© The American Society of Gene & Cell Therapy
commentary treated with concomitant administration of drug-loaded NPs than the concentration of both drugs from the nano-polypharmacy formulation, the appropriate drug ratio was not maintained. The differential delivery of drug to the tumor when coadministering NP drug formulations is perhaps due to the differential pharmaco kinetics of NPs loaded with rapamycin versus paclitaxel. Panagi et al.21 have demonstrated that the administration dose of poly (lacticco-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) NPs significantly affects their biodistribution. Because the ratio of the drugs is maintained during the coadministration of the drug-loaded NPs, the dose of each NP formulation differs and may affect the accumulation of each NP drug-loaded formulation into the tumor. There was significantly greater tumor regression by the nano-polypharmacy formulation than with the mixture of NP formulations loaded with rapamycin and paclitaxel. In addition, there was significantly greater tumor regression for nano-polypharmacy formulations delivering a rapamycin-to-paclitaxel ratio of 3:1 than with a ratio of 2:1, as predicted by the in vitro results. Further pharmacological studies are expected to be of use in determining cytotoxicity in healthy organs—one of the main challenges associated with free-drug combination treatments. NPs delivering drug combinations could also help to overcome challenges associated with drug resistance. High levels of p-Akt in breast cancer patients treated with tamoxifen are associated with reduced survival time. Additional studies using cell lines resistant to rapamycin could provide a better understanding of the potential of nano-polypharmacy to overcome drug resistance. It is possible that the drug combination ratio for cell sensitivity and cell resistance may differ—a possibility that will support personalized therapy using nanopolypharmacy formulations.22,23 It is unclear, however, whether nano-polypharmacy will be useful for drug combinations that do not require a very precise drug ratio concentration for synergistic efficacy. Although coencapsulation of drugs into nanomaterials is expected to progress from preclinical to clinical studies, regulatory issues must also be addressed. However, it is not expected that regulatory challenges will be significantly greater than for single-drug delivery nanomaterials,24,25 in that the review process 1240
is similar to that for other nanomaterials except that combination products are regulated by the Office of Combination Products at the FDA. REFERENCES 1.
Borisy, AA, Elliott, PJ, Hurst, NW, Lee, MS, Lehar, J, Price, ER et al. (2003). Systematic discovery of multicomponent therapeutics. Proc Natl Acad Sci USA 100: 7977–7982. 2. Al-Lazikani, B, Banerji, U and Workman, P (2012). Combinatorial drug therapy for cancer in the postgenomic era. Nat Biotechnol 30: 679–692. 3. Shah, PS and Schaffer, DV (2010). Gene therapy takes a cue from HAART: combinatorial antiviral therapeutics reach the clinic. Sci Transl Med 2: 36ps30. 4. Aliabadi, HM, Mahdipoor, P and Uludag, H (2013). Response of drug sensitive and resistant breast cancer cells to combinatorial siRNA therapy. Mol Ther 21: S82–S83. 5. Jia, J, Zhu, F, Ma, X, Cao, Z, Li, Y and Chen, YZ (2009). Mechanisms of drug combinations: interaction and network perspectives. Nat Rev Drug Discov 8: 111–128. 6. Keith, CT, Borisy, AA and Stockwell, BR (2005). Multicomponent therapeutics for networked systems. Nat Rev Drug Discov 4: 71–78. 7. Dancey, JE and Chen, HX (2006). Strategies for optimizing combinations of molecularly targeted anticancer agents. Nat Rev Drug Discov 5: 649–659. 8. Blanco, E, Sangai, T, Wu, S, Hsiao, A, Ruiz-Esparza, GU, Gonzalez-Delgado, CA et al. (2014). Colocalized delivery of rapamycin and paclitaxel to tumors enhances synergistic targeting of the PI3K/Akt/mTOR pathway. Mol Ther 22: 1310–1319. 9. Faivre, S, Kroemer, G and Raymond, E (2006). Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 5: 671–688. 10. Alexis, F, Rhee, JW, Richie, JP, Radovic-Moreno, AF, Langer, R and Farokhzad, OC (2008). New frontiers in nanotechnology for cancer treatment. Urol Oncol 26: 74–85. 11. Sprintz, M (2004). Nanotechnology for advanced therapy and diagnosis. Biomed Microdevices 6: 101–103.
12. Wang, X, Yang, L, Chen, ZG and Shin, DM (2008). Application of nanotechnology in cancer therapy and imaging. CA Cancer J Clin 58: 97–110. 13. Davis, SS (1997). Biomedical applications of nanotechnology—implications for drug targeting and gene therapy. Trends Biotechnol 15: 217–224. 14. Farokhzad, OC (2008). Nanotechnology for drug delivery: the perfect partnership. Expert Opin Drug Deliv 5: 927–929. 15. Alexis, F, Pridgen, E, Molnar, LK and Farokhzad, OC (2008). Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 5: 505–515. 16. Peer, D, Karp, JM, Hong, S, Farokhzad, OC, Margalit, R and Langer, R (2007). Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2: 751–760. 17. Alexis, F, Pridgen, E, Langer, R and Farokhzad, O (2010). Nanoparticle technologies for cancer therapy. In: Schäfer-Korting, M (ed) Drug Delivery (Handbook of Experimental Pharmacology, vol 197). Springer: Berlin, Germany. pp 55–86. 18. Davis, ME, Chen, Z and Shin, DM (2008). Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat Rev Drug Discov 7: 771–782. 19. Ferrari, M (2005). Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer 5: 161–171. 20. Petros, RA and DeSimone, JM (2010). Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 9: 615–627. 21. Panagi, Z, Beletsi, A, Evangelatos, G, Livaniou, E, Ithakissios, DS and Avgoustakis, K (2001). Effect of dose on the biodistribution and pharmacokinetics of PLGA and PLGA–mPEG nanoparticles. Int J Pharmaceutics 221: 143–152. 22. Chiang, A and Million, RP (2011). Personalized medicine in oncology: next generation. Nat Rev Drug Discov 10: 895–896. 23. Schilsky, RL (2010). Personalized medicine in oncology: the future is now. Nat Rev Drug Discov 9: 363–366. 24. Desai, N (2012). Challenges in development of nanoparticle-based therapeutics. AAPS J 14: 282–295. 25. Zolnik, BS and Sadrieh, N (2009). Regulatory perspective on the importance of ADME assessment of nanoscale material containing drugs. Adv Drug Deliv Rev 61: 422–427.
Large Stem Cell–Derived Cardiomyocyte Grafts: Cellular Ventricular Assist Devices? Sian E Harding1 doi:10.1038/mt.2014.97
W
hile the battle rages on about the endogenous repair capacity in the heart, solid work is progressing to take the promising exogenous pluripotent stem cells toward a clinical application. We
Imperial College, London, UK Correspondence: Sian E Harding, NHLI, Imperial College, ICTEM, Hammersmith Campus, Du Cane Road, London W12 0NN, UK. E-mail: [email protected] 1
know that the human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) can reliably be differentiated into cardiac myocytes; protocols are being defined, refined, and scaled up continuously. However, the hard grind and expense of producing the bulk of cardiomyocytes required, delivering them in a way that enables retention and using a clinically relevant model, is demanding and, arguably, unglamorous work. Certainly, Geron appears to have held that www.moleculartherapy.org vol. 22 no. 7 july 2014
© The American Society of Gene & Cell Therapy
opinion when it abandoned the effort as unprofitable in 2011. In their recent Nature paper,1 Murry and Laflamme’s group report important progress toward this goal by producing significant quantities of muscle in the hearts of macaque monkeys using hESC-derived cardiomyocytes (hESC-CMs). Although this is not a definitive study in that the small numbers of animals did not allow statistical analysis of functional outcomes, the authors made several key observations. First, it was logistically possible to introduce the hESC-CMs via the relatively straightforward method of administering multiple intramyocardial injections and to produce a functionally relevant amount of tissue. The issue of cell loss was addressed simply by massively increasing the number of hESCCMs used (to more than 1 billion cells), and the authors were able to scale up their processes sufficiently to do this with the aid of cryopreservation protocols. They demonstrated coupling to the host heart in terms of both cardiomyocyte excitation–contraction coupling and infiltration of the host vasculature to support the graft. Less encouragingly, they detected arrhythmias in the presence of the graft; however, this is not an unexpected event, and the nature and trajectory of the arrhythmias observed give important information that can lead to a more measured response than simply rejecting the entire hESC-CM strategy. A central feature of the study was the choice of the monkey as a model; excitation–contraction coupling varies widely between species, and the use of rodents (with heart rates 5- to 10-fold higher than humans) to assess integration of human stem cell–derived cardiomyocytes has always been a concern. Reports of grafts being kept isolated from surrounding mouse myocardium by encapsulating layers of fibrous tissue may represent the death of those human cardiomyocytes that did couple to the host.2 One of the main aims of grafting experiments is to detect potential arrhythmic events so as to study their underlying mechanisms. Arrhythmias may arise from spontaneous pacemaker activity from the graft, poor coupling of host/graft cells, or mismatched properties due to immaturity of hESC-CMs or species differences. In this Molecular Therapy vol. 22 no. 7 july 2014
commentary study, the elegant use of an hESC line engineered to express a calcium reporter clearly showed the coupling of graft hESCCMs to the host heart. To pinpoint the contribution from the known immaturity of the hESC-CMs, it is necessary to reduce the differences attributable to species incompatibility. Nonhuman primates are the most appropriate choice for this—and were vital before the transition to humans. Murry and Laflamme’s team previously used an intermediate model, the guinea pig, which has an action-potential morphology and calcium-cycling properties more similar than those of the rat/mouse to human heart.3 Notably, this model did not display the same postgrafting arrhythmic tendencies that the primate model did. This difference was ascribed to the greater possibility for arrhythmia-generating re-entrant circuits in larger hearts. Another difference is that, for the guinea pig, the model of disease used was cryoinjury, which produces a discrete area of tissue death. The guinea pig has a high degree of collateral circulation, so tying off a vessel to mimic the occluding event in human does not produce an infarct. This model in the primate was produced by transient occlusion and so was closer to the clinical situation, producing a scar that (as in humans) has residual transmural islands of cardiomyocyte tissue. This underlines the importance of matching not only the species but the pathology to produce a true model of clinical events. The choice of embryonic over induced pluripotent stem cells (hiPSCs) might be considered scientifically less than ideal as well as unnecessarily contentious from an ethical standpoint, especially when combined with nonhuman primate studies. However, this group’s long experience with the hESC lines in question, the assurance of genetic stability, and the work that went into differentiation protocols are powerful arguments supporting these cell lines for first proof-of-concept studies. Once differentiated, hESC-CMs and hiPSC-CMs share an immature phenotype, so the use of hESC-CMs is likely to be a good predictor for both cell types. Advantages of autologous use of hiPSCs in terms of overcoming immune rejection remain theoretical (and contested), and there is anecdotal experience that differentiation
protocols have more variable outcomes on hiPSC lines. Looking to the future, it would seem more likely that the hiPSC would be the final product for clinical use, but in fact clinical trials for spinal cord injury and retinal degeneration are already proceeding with hESCs.4,5 It is further notable for this study that, although the hESC-CMs were pretreated briefly with a prosurvival cocktail, no attempt was made to improve their maturity. This may reflect the sheer difficulty of applying to these enormous quantities of cells the various proposed maturation strategies, such as aging, transfection, or development of three-dimensional constructs. Alternatively, it might be taking advantage of the immature phenotype that confers a resistance to the initially hypoxic conditions of the graft before vascularization has occurred. An interesting side note is the authors’ mention that tissue-engineered patches were used in different areas at the same time that the dissociated hESC-CMs were injected but were less successful. The possibility of comparing multiple strategies within an experiment is a powerful advantage of the use of large hearts. Is the occurrence of arrhythmia an insuperable barrier to progression toward the clinic? The evidence collected by Murry and Laflamme’s team suggests that, even with the best species match and the appropriate disease model, intrinsic properties of the graft will produce arrhythmic events. Indeed, it is difficult to imagine that introducing such a large amount of active beating tissue to the host heart would not have consequences. It is important to note, however, that none of these events was fatal. Unlike the previous experience with skeletal myoblasts,6 there is at least the potential for hESCCMs or hiPSC-CMs to mature toward an adult cardiomyocyte phenotype in the in vivo environment. There are indications in this study that maturation had begun to occur, and arrhythmias declined toward the end of the 3-month study period. One key goal arising from the study for the scientific community could be the development of strategies to mitigate arrhythmic events during the initial period of vulnerability while the hESC-CMs or hiPSC-CMs mature in vivo. From the clinical side, protection of a patient from 1241
© The American Society of Gene & Cell Therapy
commentary arrhythmia or other adverse events is increasingly being managed by implantable devices. The patient population chosen for the first human trials might be patients with implanted ventricular assist devices (VADs), in which the indwelling pumps remove load from the heart and preserve blood flow despite poor myocardial function and arrhythmic events. Interestingly, the presence of VADs can result in some improvement of the host myocardium.7,8 We might even think of the large cell grafts, as used in this
1242
study, as cellular VADs, replacing metal and plastic with living tissue and both providing new muscle and promoting recovery of the old and damaged heart. REFERENCES 1.
2.
3.
Chong, JJ, Yang, X, Don, CW, Minami, E, Liu, YW, Weyers, JJ et al. (2014). Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature, e-pub ahead of print 30 April 2014. van Laake, LW, Passier, R, Doevendans, PA and Mummery, CL (2008). Human embryonic stem cell–derived cardiomyocytes and cardiac repair in rodents. Circ Res 102:1008–1010. Shiba, Y, Fernandes, S, Zhu, WZ, Filice, D, Muskheli, V, Kim, J et al. (2012). Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 489: 322–325.
4.
5.
6. 7.
8.
Bretzner, F, Gilbert, F, Baylis, F and Brownstone, RM (2011). Target populations for first-in-human embryonic stem cell research in spinal cord injury. Cell Stem Cell 8: 468–475. Schwartz, SD, Hubschman, JP, Heilwell, G, FrancoCardenas, V, Pan, CK, Ostrick, RM et al. (2012). Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet 379: 713–720. Murry, CE, Field, LJ and Menasche, P (2005). Cellbased cardiac repair: reflections at the 10-year point. Circulation 112: 3174–3183. Heerdt, PM, Holmes, JW, Cai, B, Barbone, A, Madigan, JD, Reiken, S et al. (2001). Chronic unloading by left ventricular assist device reverses contractile dysfunction and alters gene expression in end-stage heart failure. Circulation 102: 2713–2719. Birks, EJ, Tansley, PD, Hardy, J, George, RS, Bowles, CT, Burke, M et al. (2006). Left ventricular assist device and drug therapy for the reversal of heart failure. N Engl J Med 355: 1873–1884.
www.moleculartherapy.org vol. 22 no. 7 july 2014
commentary treated with concomitant administration of drug-loaded NPs than the concentration of both drugs from the nano-polypharmacy formulation, the appropriate drug ratio was not maintained. The differential delivery of drug to the tumor when coadministering NP drug formulations is perhaps due to the differential pharmaco kinetics of NPs loaded with rapamycin versus paclitaxel. Panagi et al.21 have demonstrated that the administration dose of poly (lacticco-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) NPs significantly affects their biodistribution. Because the ratio of the drugs is maintained during the coadministration of the drug-loaded NPs, the dose of each NP formulation differs and may affect the accumulation of each NP drug-loaded formulation into the tumor. There was significantly greater tumor regression by the nano-polypharmacy formulation than with the mixture of NP formulations loaded with rapamycin and paclitaxel. In addition, there was significantly greater tumor regression for nano-polypharmacy formulations delivering a rapamycin-to-paclitaxel ratio of 3:1 than with a ratio of 2:1, as predicted by the in vitro results. Further pharmacological studies are expected to be of use in determining cytotoxicity in healthy organs—one of the main challenges associated with free-drug combination treatments. NPs delivering drug combinations could also help to overcome challenges associated with drug resistance. High levels of p-Akt in breast cancer patients treated with tamoxifen are associated with reduced survival time. Additional studies using cell lines resistant to rapamycin could provide a better understanding of the potential of nano-polypharmacy to overcome drug resistance. It is possible that the drug combination ratio for cell sensitivity and cell resistance may differ—a possibility that will support personalized therapy using nanopolypharmacy formulations.22,23 It is unclear, however, whether nano-polypharmacy will be useful for drug combinations that do not require a very precise drug ratio concentration for synergistic efficacy. Although coencapsulation of drugs into nanomaterials is expected to progress from preclinical to clinical studies, regulatory issues must also be addressed. However, it is not expected that regulatory challenges will be significantly greater than for single-drug delivery nanomaterials,24,25 in that the review process 1240
is similar to that for other nanomaterials except that combination products are regulated by the Office of Combination Products at the FDA. REFERENCES 1.
Borisy, AA, Elliott, PJ, Hurst, NW, Lee, MS, Lehar, J, Price, ER et al. (2003). Systematic discovery of multicomponent therapeutics. Proc Natl Acad Sci USA 100: 7977–7982. 2. Al-Lazikani, B, Banerji, U and Workman, P (2012). Combinatorial drug therapy for cancer in the postgenomic era. Nat Biotechnol 30: 679–692. 3. Shah, PS and Schaffer, DV (2010). Gene therapy takes a cue from HAART: combinatorial antiviral therapeutics reach the clinic. Sci Transl Med 2: 36ps30. 4. Aliabadi, HM, Mahdipoor, P and Uludag, H (2013). Response of drug sensitive and resistant breast cancer cells to combinatorial siRNA therapy. Mol Ther 21: S82–S83. 5. Jia, J, Zhu, F, Ma, X, Cao, Z, Li, Y and Chen, YZ (2009). Mechanisms of drug combinations: interaction and network perspectives. Nat Rev Drug Discov 8: 111–128. 6. Keith, CT, Borisy, AA and Stockwell, BR (2005). Multicomponent therapeutics for networked systems. Nat Rev Drug Discov 4: 71–78. 7. Dancey, JE and Chen, HX (2006). Strategies for optimizing combinations of molecularly targeted anticancer agents. Nat Rev Drug Discov 5: 649–659. 8. Blanco, E, Sangai, T, Wu, S, Hsiao, A, Ruiz-Esparza, GU, Gonzalez-Delgado, CA et al. (2014). Colocalized delivery of rapamycin and paclitaxel to tumors enhances synergistic targeting of the PI3K/Akt/mTOR pathway. Mol Ther 22: 1310–1319. 9. Faivre, S, Kroemer, G and Raymond, E (2006). Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 5: 671–688. 10. Alexis, F, Rhee, JW, Richie, JP, Radovic-Moreno, AF, Langer, R and Farokhzad, OC (2008). New frontiers in nanotechnology for cancer treatment. Urol Oncol 26: 74–85. 11. Sprintz, M (2004). Nanotechnology for advanced therapy and diagnosis. Biomed Microdevices 6: 101–103.
12. Wang, X, Yang, L, Chen, ZG and Shin, DM (2008). Application of nanotechnology in cancer therapy and imaging. CA Cancer J Clin 58: 97–110. 13. Davis, SS (1997). Biomedical applications of nanotechnology—implications for drug targeting and gene therapy. Trends Biotechnol 15: 217–224. 14. Farokhzad, OC (2008). Nanotechnology for drug delivery: the perfect partnership. Expert Opin Drug Deliv 5: 927–929. 15. Alexis, F, Pridgen, E, Molnar, LK and Farokhzad, OC (2008). Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 5: 505–515. 16. Peer, D, Karp, JM, Hong, S, Farokhzad, OC, Margalit, R and Langer, R (2007). Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2: 751–760. 17. Alexis, F, Pridgen, E, Langer, R and Farokhzad, O (2010). Nanoparticle technologies for cancer therapy. In: Schäfer-Korting, M (ed) Drug Delivery (Handbook of Experimental Pharmacology, vol 197). Springer: Berlin, Germany. pp 55–86. 18. Davis, ME, Chen, Z and Shin, DM (2008). Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat Rev Drug Discov 7: 771–782. 19. Ferrari, M (2005). Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer 5: 161–171. 20. Petros, RA and DeSimone, JM (2010). Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 9: 615–627. 21. Panagi, Z, Beletsi, A, Evangelatos, G, Livaniou, E, Ithakissios, DS and Avgoustakis, K (2001). Effect of dose on the biodistribution and pharmacokinetics of PLGA and PLGA–mPEG nanoparticles. Int J Pharmaceutics 221: 143–152. 22. Chiang, A and Million, RP (2011). Personalized medicine in oncology: next generation. Nat Rev Drug Discov 10: 895–896. 23. Schilsky, RL (2010). Personalized medicine in oncology: the future is now. Nat Rev Drug Discov 9: 363–366. 24. Desai, N (2012). Challenges in development of nanoparticle-based therapeutics. AAPS J 14: 282–295. 25. Zolnik, BS and Sadrieh, N (2009). Regulatory perspective on the importance of ADME assessment of nanoscale material containing drugs. Adv Drug Deliv Rev 61: 422–427.
Large Stem Cell–Derived Cardiomyocyte Grafts: Cellular Ventricular Assist Devices? Sian E Harding1 doi:10.1038/mt.2014.97
W
hile the battle rages on about the endogenous repair capacity in the heart, solid work is progressing to take the promising exogenous pluripotent stem cells toward a clinical application. We
Imperial College, London, UK Correspondence: Sian E Harding, NHLI, Imperial College, ICTEM, Hammersmith Campus, Du Cane Road, London W12 0NN, UK. E-mail: [email protected] 1
know that the human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) can reliably be differentiated into cardiac myocytes; protocols are being defined, refined, and scaled up continuously. However, the hard grind and expense of producing the bulk of cardiomyocytes required, delivering them in a way that enables retention and using a clinically relevant model, is demanding and, arguably, unglamorous work. Certainly, Geron appears to have held that www.moleculartherapy.org vol. 22 no. 7 july 2014
© The American Society of Gene & Cell Therapy
opinion when it abandoned the effort as unprofitable in 2011. In their recent Nature paper,1 Murry and Laflamme’s group report important progress toward this goal by producing significant quantities of muscle in the hearts of macaque monkeys using hESC-derived cardiomyocytes (hESC-CMs). Although this is not a definitive study in that the small numbers of animals did not allow statistical analysis of functional outcomes, the authors made several key observations. First, it was logistically possible to introduce the hESC-CMs via the relatively straightforward method of administering multiple intramyocardial injections and to produce a functionally relevant amount of tissue. The issue of cell loss was addressed simply by massively increasing the number of hESCCMs used (to more than 1 billion cells), and the authors were able to scale up their processes sufficiently to do this with the aid of cryopreservation protocols. They demonstrated coupling to the host heart in terms of both cardiomyocyte excitation–contraction coupling and infiltration of the host vasculature to support the graft. Less encouragingly, they detected arrhythmias in the presence of the graft; however, this is not an unexpected event, and the nature and trajectory of the arrhythmias observed give important information that can lead to a more measured response than simply rejecting the entire hESC-CM strategy. A central feature of the study was the choice of the monkey as a model; excitation–contraction coupling varies widely between species, and the use of rodents (with heart rates 5- to 10-fold higher than humans) to assess integration of human stem cell–derived cardiomyocytes has always been a concern. Reports of grafts being kept isolated from surrounding mouse myocardium by encapsulating layers of fibrous tissue may represent the death of those human cardiomyocytes that did couple to the host.2 One of the main aims of grafting experiments is to detect potential arrhythmic events so as to study their underlying mechanisms. Arrhythmias may arise from spontaneous pacemaker activity from the graft, poor coupling of host/graft cells, or mismatched properties due to immaturity of hESC-CMs or species differences. In this Molecular Therapy vol. 22 no. 7 july 2014
commentary study, the elegant use of an hESC line engineered to express a calcium reporter clearly showed the coupling of graft hESCCMs to the host heart. To pinpoint the contribution from the known immaturity of the hESC-CMs, it is necessary to reduce the differences attributable to species incompatibility. Nonhuman primates are the most appropriate choice for this—and were vital before the transition to humans. Murry and Laflamme’s team previously used an intermediate model, the guinea pig, which has an action-potential morphology and calcium-cycling properties more similar than those of the rat/mouse to human heart.3 Notably, this model did not display the same postgrafting arrhythmic tendencies that the primate model did. This difference was ascribed to the greater possibility for arrhythmia-generating re-entrant circuits in larger hearts. Another difference is that, for the guinea pig, the model of disease used was cryoinjury, which produces a discrete area of tissue death. The guinea pig has a high degree of collateral circulation, so tying off a vessel to mimic the occluding event in human does not produce an infarct. This model in the primate was produced by transient occlusion and so was closer to the clinical situation, producing a scar that (as in humans) has residual transmural islands of cardiomyocyte tissue. This underlines the importance of matching not only the species but the pathology to produce a true model of clinical events. The choice of embryonic over induced pluripotent stem cells (hiPSCs) might be considered scientifically less than ideal as well as unnecessarily contentious from an ethical standpoint, especially when combined with nonhuman primate studies. However, this group’s long experience with the hESC lines in question, the assurance of genetic stability, and the work that went into differentiation protocols are powerful arguments supporting these cell lines for first proof-of-concept studies. Once differentiated, hESC-CMs and hiPSC-CMs share an immature phenotype, so the use of hESC-CMs is likely to be a good predictor for both cell types. Advantages of autologous use of hiPSCs in terms of overcoming immune rejection remain theoretical (and contested), and there is anecdotal experience that differentiation
protocols have more variable outcomes on hiPSC lines. Looking to the future, it would seem more likely that the hiPSC would be the final product for clinical use, but in fact clinical trials for spinal cord injury and retinal degeneration are already proceeding with hESCs.4,5 It is further notable for this study that, although the hESC-CMs were pretreated briefly with a prosurvival cocktail, no attempt was made to improve their maturity. This may reflect the sheer difficulty of applying to these enormous quantities of cells the various proposed maturation strategies, such as aging, transfection, or development of three-dimensional constructs. Alternatively, it might be taking advantage of the immature phenotype that confers a resistance to the initially hypoxic conditions of the graft before vascularization has occurred. An interesting side note is the authors’ mention that tissue-engineered patches were used in different areas at the same time that the dissociated hESC-CMs were injected but were less successful. The possibility of comparing multiple strategies within an experiment is a powerful advantage of the use of large hearts. Is the occurrence of arrhythmia an insuperable barrier to progression toward the clinic? The evidence collected by Murry and Laflamme’s team suggests that, even with the best species match and the appropriate disease model, intrinsic properties of the graft will produce arrhythmic events. Indeed, it is difficult to imagine that introducing such a large amount of active beating tissue to the host heart would not have consequences. It is important to note, however, that none of these events was fatal. Unlike the previous experience with skeletal myoblasts,6 there is at least the potential for hESCCMs or hiPSC-CMs to mature toward an adult cardiomyocyte phenotype in the in vivo environment. There are indications in this study that maturation had begun to occur, and arrhythmias declined toward the end of the 3-month study period. One key goal arising from the study for the scientific community could be the development of strategies to mitigate arrhythmic events during the initial period of vulnerability while the hESC-CMs or hiPSC-CMs mature in vivo. From the clinical side, protection of a patient from 1241
© The American Society of Gene & Cell Therapy
commentary arrhythmia or other adverse events is increasingly being managed by implantable devices. The patient population chosen for the first human trials might be patients with implanted ventricular assist devices (VADs), in which the indwelling pumps remove load from the heart and preserve blood flow despite poor myocardial function and arrhythmic events. Interestingly, the presence of VADs can result in some improvement of the host myocardium.7,8 We might even think of the large cell grafts, as used in this
1242
study, as cellular VADs, replacing metal and plastic with living tissue and both providing new muscle and promoting recovery of the old and damaged heart. REFERENCES 1.
2.
3.
Chong, JJ, Yang, X, Don, CW, Minami, E, Liu, YW, Weyers, JJ et al. (2014). Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature, e-pub ahead of print 30 April 2014. van Laake, LW, Passier, R, Doevendans, PA and Mummery, CL (2008). Human embryonic stem cell–derived cardiomyocytes and cardiac repair in rodents. Circ Res 102:1008–1010. Shiba, Y, Fernandes, S, Zhu, WZ, Filice, D, Muskheli, V, Kim, J et al. (2012). Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 489: 322–325.
4.
5.
6. 7.
8.
Bretzner, F, Gilbert, F, Baylis, F and Brownstone, RM (2011). Target populations for first-in-human embryonic stem cell research in spinal cord injury. Cell Stem Cell 8: 468–475. Schwartz, SD, Hubschman, JP, Heilwell, G, FrancoCardenas, V, Pan, CK, Ostrick, RM et al. (2012). Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet 379: 713–720. Murry, CE, Field, LJ and Menasche, P (2005). Cellbased cardiac repair: reflections at the 10-year point. Circulation 112: 3174–3183. Heerdt, PM, Holmes, JW, Cai, B, Barbone, A, Madigan, JD, Reiken, S et al. (2001). Chronic unloading by left ventricular assist device reverses contractile dysfunction and alters gene expression in end-stage heart failure. Circulation 102: 2713–2719. Birks, EJ, Tansley, PD, Hardy, J, George, RS, Bowles, CT, Burke, M et al. (2006). Left ventricular assist device and drug therapy for the reversal of heart failure. N Engl J Med 355: 1873–1884.
www.moleculartherapy.org vol. 22 no. 7 july 2014
