1. Volume 19, Issue 9, Pages 1695-1703 (September 2011)
Directed Differentiation of Human Embryonic Stem Cells to Interrogate the Cardiac Gene Regulatory Network 
James E Dixon, Emily Dick, Divya Rajamohan, Kevin M Shakesheff, Chris Denning 
Molecular Therapy 
Volume 19, Issue 9, Pages (September 2011)
DOI: /mt
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Induction of cardiac differentiation of hESCs by 15F. Lentiviruses were used to deliver candidate transgenes to hESCs at high efficiency. (a) Schematic representation of the lentiviral construct used to force expression of transgenes in hESCs. (b) Schematic of the strategy to test candidate cardiomyocyte-inducing factors. (c–d) Morphology of beating colonies of cardiomyocytes induced from hESCs 28 days post infection of the 15F candidates (c, brightfield; d, MYH6-mRFP fluorescence; dotted lines show beating area; bar = 100 µm). (e) Beating clusters similar to those in (c) were dissociated to single cardiomyocytes and stained with α-actinin (green, α-actinin; blue, DAPI; inset shows magnified region or sacromeric striations; bar = 100 µm). (f) Dissociated, single cardiomyocytes were also used in patch clamp analysis (left, atrial waveform; right, ventricular waveform). hESCs, human embryonic stem cells; mRFP, monomeric red fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Combination of four factors induces cardiac differentiation of hESCs. Single factors were removed from the 15F pool and assessed for induction activity. (a) MYH6-mRFP+ cells were analyzed by flow cytometry at day 28 post-transduction (% is amount of total cell numbers over triplicates; bars are s.d.) and (b) by counting numbers of MYH6-mRFP+ colony clusters (values are numbers per well over triplicates; bars are s.d.). (c) Factors were removed from the 15F pool and assessed for cardiogenic activity by counting number of MYH6-mRFP+ clusters; 15-MH is 15F pool lacking MESP1 and HAND1; 15F-TBGNMI is 15F pool lacking TBX5, BAF60c, GATA4, NKX2.5, MEF2C, and ISL1; 15F-GTNB is 15F pool lacking GATA4, TBX5, NKX2.5, and BAF60c (values are numbers per well over triplicates; bars are s.d.). (d) Permutations of the GTNB (GATA4, TBX5, NKX2.5, and BAF60c) transgenes were assessed for cardiogenic activity. MYH6-mRFP+ cells were analyzed by flow cytometry at day 28 post-transduction (% is amount of total cell numbers over triplicates; bars are s.d.) and by e) counting numbers of MYH6-mRFP+ clusters (values are numbers per well over triplicates; bars are s.d.). f–i) Morphology of cardiomyocytes induced from hESCs 28 days post infection of the GTNB pool. (f,h: brightfield; g: MYH6-mRFP fluorescence; i: propidium iodide (PI) stain fluorescence showing multinucleated cells; dotted lines shows beating area bar = 100 µm). hESCs, human embryonic stem cells; mRFP, monomeric red fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
GTNB-transduction efficiency is controlled by cell-culture environment and cell-density. Cardiac differentiation of hESCs by GTNB transduction is successful in defined hESC media. (a) hESCs were GTNB-transduced and either transferred directly into different media (CM is MEF conditioned medium; mTesR1, StemPro and HESGro are commercially available defined media). Number of MYH6-mRFP+ colony clusters (iCMs) were counted at day 28 post-transduction (values are numbers per well over triplicates; bars are s.d.). (b) Morphology of cardiomyocytes induced from GTNB-transduction in different culture media (bar = 100 µm). (c) Cardiac differentiation by GTNB-transduction is dependent on cell density. hESCs were GTNB-transduced at different densities in six-well plates (50,000–500,000/well) and MYH6-mRFP+ cells were analyzed by flow cytometry at day 28 post-transduction (% is amount of total cell numbers over triplicates; bars are s.d.) or by (d) counting numbers of MYH6-mRFP+ clusters (values are numbers per well over triplicates; bars are s.d.). (e) Morphology of GTNB-transduced cultures at different starting densities (bar = 100 µm). hESCs, human embryonic stem cells; MEF, mouse embryonic fibroblast; mRFP, monomeric red fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
GTNB-transduction efficiency generates cardiac progenitors and terminally differentiated cardiomyocytes from hESCs. Characterization of the differentiation process after GTNB-transduction of hESCs. (a) Morphology and phenotype of hESC cultures at different times after GTNB-transduction (mRFP, control transduction culture on day 1 post infection; 6H, 6 h post infection; bar = 100 µm). (b) MYH6-mRFP+ cells were analyzed by flow cytometry at different time-points post-transduction (% is amount of total cell numbers over triplicates; bars are s.d.). Beating cardiomyocytes appear from day 12 post-transduction. (c) Number of MYH6-mRFP+ colony clusters (iCMs) were counted before flow cytometry (values are numbers per well over triplicates; bars are s.d.). (d) Mesoderm and endoderm differentiation in hESCs after GTNB transduction: immunofluorescence detection of T (BRACHYURY) and SOX17 expression (green: T/SOX17; blue: DAPI; bar = 100 µm). (e–h) Pluripotency and differentiation marker gene expression was analyzed by quantitative real time polymerase chain reaction (qPCR) analysis over a time-course post GTNB-transduction: (e) Pluripotency; (f) Germ-layer; (g) Cardiac-progenitor; and (h/i) terminally differentiated gene expression markers. Pluripotency is lost with reciprocal induction of mesoderm and endoderm markers. Following mesoderm induction, cardiac progenitors and then terminal cardiac differentiation markers are expressed. hESCs, human embryonic stem cells; mRFP, monomeric red fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
GTNB-transduction produces cardiac progenitors. Isolation of cardiac progenitor cells produced from GTNB-transduced hESCs. (a) Schematic of the strategy to isolate cardiac progenitors. (b) Phenotype of GTNB-transduced hESC cultures at day 7 from which iCPs were manually dissected. (c) iCPs express ISL1 (green: ISL1; blue: DAPI; bar = 100 µm). (d) Morphology of iCPs plated one day after manual dissection. (e) During extended differentiation to day 15 post-transduction (8 days post dissection), iCPs produced MYH6-mRFP+ cells and this time-course was followed by flow cytometry (in f, % is amount of total cell numbers over triplicates; bars are s.d.) (g–i) Gene expression was compared between iCPs enriched by dissection (isolated iCPs day 7) and GTNB-transduced hESC cultures (main GTNB day 7). Expression profiles were for (g) pluripotency; (h) mesoderm and cardiac-progenitor; and (i) terminally differentiated cardiomyocytes. iCPs, induced cardiac progenitors; hESCs, human embryonic stem cells; mRFP, monomeric red fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
GTNB-transduction induces cardiogenesis in different pluripotent stem cell lines. Fluorescence-activated cell sorting (FACS) analysis 28 days post GTNB-transduction of (a) HUES7 MYH6-mRFP cells stained with cTnI, (b) HUES7 parental cells, H9 or FIB-hiPSC with cTnI or α-actinin, (c) HUES7 with cTnI and α-actinin. In (d) HUES7 MYH6-mRFP, HUES7 parental or H9 cells were transduced with GTNB. On day 10 post-transduction, Ara-C was added at 0.1 µmol/l (see also Supplementary Figure S4) and maintained for the next 18 days (to day 28), at which time FACS was carried out with cTnI staining. Open histograms show isotype control. mRFP, monomeric red fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Early GTNB function is required to specify the cardiac lineage. The temporal requirement of GTNB transgene activity was assessed by using Tet-On inducible transgenes delivered by lentivirus. (a) Schematic of the strategy to test the timing of GTNB expression to induce cardiac differentiation of hESCs. Doxycyclin (DOX) was added to cultures (1 µg/ml) directly following transduction and removed at different time-points throughout the 28 culture period. (b) Addition of DOX throughout the 28 days yielded MYH6-mRFP+ cells (bar = 100 µm). (c–e) The morphology (c, bar = 100 µm) and appearance of MYH6-mRFP+ cells were measured by flow cytometry (d, % is amount of total cell numbers over triplicates; bars are s.d.) and counting number of MYH6-mRFP+ clusters (e, values are numbers/well over triplicates; bars are s.d.) was evaluated on day 28 of the experiment in which cells were exposed to different durations of DOX. mRFP, monomeric red fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions