gene ontologies associated with G1-S transition and DNA replication, but only compounds 3 and 65 induced G2-M transition, consistent with the induction of pH3-positive cardiomyocytes in Figure 3D.
(G) Each compound activated distinct “cell cycle” proteins in the proteome.
Significantly regulated for RNA-seq analyses with a false discovery rate (FDR) < 0.10 or p LIMMA < 0.05 for proteomics. For bubble plots, size correlates to p value (see legend in the top corner) and gene ontologies are represented in x and y in a principle-component analysis showing relatedness.
Cell Stem Cell  , e6DOI: ( /j.stem )
Copyright © 2019 Elsevier Inc. Terms and Conditions

7. Figure 5
The Core Cell-Cycle Program Is Correlated with Activation of a Cell-Cycle Network and the Mevalonate Pathway
(A) Proteins upregulated in all proliferative conditions. Note that aurora kinase B (AURKB) was also added to the cell-cycle network, as it was consistently upregulated, but failed to reach significance for some treatments.
(B) Expression of proliferation signature proteins (plus HMGCS1) in different conditions.
(C) Cholesterol pathway proteins that increase with the strongest inducers, compounds 51 and 65.
(D) Regulation of mevalonate and cholesterol biosynthesis genes HMGCR, HMGCS1, and CYP51A1 (Cyp51 in the mouse) and SQLE expression during both mouse and human maturation in vivo. RNA-sequ data were extracted from data generated in previous studies (Kuppusamy et al., 2015; Mills et al., 2017a; Quaife-Ryan et al., 2017). Mouse data are for purified cardiomyocytes and all are significantly regulated (FDR < 0.05). Human data are for whole-heart or hPSC-CM differentiation cultures and are significantly regulated from 20-day-old hPSC-CMs (adjusted p < 0.05, using 2-way ANOVA with Tukey’s post-test).
(E) Cholesterol proteins Hmgcr, Hmgcs1, Cyp51, and Sqle showing a trend or statistically regulated (∗) increases during heart regeneration in mice following delivery of constitutively active YAP1(S127A). Data from Lin et al. (2014). MI, myocardial infarction.
(F) Schematic showing that the cell-cycle network is correlated with co-activation of the mevalonate pathway for full cell-cycle progression.
Cell Stem Cell  , e6DOI: ( /j.stem )
Copyright © 2019 Elsevier Inc. Terms and Conditions

8. Figure 6
Mevalonate Metabolic Products Are Required for Proliferation in hPSC-CMs and Mature hCOs
(A) Experiments in 2D proliferative hPSC-CMs (C–E).
(B) Representative image from 2D experiments used for image cytometry and analysis.
(C) Cardiomyocyte proliferation after 3 days of simvastatin treatment. n = 12.
(D) Cardiomyocyte number after 3 days of simvastatin treatment. n = 12.
(E) Cardiomyocyte size after 3 days of simvastatin treatment. n = 12.
(F) The mevalonate pathway. Red displays enzymes inhibited or metabolites added in (G).
(G) Cardiomyocyte proliferation after 24 h. Mev, mevalonate; GGPP, geranyl-geranyl pyrophosphate; FPP, farnesyl pyrophosphate. n = 12, 3 experiments.
(H) Experiment in mature hCOs (I and J).
(I) Ki-67 intensity in mature hCOs treated with 1 μM compound 65, 3 μM compound 3, or 10 μM compound 6.28 is abolished by 10 μM simvastatin. n = 11–14, 2 experiments (compound 65) and n = 8–22, 3 experiments (compounds 3 and 6.28).
(J) Simvastatin abolishes 3 μM compound 3 induced Ki-67+ cardiomyocytes in hCOs. n = 5–6.
Scale bars, 20 μm (except for the full image in B, where the scale bar represents 200 μm). Data are presented as mean ± SEM. ∗, ∗∗, ∗∗∗, and ∗∗∗∗ denote p < 0.05, p < 0.01, p < 0.001, and p < , respectively, using a t test (C–E and I) and a one-way ANOVA with Tukey’s post-test (G and J).
Cell Stem Cell  , e6DOI: ( /j.stem )
Copyright © 2019 Elsevier Inc. Terms and Conditions

9. Figure 7 The Mevalonate Pathway Controls Proliferation In Vivo in Mice
(A) Schematic overview of neonatal mouse experiments (B–D).
(B) Cardiomyocyte (MLC2v) proliferation (BrdU). n = 7.
(C) Heart size. n = 7.
(D) Cardiomyocyte cross-sectional area. n = 140 from 7 hearts.
(E) Schematic overview of adult mouse experiments (F–I).
(F) Striated cardiomyocyte following a fix-dissociation method.
(G) Representative proliferation (BrdU+) and cardiomyocyte (MLC2v) staining.
(H) DNA synthesis (BrdU+) in adult cardiomyocytes (MLC2v) in vivo. n = 5–6 mice.
(I) DNA synthesis (BrdU+) in adult cardiomyocytes (MLC2v) in vivo. n = 4–6 mice (data from H were used).
Scale bars, 20 μm. Data are presented as mean ± SEM. ∗ and ∗∗∗ denote p < 0.05 and p < 0.001, respectively, using a t test (B and D) and a Kruskal-Wallis test with Dunn’s post-test for (H and I).
Cell Stem Cell  , e6DOI: ( /j.stem )
Copyright © 2019 Elsevier Inc. Terms and Conditions