Secreto, Frank J.

Cardiac ventricular pressure overload affects patients with congenital heart defects and can cause cardiac insufficiency. Grafts of stem cell–derived cardiomyocytes are proposed as a complementary treatment to surgical repair of the cardiac defect, aiming to support ventricular function. Here, we report successful engraftment of human induced pluripotent stem cell–derived cardiac lineage cells into the heart of immunosuppressed rhesus macaques with a novel surgical model of right ventricular pressure overload. The human troponin+ grafts were detected in low-dose (2 × 10 6 cells/kg) and high-dose (10 × 10 6 cells/kg) treatment groups up to 12 weeks post-injection. Transplanted cells integrated and progressively matched the organization of the surrounding host myocardium. Ventricular tachycardia occurred in five out of 16 animals receiving cells, with episodes of incessant tachycardia observed in two animals; ventricular tachycardia events resolved within 19 days. Our results demonstrate that grafted cardiomyocytes mature and integrate into the myocardium of nonhuman primates modeling right ventricular pressure overload.

The functional maturation status of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has a notable impact upon their use in pharmacological studies, disease modeling, and therapeutic applications. Non-cardiomyocytes (non-CMs) produced in the differentiation process have previously been identified as having an extrinsic influence upon hiPSC-CM development, yet the underlying mechanisms are not fully understood. Herein, we aimed to modulate electrophysiological properties of hiPSC-CMs within co-cultures containing varied proportions of non-CMs and investigate the nature of interactions between these different cell types. Therefore, we sorted cardiac differentiations on day 10 and subsequently replated the cells at ratios of 7:3, 1:1, 3:7, and 1:9 non-CMs to CMs. After a month of co-culture, we evaluated electrophysiological properties through the genetically encoded voltage indicator ArcLight. We ultimately identified that co-cultures with approximately 70%–90% CM purity demonstrated the highest action potential (AP) amplitude and maximum upstroke velocity by day 40 of differentiation, indicative of enhanced electrophysiological maturation, as well as more ventricular-like AP morphologies. Notably, these findings were distinct from those observed for co-cultures of hiPSC-CMs and dermal fibroblasts. We determined that the co-culture phenotypes could not be attributed to paracrine effects of non-CMs due to the inability of conditioned media to recapitulate the observed effects. This led to the further observation of a distinctive expression pattern of connexin 43 (Cx43) at cell-cell interfaces between both CMs and non-CMs. Depletion of Cx43 by short hairpin RNA (shRNA) specifically in the non-CM population within a co-culture environment was able to recapitulate electrophysiological phenotypes of a purer hiPSC-CM population. Collectively, our data demonstrate that abundant non-CM content exerts a significant negative influence upon the electrophysiological maturation of hiPSC-CMs through Cx43-mediated cell-cell-contacts, and thus should be considered regarding the future production of purpose-built hiPSC-CM systems.

Abstract Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHL) including the rare lymphoplasmacytic lymphoma, Waldenström’s macroglobulinemia (WM). Using whole exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). MYD88L265P was detected at lower frequencies in other indolent lymphomas including LPL (0%), MALT (4%), nodal MZL (5%) and splenic MZL (8%); all but one MYD88L265P was heterozygous. Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B cell survival, therapeutic targeting of MYD88 signaling pathways may be useful clinically. However, while the effects of MYD88L265P on the activity of IRAK1/4 and NF-κB are have been studied previously, we are lacking a thorough characterization of the role of intermediary signaling proteins such as TRAF6 and TAK1 on the biology of MYD88L265P-expressing B cells. A better understanding of the proteins involved in MYD88L265P signaling may lead to the development of more targeted and effective therapeutic approaches. In an attempt to identify MYD88L265P –specific therapeutic targets we first wanted to characterize the role of intermediary signaling proteins that facilitate the downstream activation of NF-κB. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodimer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-κB. We monitored the formation of a complex comprised of MYD88, IRAK1, IRAK4 and TRAF6 and immunoprecipitation of either endogenous IRAK4 or IRAK1 revealed constitutive association of IRAK with TRAF6 and MYD88L265P. To assess if the formation of a MYD88L265P/IRAK/TRAF6 complex results in downstream activation of TAK1, constitutive TAK1 phosphorylation was measured and detected in all three cell lines that express MYD88L265P. An association between TAK1 and TRAF6, another measure of TAK1 activation, was also detectable. When a similar analysis of TAK1 was performed in DLBCL cells expressing wild-type MYD88, no phosphorylation of TAK1 was detected, nor was TAK1 associated with TRAF6. IRAK1, IRAK4, TAK1, TRAF6, and MYD88 were expressed at similar levels in all cell lines studied and therefore did not contribute the differences in MYD88 complex formation observed between cell lines. These studies were further confirmed using HEK 293T cells that were transduced with either a vector control plasmid or HA-tagged MYD88WT or MYD88L265P expression plasmids. Together, these studies suggest that MYD88L265P forms a complex with IRAK and TRAF6 resulting in constitutive activation of TAK1 and NF-κB. To confirm the significance of TAK1-mediated MYD88L265P signaling on lymphoma cell growth, the effect of the selective TAK1 inhibitor, (5Z)-7-Oxozeaenol, on cell proliferation was tested. All MYD88L265P-expressing cell lines were sensitive to TAK1 inhibition in a dose-dependent manner (0-10 μM). In contrast, NHL cells expressing MYD88WT were found to be insensitive to TAK1 inhibition. We next tested the impact of the TAK1 inhibitor on a MYD88L265P positive WM patient sample. Similar to what was seen in the WM cell lines, the TAK1 inhibitor inhibited WM cell growth and survival in a dose dependent manner. Additionally, the TAK1 inhibitor significantly reduced the level of IL-10 secreted by each of the cell lines. Together, these data suggest that MYD88L265P drives cell proliferation and cytokine secretion through a TAK1-dependent mechanism. In conclusion, we are the first to validate by NGS in a large patient cohort the high prevalence and specificity of MYD88L265P in WM. Cells harboring the L265P mutation but not wild-type MYD88 exhibit constitutive signaling leading to the hyperactivation of NF-κB. We have established the role of TAK1 as an integral component of MYD88L265P signaling in both WM and DLBCL cell. Our data suggest that targeting TAK1 clinically may be an effective strategy for the treatment of WM and other lymphomas driven by MYD88L265P signaling. Disclosures: Fonseca: millennium: Consultancy; amgen: Consultancy; Binding site: Consultancy; onyx: Consultancy; medtronic: Consultancy; Genzyme: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; lilly: Consultancy; Onyx: Research Funding; cylene: Research Funding.

Abstract Abstract 2642 BAFF is essential for B cell maturation, and a lack of either BAFF or its primary receptor, BAFF-R, results in a severe depletion of T2 marginal zone and follicular B cells. Elevated serum BAFF levels have been correlated with an increased risk of developing non-Hodgkin's lymphoma (NHL), along with a more aggressive phenotype. A growing body of genetic evidence points toward an association between the development of human disease and variation in genes encoding BAFF and its receptors. Recently, we characterized a novel lymphoma-associated mutation in TNFRSF13C, the gene encoding BAFF-R. This mutation (BAFF-R H159Y) encodes a His159Tyr substitution in the C-terminus of BAFF-R adjacent to the TRAF3 binding motif. Signaling through BAFF-R H159Y results in increased NF-κB activity, elevated immunoglobulin production and increased association with TRAF2, TRAF3 and TRAF6 compared to wild type (WT) BAFF-R. We have detected this mutation in 6% of total NHL cases (n=129), and in 10% of follicular lymphoma (FL) cases (n=41) evaluated thus far. We previously reported that BAFF-R H159Y expressing mouse B cells exhibited significantly more resistance to Fas ligand (FasL) induced apoptosis compared to their cells expressing BAFF-R WT, and we propose here that BAFF-R H159Y mediated increases in PI3K activity may explain such an enhanced anti-apoptotic response. In this study we now show that BAFF stimulated HEK 293 cells stably expressing BAFF-R H159Y not only display significantly increased Akt phosphorylation when compared to BAFF-R WT expressing cells, but also demonstrate robust Akt phosphorylation in the absence of BAFF. BAFF-R H159Y-dependent Akt activation also led to activation of the downstream Akt targets mTOR and GSK3β and their phosphorylation was inhibited following treatment with the PI3- kinase inhibitor wortmannin. We next examined the impact of the BAFF-R H159Y mutation on expression of BAFF-target genes. Quantitative PCR analyses revealed that BAFF-R H159Y cells exhibited a pattern of gene expression indicative of promoting cell survival, displaying significantly higher levels of BCL2, BCL2L1 and PIN1, while down-regulating expression of the pro-apoptotic gene BIM. We recently reported that TRAF6 associates with BAFF-R, and that such binding is more pronounced in cells expressing BAFF-R H159Y. In order to investigate the role TRAF6 plays in mediating BAFF-R-dependent PI3K activity, we silenced TRAF6 expression in HEK 293 and Karpas 422 lymphoma cells using TRAF6 shRNA. Reduced TRAF6 protein expression resulted in a parallel decrease in BAFF-R WT mediated phosphorylation of mTOR in Karpas 422 cells and phosphorylation of both Akt and GSK3β was markedly reduced in BAFF-R H159Y expressing HEK 293 cells. Interestingly, TRAF6 knock-down did not affect NF-kB2 activation in either Karpas 422 or HEK BAFF-R expressing cells suggesting that Akt does not play a role in BAFF-R mediated activation of non-canonical NF-kB. Finally, preliminary co-precipitation studies indicate that Akt can be recruited to BAFF-R itself, and our initial observations suggest that such an association is significantly reduced in cells expressing BAFF-R H159Y. Taken together, these studies suggest that the BAFF-R H159Y mutation confers enhanced BAFF-R-dependent PI3K signaling and pro-survival gene expression independent of BAFF. Moreover, such enhanced P13K activation is partly dependent upon TRAF6, and decreased recruitment of Akt to BAFF-R H159Y may function to increase the amount of this PI3K target for activation. Thus, BAFF-R H159Y likely contributes to BAFF signaling irregularities in NHL patients harboring this mutation, and may predispose individuals to developing lymphoma regardless of their serum BAFF concentration. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2688 Members of the signal transducers and activators of transcription (STAT) family of proteins function as secondary messengers mediating cellular responses to various cytokines. Aberrant activation of STAT5 protein has been implicated in the pathogenesis of hematologic malignancies due to the ability of these transcription factors to regulate genes involved in cellular proliferation and survival. The two highly homologous transcription factors collectively known as STAT5, namely STAT5A and STAT5B, display both redundant and distinct physiologic functions in non-malignant B cells. Yet, despite evidence suggesting independent functions for STAT5A and STAT5B in the pathophysiology of solid tumors, the precise roles of these isoforms in hematologic malignancies such as Waldenstrom's macroglobulinemia (WM) are not known. Initial immunohistochemical staining of WM bone marrow biopsies (n=6) revealed higher levels of STAT5 phosphorylation compared to bone marrows from normal controls (n=6). Additionally, phosphorylated STAT5 was highly expressed in CD19/CD138+ sorted cells obtained from the bone marrow of patients with WM (average ΔMFI compared to isotype control, 4.64 +/− 1.7, n=6) as determined by FACS analysis. Furthermore, western blotting demonstrated baseline STAT5 phosphorylation in both WM and IgM-secreting cell lines (MWCL-1 and BCWM-1, respectfully). The expression of other phosphorylated STAT proteins (1, 3, 4, and 6) was not detected. To better understand the overall role of STAT5 biologically in WM, MWCL-1 and BCWM.1 cells were treated with a specific inhibitor of STAT5, N′-((4-Oxo-4H-chromen-3-yl)methylene) nicotinohydrazide. Following 72 hours in culture, this inhibitor significantly decreased IgM secretion at lower doses when compared to controls (10 μM, MWCL-1: 9413 +/− 511 ng/mL vs. 6166 +/− 160 ng/mL, p<0.001; BCWM.1: 13,380 +/− 393 ng/mL +/− 10970 +/− 240 ng/mL, p<0.001) and nearly abolished IgM secretion at higher doses. Significant effects on proliferation and viability were also observed, but only at higher concentrations of drug where the specificity toward STAT5 may have diminished. To characterize the roles of the individual STAT5 isoforms in WM, we have developed doxycycline-inducible STAT5A-shRNA and STAT5B-shRNA and used these to create stable knockdowns in the MWCL-1 cell line. Addition of doxycycline to these cell lines for 72h nearly abolishes STAT5A or STAT5B protein expression, respectively, without affecting expression of the other isoform, while no change in STAT5 expression is detected in cells transduced with the empty vector (EV) control. The functional effects of isoform-specific STAT5 inhibition on WM tumor biology were assessed through proliferation assays and ELISA to determine the utility of therapeutically targeting a single STAT5 isoform. No difference in proliferation was observed in the STAT5A- or STAT5B-shRNA cells following 72h induction with doxycycline as compared to the EV control cells. However, specific knockdown of STAT5A following doxycycline induction significantly decreased IgM secretion as compared to the EV control (EV, 91.8 +/− 10.2% of baseline secretion, STAT5A, 66.7 +/− 15.1% of baseline, p<0.05), whereas IgM secretion was decreased by knockdown of STAT5B but not significantly. Similarly, IL-6 secretion was only diminished in cells lacking STAT5A as compared to EV cells (EV, 94 +/− 14.16% of baseline secretion; STAT5A, 64% +/− 7.6% of baseline, p<0.001). Overall these initial data indicate a role for isoform-specific STAT5 activation in the maintenance of WM tumors, and future studies will further our understanding of the specific roles of STAT5A and STAT5B in WM tumors. Disclosures: Ansell: Seattle Genetics, Inc.: Research Funding.

Abstract Abstract 2699 Myeloid differentiation factor 88 (MYD88) is an adaptor protein that plays a key role in mediating innate immune response. Upon activation of toll-like (TLRs) or IL-1b receptors, MYD88 activates a signaling cascade that culminates in activation of NF-kB and expression of proinflammatory genes. Activating mutations in MYD88 have been reported in non-Hodgkin's lymphoma (NHL) tumors, including diffuse large B cell and marginal zones lymphoma. The most common MYD88 mutation described thus far is a single base pair mismatch resulting in an amino acid switch from lysine to proline at position 265 (MYD88L265P). MYD88L265P functions as a constitutively active MYD88 protein resulting in dysregulated NF-kB and STAT3 signaling. This mutation has recently been reported to be present in lymphomoplasmacytic lymphoma/Waldenstrom macroglobulinemia (LPL/WM) at a high frequency, ∼ 90% (Xu et al, ASH, #261, 2011). The discovery of MYD88L265P in a high percentage of LPL/WM tumors, and other subtypes of NHL, provides the basis for the identification and targeting of MYD88 dependent pathways. Additionally, the ability of this mutation to drive NF-kB-mediated cytokine expression combined with our finding that LPL/WM patients have elevated serum cytokines suggests that the MYD88 mutation may play a significant role in LPL/WM pathogenesis. We first wanted to confirm the incidence of the MYD88L265P mutation in LPL/WM and other subtypes of indolent lymphoma. Using Sanger sequencing we found that 57% (n=58) of LPL, 4% of MALT (n=23), 5% of nodal marginal zone (n=34), and 6% of splenic marginal zone (n=34) lymphomas harbor the MYD88L265P mutation, with all but 1 case being heterozygous. When we specifically looked at the LPL cases with WM, we found that 70% of WM tumors have the MYD88L265P mutation. In a validation cohort of WM (n=41), we found a similar mutation rate of 68%. Additionally, the WM cell line MWCL1 and the IgM secreting cell line BCWM.1 also harbor the MYD88L265P mutation. The prevalence of the MYD88L265P mutation in NHL, specifically WM and LPL, suggests that it may be a novel driver of lymphoma pathogenesis and a potential therapeutic target. However, it is not yet clear if this mutation correlates with activation of signaling pathways, or cell growth and survival in LPL/WM. To address these questions we first explored the possibility that the MYD88 signaling pathway is constitutively active in cells that express MYD88L265P. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodiamer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-kB. Using three cell lines that express MYD88L265P (BCWM, MWCL1, and OCI-Ly3), we looked for complex formation of MYD88, IRAK1/4, TRAF6, and TAK1. Immunoprecipitation of either endogenous IRAK1 or TAK1 revealed constitutive association with both TRAF6 and MYD88. We also detected phosphorylation of TAK1, and taken together, these data suggest that the MYD88 pathway is activated in cells that harbor the MYD88L265P mutation. We next wanted to confirm the significance of the MYD88 pathway on lymphoma cell growth. Using our cell lines we tested the effect of IRAK1/4, TAK1, and NF-kB inhibitors on cell proliferation. All MYD88L265P expressing cell lines were sensitive to the TAK1 and NF-kB inhibitors in a dose dependent manner (0–10 uM), however, we only saw inhibition of cell proliferation with the IRAK1/4 inhibitor at the highest dose (10 uM) in the BCWM cell line. These data suggest that cell proliferation is dependent on the MYD88 signaling pathway, with some variation being detected between cell line models and inhibitors. Because the MYD88 pathway has also been shown to drive cytokine secretion we next tested the impact of the IRAK1/4, TAK1, and NF-kB inhibitors on expression of IL-10. Using drug doses that were shown to inhibit cell proliferation, but not impact survival, we found that all three inhibitors reduced the level of IL-10 secreted by each of cell lines. In summary, our data confirm the high incidence on the MYD88L265P mutation in LPL/WM, we find that the MYD88 signaling pathway is constitutively activated in cells the harbor the mutation, and we show that cell proliferation and cytokine secretion are dependent on MYD88 signaling targets. These studies highlight the biologic significance of the MYD88L265P mutation in LPL/WM and suggest that the MYD88 signaling pathway may be a novel therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p<0.05) and IgM secretion by 80% (p<0.005). Similarly, in primary CD19+CD138+ WM cells (n=5), proliferation increased on average by 38% and IgM secretion by 71%. No apoptotic effects were associated with IL-21 in WM. Characterization of STAT activation in response to IL-21 revealed significant phosphorylation of STAT3 in both CD19+CD138+ WM cells and MWCL-1 cells and was associated with increases in BLIMP-1 and XBP-1 protein and decreases in PAX5. As STAT3 activation is known to regulate IL-6, we assessed the effect of IL-21 on B cell-mediated IL-6 secretion using ELISA. IL-21 significantly increased IL-6 secretion by both primary CD19+CD138+ WM cells (n=4) and MWCL-1 cells (87.9 +/− 10.9 ng/mL vs. 297.8 +/− 129.2 ng/mL, p<0.05). Treatment with IL-6 and IL-21 together had no additional effect over IL-21 alone on proliferation or IgM secretion in MWCL-1 cells, but culturing anti-CD3/anti-CD28-activated CD19−CD138−cells from WM bone marrows with IL-6 significantly increased IL-21 secretion (n=3). Overall, these data indicate that T-cell derived IL-21 significantly promotes growth and immunoglobulin production by malignant WM B cells and that subsequent IL-6 secretion by malignant B cells may enhance the secretion of IL-21 by T cells within the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Abstract Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high-throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time-consuming, and incompatible with high-throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells’ documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419-42-0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of &lt;300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells.

Background—Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors may desynchronize natural developmental schedules. This study aims to evaluate the effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells.Methods and Results—Targeted inclusion and exclusion of reprogramming transgenes (c-MYC,KLF4,OCT4, andSOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system. Pulsed transgene overexpression, before iPS cell differentiation, hindered cardiogenic outcomes. Delayed in counterparts with maintained integrated transgenes, transgene removal enabled proficient differentiation of iPS cells into functional cardiac tissue. Transgene-free iPS cells generated reproducible beating activity with robust expression of cardiac α-actinin, connexin 43, myosin light chain 2a, α/β-myosin heavy chain, and troponin I. Although operational excitation–contraction coupling was demonstrable in the presence or absence of transgenes, factor-free derivatives exhibited an expedited maturing phenotype with canonical responsiveness to adrenergic stimulation.Conclusions—A disproportionate stemness load, caused by integrated transgenes, affects the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of nonintegrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics.