Clinical Translation of Pluripotent Stem Cell Therapies: Challenges and Considerations

Cell Stem Cell

Review Clinical Translation of Pluripotent Stem Cell Therapies: Challenges and Considerations Manon Desgres1 and Philippe Menasche´1,2,* 1Universite ´

de Paris, PARCC, INSERM, 75015 Paris, France of Cardiovascular Surgery, Hoˆpital Europe´en Georges Pompidou 20, rue Leblanc, 75015 Paris, France *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2019.10.001 2Department

Although the clinical outcomes of cell therapy trials have not met initial expectations, emerging evidence suggests that injury-mediated tissue damage might benefit from the delivery of cells or their secreted products. Pluripotent stem cells (PSCs) are promising cell sources primarily because of their capacity to generate stage- and lineage-specific differentiated derivatives. However, they carry inherent challenges for safe and efficacious clinical translation. This Review describes completed or ongoing trials of PSCs, discusses their potential mechanisms of action, and considers how to address the challenges required for them to become a major therapy, using heart repair as a case study. Whereas the treatment of most diseases has long relied on two main pillars, i.e., drugs and interventional procedures, biotherapies have emerged as a potential third player over the past two decades. Among them, cells (which do not always fulfill the criterion of ‘‘stemness’’ despite this common way of naming them) are receiving a continued interest. This is largely because they feature a major disruption from conventional therapies in that they do not only aim at relieving symptoms but address the root cause of the disease by targeting the repair and at most the regeneration of the damaged tissue. Historically, cells from adult tissue sources have been the most commonly tested in clinical trials encompassing almost all kinds of diseases. However, the recognition of their drawbacks, such as limited differentiation potential and poor scalability, has led the field to increasingly consider more immature cells, particularly pluripotent stem cells (PSCs) as attractive alternates. Indeed, the therapeutic interest of these cells is not so much their theoretically unlimited availability or scalability (as these properties can be shared by some adult cells such as mesenchymal stromal cells [MSCs]) but rather lies on the unique possibility to leverage their intrinsic pluripotentiality to drive them toward any cell type, provided they receive the appropriate lineage-specific cues and to control this differentiation process by way of ‘‘freezing’’ it at the desired stage (early progenitors or more mature cells)—all properties that adult cells lack. In this article, we will review the main PSC trials and discuss the safety challenges they may raise. ‘‘Tissue repair’’ for heart failure will be taken as a case study since it illustrates most of these challenges and allows discussion of some of the solutions based on the presumed mechanism of action of the transplanted cells. Human PSC Clinical Trials: Overview of the Current Landscape Chronologically, the first PSCs to enter the clinical arena have targeted traumatic spinal cord injury. The program was initiated by Geron Corporation, which launched a phase I trial of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells in October 2010 but stopped it 1 year later when it shifted 594 Cell Stem Cell 25, November 7, 2019 ª 2019 Elsevier Inc.

focus toward cancer therapeutics. The technology assets were then acquired by Asterias Biotherapeutics, which subsequently initiated the SciStar trial in which 35 patients with subacute cervical spinal cord injury were transplanted with the same hESCderived progenitor cells according to a dose-escalating regimen (up to 20 3 106 cells). In January, 2019, the company reported 12-month results in 25 patients showing the absence of safety issues and encouraging improvements in upper extremity motor function. The second organ that has been targeted by hESC-differentiated derivatives is the eye, which features several attractive characteristics for stem cell therapy, including easy access, presumed immune privilege, requirements for low dosages, and high compartmentalization reducing the risk of systemic spread. Age-related macular degeneration (AMD) has been so far the primary indication. The first trial (Schwartz et al., 2015) enrolled 18 patients (9 with AMD and 9 with Stargardt’s macular dystrophy) who were followed up for 22 months after subretinal transplantation of increasing doses of hESC-derived retinal pigment epithelial (RPE) cells (from 50,000 to 150,000). The only adverse events were related to surgery and immunosuppression, but improvements in visual acuity were observed in more than half of the treated eyes. A similar trend was reported in a smaller series of 4 patients (Song et al., 2015), while another trial including 12 patients with advanced Stargardt’s disease failed to show a significant benefit on retinal function at 12 months, possibly because of the severity of established retinal degeneration (Mehat et al., 2018). In contrast, a visual acuity gain was recently reported in two small-sized trials in which injections of cell suspensions were replaced by transplantation of a bioengineered construct whereby hESC-derived RPE were seeded onto a polyester (da Cruz et al., 2018) or a parylene scaffold (Kashani et al., 2018) mimicking a Bruch’s membrane. This delivery modality may have contributed to a more successful outcome by preventing a de-differentiation of RPE cells that may occur when they are dissociated and requires their challenging re-differentiation in vivo to form a functional monolayer (Carr et al., 2013). As shown in Table S1, several other trials are currently enrolling patients, and their outcomes will allow us to better define the place

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Review of PSC-derived RPE in comparison with other cell types (Higuchi et al., 2017). The brain of patients with Parkinson’s disease is another potential indication for PSC derivatives because of their ability to be differentiated into midbrain dopaminergic neurons and the capacity of these cells, following intracerebral transplantation, to rescue motor behavior deficits in animal models of the disease (Sonntag et al., 2018), including those involving nonhuman primates (Kikuchi et al., 2017). Whether these encouraging preclinical data can translate into similar improvements in patients is being currently tested in a Chinese trial (Table S1). Likewise, a deeper understanding of the signaling pathways driving the endodermal layer toward the generation of pancreatic islet cells has allowed the in vitro recapitulation of this stepwise process and paved the way for a diabetes treatment based on the use of beta-like cells differentiated from human PSCs (hPSCs) (Sneddon et al., 2018). To mitigate their rejection and reduce (or at most eliminate) the need for immunosuppressive drugs, these cells have usually been encapsulated in alginatebased microparticles with the premise that the capsule membrane would protect the cells from the host’s immune system while allowing an influx of oxygen and nutrients and an appropriate release of insulin. A major limitation of this encapsulation strategy has been a foreign-body reaction and a subsequent fibrosis around the capsules limiting the long-term function of the graft. Efforts have thus been made to optimize the formulation of the alginate particles (Strand et al., 2017). In August 2014, the company ViaCyte launched the STEP ONE* trial (NCT02239354) for evaluating the safety and efficacy of ViaCyte’s PEC-Encap in type I diabetes. The product consists of pancreatic progenitor cells delivered in a subcutaneously implanted and retrievable immune-encapsulation device (Encaptra). In June, 2018, the company reported encouraging 2-year outcomes in 19 patients with no safety issues, no evidence for allo-immunization to donor cells in the absence of immunosuppression, and differentiation into insulin-producing beta cells and glucagon-producing alpha cells in several explants. The delivery system is now undergoing changes to enhance long-term cell engraftment, and the company hopes to resume the STEP ONE trial enrolment in 2019. While islet survival may be compromised by their destruction by endogenous immune cells, hypovascularization of the graft can further contribute to hamper their sustained therapeutic benefit. Efforts have thus been made to enhance microvascularization of the encased islets, which has translated clinically into two different approaches. In 2017, ViaCyte has started the 55-patient PEC-Direct trial (NCT03163511) in which the company’s ESC-derived pancreatic progenitors are delivered in a device designed to allow their direct vascularization (Table S1). A potential drawback of the open design of this device is that the patients need to be immunosuppressed. Alternatively, Sernova has developed a cell pouch that is implanted subcutaneously to enable the creation of a vascularized environment intended to nurture islets, which are then transplanted in the pouch 2–12 weeks later. A trial testing this concept (NCT01652911) has included 3 patients and is now terminated, but no outcome data have been reported yet. A subsequent phase I/II nonrandomized, unblinded, singlearm study was announced in May 2018. A last challenge is that, in vivo, beta cells do not exist isolated but clustered within three-

dimensional niches also harboring nonendocrine vascular cells and extracellular matrix components, hence the possible interest of an in vitro co-aggregation of allogeneic (human) islet cells with MSCs, which has resulted in their engraftment in the omentum of immunocompetent mice and allowed glycemic control without immunosuppression (Westenfelder et al., 2017). In the case of heart failure, the first cells that have been implanted clinically in the myocardium have been noncardiac, i.e., skeletal myoblasts and bone-marrow-derived cells. Although they have globally failed to yield clinically meaningful improvements in patient outcomes (Menasche´ et al., 2018; Fisher et al., 2016), they have fuelled a burgeoning multifaceted area of research. Currently, however, only CD34-positive (Vrtovec, 2018) and, to a greater extent, mesenchymal cells (MSCs) continue to generate a clinical interest. Whereas the angiogeneic properties of the former may qualify them for treating refractory angina (Henry et al., 2018), the latter are attractive in the context of heart failure because of their alleged angiogeneic, anti-inflammatory, and immunomodulatory properties. Indeed, by March 2019, entering the keywords ‘‘chronic heart failure’’ and ‘‘mesenchymal stromal cells’’ in the ClinicalTrials. gov website retrieved 24 trials, among which the event-driven DREAM-HF study generates a strong interest because of its large sample size (566 patients randomized to receive allogeneic MSCs or placebo). The last patient has recently been dosed, and trial outcomes might become available within the next 12 months. Despite the interest in strategies employing noncardiac cell therapy, an important output of research in the area of cardiac repair has been the recognition that better outcomes might be achieved with cells phenotypically close to those of the target organ. This gave rise to the second generation of clinical trials entailing the use of cardiac-committed cells. Expectedly, the first ones to enter the clinical arena have used adult sources, but, so far, they have equally failed to result in substantial clinical benefits. The CHART-1 trial, which randomized 315 patients, of whom 157 received transendocardial injections of autologous MSCs engineered in vitro to express cardiac transcription factors, missed its primary endpoint (all-cause mortality, worsening heart failure, Minnesota Living with Heart Failure Questionnaire score, 6-min walk distance, left ventricular end-systolic volume, and ejection fraction at 39 weeks) (Bartunek et al., 2017) even though a post hoc data analysis provided evidence for reverse remodeling in a subset of patients (Teerlink et al., 2017). The ALLSTAR trial tested cells derived from cardiospheres, which are composed of several cell types, predominantly MSCs, harvested from the right ventricle by an endomyocardial biopsy (Smith et al., 2007); this trial was prematurely terminated in April 2017 for futility. A third small-sized study (SCIPIO) tested c-kit+ ‘‘cardiac stem cells’’ grown from a right atrial biopsy taken during a coronary artery bypass grafting operation and injected (at the very low dose of 1 million) into the coronary arteries an average of 113 days later in 16 patients (while 7 served as controls). The authors’ initial enthusiastic claims (Bolli et al., 2011; Chugh et al., 2012) are seriously questioned by the recent retraction by Harvard Medical School of 31 papers, which had overall rationalized the use of these cells (Science, 2018), now largely recognized as unable to give rise to cardiomyocytes (van Berlo et al., 2014). Cell Stem Cell 25, November 7, 2019 595

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Review In parallel, other groups, including ours, have taken a different approach consisting of starting from PSCs, in practice hESCs, and coaxing them to generate cardiac cells closer to the native ones, either at a progenitor state (Bellamy et al., 2015) or more fully differentiated into cardiomyocytes (Liu et al., 2018). Thus, in our clinical study (ESCORT, NCT02057900), we have used early-committed Isl-1+ progenitors embedded in a fibrin gel, which was then delivered onto the epicardium of the infarct area during a coronary artery bypass grafting in 6 patients with severe left ventricular dysfunction (Menasche´ et al., 2018). Aside from demonstrating that generation of such a composite construct was doable under Good Manufacturing Practice (GMP) standards, our trial successfully met its primary safety endpoint in that, with a maximal follow-up of 4 years and 5 months in March 2019, none of the patients has presented complications that could be specifically ascribed to the cells (arrhythmias, tumor, clinically relevant allo-immunization). By virtue of its design, the trial cannot answer the question of efficacy and the improved systolic thickening of the cell-treated (and nonrevascularized) myocardial areas seen in some patients can, at most, be considered as an encouraging hint. Other trials using hESC-derived more differentiated cardiomyocytes are currently in preparation in the United States and Canada. While ESCs have been the first PSC to be tested clinically and continue to be evaluated across a wide range of diseases (summarized in Table S1), they are now followed by the use of induced pluripotent stem cells (iPSCs). The first trial, initiated in Japan, used iPSC-differentiated RPE derivatives for treating macular degeneration. The study, however, was stopped after one patient had been injected because of concerns about genetic changes in the cells prepared for the second one (Mandai et al., 2017). This has led the investigators to redesign the protocol and switch to the use of allogeneic banked iPSCs, which have then successfully passed all quality controls. So far, one patient has been transplanted and there are 4 more to come. Another trial testing allogeneic iPSC-derived dopamine precursor cells in patients with Parkinson’s disease was started in October 2018, and six more patients are planned for inclusion (Cyranoski, 2018), while two additional studies in spinal cord injury (Cyranoski, 2018) and corneal diseases (Cyranoski, 2019) have been approved (these three trials in Japan). Heart failure is also targeted by iPSC therapies and two trials are in preparation, one in Japan and the other in Germany, in which iPSCderived cardiomyocytes will be transplanted in combination with a scaffold through a limited surgical approach. A third trial (HEAL-CHF), conducted in China, is already registered (NCT 03763136) and should include 5 patients with heart failure receiving intramyocardial injections of allogeneic iPSCs at the time of coronary artery bypass grafting. It is noteworthy that for practical and economic reasons, the initially claimed advantage of iPSCs over ESCs, i.e., the possibility of using them in an autologous fashion, has been abandoned in favor of allogeneic banked iPSCs. The balance between the advantages (consistency of the final cell product, streamlined logistics, and lower costs) and drawbacks (rejection) of this allogeneic strategy will be discussed in another section of this article. To summarize, taken together, all these cell therapy trials have primarily demonstrated the safety of the procedure (5 patients of the initial spinal cord injury trial have now reached 8 years of 596 Cell Stem Cell 25, November 7, 2019

follow-up without reporting serious adverse events), while few of them have yet been designed and powered to really show efficacy. Although the interpretation of their results is further complicated by the diversity of patient disease, cell processing protocols, dosing, timing and route of delivery, duration of follow-up, and outcome measures, some encouraging hints are emerging, which makes credible that PSC-derived differentiated progenies or their secreted products (see below) can find a place within the armamentarium of therapies against a variety of life-threatening or functionally invalidating human diseases. Of note, the outcome metrics of current and upcoming PSC trials will have to be analyzed in light of those of the competing therapies, which are also extensively investigated for treating the same disorders such as gene therapy for ocular diseases (Ramlogan-Steel et al., 2018), automated insulin delivery devices for diabetes (Latres et al., 2019), or direct intracerebral administration of growth factors for Parkinson’s disease (Whone et al., 2019). Delivery Issues Cells are usually injected directly into the target organ, but this procedure can damage both the host tissue and the cells themselves as they incur a dramatic increase in flow velocity and shear stresses as they flow from the syringe to the small bore needle (Aguado et al., 2012). In addition, unlike fluids, cells in suspension tend to sediment in a time-dependent manner, which may induce a density gradient leading to uneven cell dispersion (and thus variable cell dosing) when multiple injections are performed from the same syringe with the highest cell densities during the first injection (Potts et al., 2013). Efforts are thus made to improve the accuracy of targeting while minimizing its invasiveness. Typical examples are the use of MRI to provide real-time imaging for stereotactic procedures and of a radially branched deployment device for intra-cerebral (Potts et al., 2013) or the development of a device for delivering RPE without creating a hole in the retina (Orbit Biomedical). In the heart, the main routes for cell injections have usually been trans-epicardial, trans-endocardial, or intra-coronary. Even though the direct intra-myocardial approach seems the most effective (Hou et al., 2005), it is still fraught with a poor retention rate. This likely hinders a successful therapeutic outcome because, as supported by the experimental (Levit et al., 2013) and clinical (Vrtovec et al., 2013) links between cell retention and functional improvements. Even though early retention of cells or cell-derived biologics can be improved by dedicated catheters (Tabei et al., 2019) or combination with biomaterials (as discussed below), the above-mentioned routes of administration share in common several limitations: they require dedicated facilities and highly trained staff, they are costly, and, more importantly, they are invasive and, as such, cannot be readily repeated. This could have contributed to the failure of many clinical trials entailing a single shot of cells, an assumption supported by the finding that repeated administrations of transplanted cells are superior to the single administration of an equivalent cumulative dose (Tang et al., 2018). In an attempt to address this issue, there is an increasing interest in the intravenous route. At first glance, this approach may look paradoxical because bio-distribution studies of intravenously injected cells have shown that most of them fail to reach

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Review the heart but rather accumulate remotely in the liver, lung, and spleen (Lee et al., 2009). However, such a distribution is still associated with an improvement in heart function after both cell-based therapy in preclinical (Luger et al., 2017) and clinical (Bartolucci et al., 2017) ischemic settings, a secretome-based therapy in a large animal infarction model (Timmers et al., 2011), and an exosome-based therapy in a nonischemic cardiomyopathy model (Sun et al., 2018). Put together, these data raise the possibility that the biomolecules secreted by the sequestered cells (or infused directly) act at a distance in an endocrine fashion (Lee et al., 2009) and/or induce phenotypic changes in immune-inflammatory cells. The beneficial effects on the exogenously infused cells might thus be indirectly due to a systemic modulation of inflammation (Wysoczynski et al., 2018), which is a major component of advanced cardiomyopathies and other diseases, as suggested by the data of the MASTERS trial in which patients were intravenously injected with multipotent adult stem cells shortly after stroke and demonstrated an early postinjection reduction in blood levels of CD3 lymphocytes and pro-inflammatory cytokines (Hess et al., 2017). It is clear that these protective effects of systemically infused adult cells cannot be readily extrapolated to PSCs. Should these cells be injected intravenously, it would be still more mandatory to generate pure populations of differentiated cells and to validate the absence of side effects due to their trapping in remote organs. However, some reassuring data have already been provided by preclinical studies using intravenously injected ESCderived endothelial cells or iPSC-derived neural stem cells in ischemic hindlimb (Huang et al., 2010) and amyotrophic lateral sclerosis (Nizzardo et al., 2014) models, respectively, and which have reported the absence of short-term adverse events along with a therapeutic benefit. The exclusive use of the PSC secretome could further increase the safety of this systemic approach without compromising its efficacy as roughly similar bio-distribution patterns have been reported after intravenous infusion of either MSCs or their exosomes in a stroke model where both treatments resulted in neurological improvement (Moon et al., 2018). Indeed, homing of extracellular vesicles (EVs) present in the secretome and thought to largely mediate its effects might even be enhanced by engineering the mother cells with ligands for adhesion receptors expressed in the target tissue (Ciullo et al., 2019). What is important from a clinical viewpoint is that, regardless of whether the administered product consists of cells or their products, the intravenous approach features definite attractive advantages, primarily its absence of invasiveness and correlatively the possibility of repeated administrations, which could be critical for a sustained therapeutic benefit. Autologous versus Allogeneic Cells The first cells that have been tested clinically were autologous for obvious reasons: ease of procurement, possibility of personalized treatment, lack of ethical issues, and, importantly, the risk of rejection due to perfect Human Leukocyte Antigen (HLA) match between host and transplanted cells. With the accumulated experience, however, it soon became evident that this model had serious limitations: variability in cell function, rates of proliferation and differentiation, and subsequent variability in effective dose between patients, making it difficult to produce a consistent product; complexity of logistics when there is no point-of-care

processing station requiring round-trip transportation (from the procurement center to the cell production facility and back); and cost of individualized quality controls. Recognition of these drawbacks then led to testing allogeneic cells, which can be stored in fully qualified banks and offer the clinicians the advantage of an off-the-shelf readily available product. The finding that in preclinical studies allogeneic cells yielded similar outcomes as their autologous counterparts (Jansen Of Lorkeers et al., 2015) tends to further boost the field in this direction, particularly if the objective is to treat large numbers of patients as the allogeneic model is the only one that looks realistic from a manufacturing and economic standpoint (Smith, 2012). By definition, this allogeneic model is the only one that is relevant to the clinical use of PSCs, including iPSCs, as mentioned above. The obvious issue raised by allogeneic cells is the risk of rejection. To prevent it, classic immunosuppressants (cyclosporine/ FK-506/dexamethasone/antibodies against co-stimulatory molecules) have been effectively used for a long time, but many side effects are associated with these protracted immunosuppressive therapies (reviewed in Malat and Culkin, 2016), and, given the common comorbidities of these patients, it can be anticipated that many of them will develop complications over time. In addition, these agents can modulate metabolism of the grafted cells (in terms of survival, proliferation, and differentiation) making them biologically less attractive or requiring to increase their dosing (Fo¨ldes et al., 2014). The use of a prolonged immunosuppression regimen might thus be a serious obstacle to a wide clinical dissemination of PSC use. This awareness has led to efforts for inducing immunological tolerance through host conditioning by activation/adoptive transfer of regulatory T cells (Pan et al., 2015) and, more recently, administration of nanobiologics targeting macrophages and shown effective in enabling a long-term tolerance to heterotopically transplanted hearts (Braza et al., 2018). To mitigate rejection, other strategies have been tested, including somatic nuclear transfer for generating genetically matched stem cells (Chung et al., 2014), donor-recipient match by using cells from HLA-haplotyped lines (Neofytou et al., 2015), epitope-based HLA matching (Duquesnoy, 2017), and PSC line genetic engineering to knock out HLA class I and II genes with a concomitant overexpression of HLA-E (Gornalusse et al., 2017) or CD47 (Deuse et al., 2019) or knockout of HLA-A and -B while retaining HLA-C to suppress the natural killer (NK) cell response (Xu et al., 2019). Currently, HLA-matched cells were already transplanted in a Parkinson trial (https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi? recptno=R000038278), and the next step could be the use of a gene-editing-based ‘‘universal cell line,’’ which, if successful, would profoundly transform the landscape by allowing at least the reduction of the level of immunosuppression and keeping it at the minimum required for addressing the adaptive allo-immune response, which might still be triggered by the mitochondrial content of the graft (Deuse et al., 2015). Regardless of the strategy that may ultimately be used, we think that overcoming the immunosuppression-associated safety issues is a prerequisite for shifting the risk to benefit ratio in a way that can facilitate the clinical acceptance of PSCs in the real world. However, as discussed in the next section, this rejection issue is manageable in totally different ways depending of the targeted mechanism of action of the transplanted cells. Cell Stem Cell 25, November 7, 2019 597

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Review Figure 1. Putative Mechanisms of Action of Stem Cells Tx, transplantation; PSC, pluripotent stem cell; IV, intravenous; EV, extracellular vesicle.

Mechanisms of Action Historically, the primary objective of cell therapy has been to replace tissue-specific damaged cells by new, exogenously supplied cells endowed with the capacity of generating a functional neo-tissue. This objective remains unchanged when the specific role assigned to the grafted cells is to continuously supply a missing mediator underlying the disease such as dopamine or insulin in Parkinson’s and diabetes, respectively, or to prevent photoreceptor loss in macular degeneration by providing these cells with nutrients. In these situations, a critical issue is to ensure the long-term engraftment of the transplanted cells given the multiplicity of factors that contribute to their death, including stress during delivery, inflammation, hypoxia, and lack of anchorage to a matrix when they are in suspension and rejection if the grafted cells originate from allogeneic PSCs. In the specific case of the heart, ‘‘remuscularization’’ of the diseased myocardial areas, both in ischemic and nonischemic cardiomyopathies, also looks to be a sound objective that has been successfully accomplished in clinically relevant nonhuman primate infarct models of PSC-derived cardiomyocyte transplantation (Chong et al., 2014; Liu et al., 2018; Shiba et al., 2016). Of note, these cells have recently been shown to be superior to other somatic cell sources such as skeletal myoblasts and MSCs with regard to improvement of post-infarction ventricular function (Ishida et al., 2019). However, the clinical translatability of this approach is fraught with some specific challenges. The first is the requirement for large numbers of exogenously supplied cardiomyocytes, in the range of one billion, given the usual extent of myocardial damage that may warrant this type of procedure. Keeping this large number cells alive and viable over time so that they can contribute to force generation is another challenge that has motivated a myriad of cell empowering strategies, often based on genetic engineering or preconditioning (Mohsin et al., 2011) but that have failed to be translated clinically 598 Cell Stem Cell 25, November 7, 2019

because of the related increase in technical, regulatory, and economic complexity, except for the co-supply of MSCs and c-kit+ cardiac cells expected to act synergistically (Karantalis et al., 2015). Of note, the benefits of engineered cardiac tissues made of iPSC-derived cardiomyocytes are also enhanced by the concomitant presence of vascular and mural cells (Masumoto et al., 2016), and the outcome of future iPSC-cardiomyocyte trials might thus be influenced by the transplanted cell ratios—a variable that could be difficult to control in a reproducible fashion. Finally, injected cardiomyocytes need ideally to couple with the native ones in a homogeneous fashion to avoid host-graft electrical mismatch, which might trigger arrhythmias, as discussed in another section of this review. However, the consistent discrepancy between the rapid clearance of the grafted cells (regardless of their phenotype) from the transplantation sites and the maintenance of a functional benefit makes it quite unlikely that the tiny, if any, number of residual cells could account for such an improvement (Sharma et al., 2017) and has led us to hypothesize an alternate mechanism of action based on paracrine signaling (Garbern and Lee, 2013), whereby cell-secreted bioactive molecules, including those clustered in EVs, harness a multifaceted endogenous tissue repair and manifest as a stimulation of angiogenesis and a mitigation of inflammation, fibrosis, and, possibly, of apoptosis (Gnecchi et al., 2008), whereas, in the absence of unequivocal fate-mapping studies, a proliferation of host cardiomyocytes looks more uncertain (Figure 1). Additional evidence for this paracrine hypothesis comes from the observations that the cell-derived EV-enriched secretome can recapitulate the protective effects of the parent cells in preclinical models of heart (Kervadec et al., 2016), lung (Chen et al., 2014), kidney (Bruno et al., 2009), and brain (Doeppner et al., 2015) injury. Adhesion to this paradigm directly impacts on the choice of the cells. A first prerequisite is that they feature a robust secretory profile, which accounts for a common use of MSCs. However, it seems equally important that their phenotype closely matches that of the target tissue (Barile et al., 2018), which qualifies cardiac-committed cells from neonatal tissue sources (Sharma et al., 2017) or PSCs (El Harane et al., 2018). It is fair to acknowledge that the precise mechanism of action of EVs (and other soluble factors present in the secretome) is still incompletely clarified. The current view is that these particles activate signaling pathways in recipient cells that may contribute to tissue preservation. This activation is thought to occur via cell-surface interactions and/ or intracytoplasmic release of the EV cargo constituted by proteins, lipids, microRNAs (miRNAs), and other nucleic acids

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Review (Wiklander et al., 2019). Of note, the shuttling process that allows EVs to transfer this cargo has been shown to occur rapidly (Yuan et al., 2009), which could make it compatible with the well-documented short half-life of exosomes and other EVs. However, the recognition that the protective effects of transplanted cells can be duplicated by their secretome leads to the idea of only delivering the latter, which may have clinically relevant advantages at least in some indications, probably excluding diseases such as diabetes or Parkinson’s, which require the continuous supply of a missing mediator from a cellular source, as outlined above. These advantages include a standardized pharma-like manufacturing process, a likely reduced immunogenicity (at least when the secreting cells are hypoimmunogenic themselves), and a functional stability under cryo-storage compatible with off-the-shelf availability. Of note, from a regulatory viewpoint, cell-derived secretomes, or more specifically EVs, are not considered ‘‘Advanced Therapy Medicinal Products’’ but ‘‘Biological Medicines,’’ which corresponds to a different (albeit not necessarily easier) regulatory path. Along a similar a-cellular line of reasoning, others investigate bioinspired materials such as a decellularized cardiac extracellular matrix also credited for activating endogenous repair processes (the Ventrigel trial, NCT02305602). Regardless of the mechanism of action, a major issue is to ensure an effective engraftment. Multiple strategies have thus been developed that often share in common the adjunctive use of a biomaterial acting through its shielding effect (see below) with some disease-specific strategies such as a refillable oxygen tank nurturing device-encapsulated beta-cells (Carlsson et al., 2018) or the co-supply of MSCs and c-kit+ cardiac cells to be tested in heart failure patients in the future TAC-HFT-II trial. In the case of allogeneic cells, including PSCs, anti-rejection strategies have been discussed above and, in clinical practice, still remain largely based of immunosuppressive drugs, the management of which, however, is strongly dependent on the presumed mechanism of action of the grafted cells. In the paracrine paradigm, it is assumed that cells will inevitably die and that they only need to be present transiently so that they have enough time to release the biomolecules underpinning their effects. In this case, immunosuppression can only be given for a short period of time, which is usually well tolerated, as supported by our clinical experience with the ESCORT trial where patients were immunosuppressed for 1 month. Conversely, if the objective is a long-term cell engraftment, immunosuppression should theoretically be lifelong. However, this can be modulated according to the target organ. Thus, in the eye, a course of cyclosporine and mycophenolate mofetyl limited to 12 weeks did not prevent a likely a survival of injected donor hESC-derived RPE cells at 12 months (Mehat et al., 2018); likewise, with a median follow-up of 22 months, 13 out of the 18 patients treated by Schwartz and colleagues (Schwartz et al., 2015) demonstrated an increase in subretinal pigmentation consistent with a successful transplantation, although 3 had to interrupt the immunosuppression treatment because of adverse events. In the brain, the data are more conflicting as, in one study, withdrawal of immunosuppressive drugs 6 months after injection of fetal cells (which may not behave like PSC-derived cells) did not prevent long-term cell survival and function (Hallett et al., 2014), while, in another study, deterioration temporally coincided with the

discontinuation of cyclosporine (Olanow et al., 2003). The future trials of PSC-derived cardiomyocytes plan a pharmacological immunosuppression, but it is clear that there is still a need for better defining the type of drugs, their optimal dosing, and the duration of treatment. Of note, the objective of repopulating the heart with new cardiomyocytes might be achieved by alternate approaches based on stimulation of endogenous cardiomyocyte division by releasing cell-cycle checkpoints. This objective can be achieved by a variety of approaches such as genetic engineering of cells with cell-cycle activators (Zhu et al., 2018), small molecules such as neuregulin (Polizzotti et al., 2015), blockade of signaling pathways leading to cardiomyocyte cell-cycle arrest such as hippo (Lin and Pu, 2014) or GSK-3 (Singh et al., 2019), or reprogramming of in situ fibroblasts with a cocktail of cardio-instructive transcription factors (Mohamed et al., 2017). However, regardless of the strategy, the key prerequisite here could be to reverse the metabolic switch from glycolysis to oxidative phosphorylation, which seems to be a key driver for stopping cardiomyocyte proliferation (Mills et al., 2017). Furthermore, while the feasibility of these approaches has been demonstrated in small animal models of myocardial infarction, their use in patients is likely a far-fetching objective because of the still unsettled technical and safety issues related to the choice of the activating/reprogramming agents, their usually low efficiency, and the means of regulating this differentiation process to avoid an uncontrolled cell proliferative growth. Maturation of PSC-Derived Cardiac Cells While the in vitro replication of the signaling pathways that determine cell fate during embryogenesis allows generation of lineage-specific derivatives, it also raises the challenging question of the optimal stage of differentiation at which those cells should be transplanted. In a rat model of stroke, early- and mid-differentiated neuroepithelial stem cells were found more abundant in the brain compared with a late-differentiated population (Payne et al., 2018). Likewise, in a Parkinson’s disease mouse model, post-mitotic immature dopamine neurons were identified as those yielding the best outcomes compared with cells at earlier or later stages of differentiation (Qiu et al., 2017). Similar benefits of iPSC-derived dopaminergic cells at a progenitor stage have been reported in a nonhuman primate model of Parkinson’s disease (Kikuchi et al., 2017), thereby paving the way for the use of these cells in an ongoing clinical trial. In the case of diabetes, the data are more conflicting. Some groups have chosen to transplant pancreatic progenitors with the premise that they better resist to hypoxia and can complete their maturation in vivo to form an islet-like tissue capable of sensing blood glucose levels and releasing insulin accordingly (Schulz, 2015). Other investigators argue that this process is lengthy (in the order of 4 months), susceptible to be influenced by the host micro-environment, and fraught with an increased risk of contamination of the transplant by still some pluripotent cells and that, additionally, it requires larger numbers of cells; consequently, they support the use of differentiated PSCderived beta-cells, which, even though they cannot be equated to bona fide human adult beta-cells, are expected to be rapidly functional after transplantation with regard to insulin release kinetics in response to blood glucose levels (Pagliuca et al., Cell Stem Cell 25, November 7, 2019 599

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Review 2014). These benefits might be further enhanced in the future owing to the possibility of also differentiating PSCs into glucagon-expressing cells, thereby yielding mixed islet populations better replicating the kinetics of physiological insulin secretion. In the case of the heart, one approach is to target the transplantation of PSC-derived ventricular-like cardiomyocytes as close as possible to the native ones with the premise that they may generate more force contraction, be less arrhythmogenic because an extended differentiation process should get rid from pacemaker cells and the attendant presence of automaticity foci, and also be less risky because of the presumed absence of more immature cells with a high proliferation potential. This challenging objective has been targeted by a variety of strategies that are not mutually exclusive and can be broadly categorized as biophysical (electrical stimulation, mechanical strain), biochemical (hormones and small molecules), genetics (forced expression of missing ion channels), or culture conditions dependent (prolongation of culture time, growth in three-dimensional configurations, modulation of substrate chemistry/topography/stiffness, co-cultures with other cell types) (Denning et al., 2016; Veerman et al., 2015). An alternate approach consists of transplanting early progenitors cells, a choice rationalized by (1) their glycolytic metabolism that could help the cells to better withstand the hypoxic environment they are transplanted in and thus to form larger grafts (Zhang et al., 2015), (2) a greater plasticity allowing a differentiation in cardiac and vascular cells (Cao et al., 2013), and (3) a higher secretory profile allowing to optimize paracrine signaling (Agarwal et al., 2017; El Harane et al., 2018). These arguments have rationalized our use of Isl-1+ progenitor cells in patients with heart failure (Menasche´ et al., 2018), but it is fair to acknowledge that the issue of the optimal differentiation stage remains unsettled as experimentally early human myocardial Nkx2.5+ precursors have failed to provide better recoveries than fully differentiated cardiomyocytes (Ye et al., 2015). Safety The major safety concern associated with the clinical use of PSCs is the occurrence of teratomas, which, even though they are benign by nature, can cause compression of the neighboring structures and also undergo malignant transformation. A first cause is the occurrence of genetic abnormalities that cells can incur during the stepwise culture process and which makes them vulnerable to transformation (Werbowetski-Ogilvie et al., 2009); the risk seems still higher with iPSCs (as opposed to ESCs) because somatic cells from which they are derived may already harbor mutations, while the reprogramming process by itself can superimpose chromosomal aberrations and epigenetic abnormalities potentially increasing the risk of tumorigenicity (Ben-David and Benvenisty, 2011). As part of a translational process, it is thus critical to monitor cultured PSCs for the occurrence of cancer-driving mutations by a combinatorial approach including assessment of the karyotype, fluorescence in situ hybridization (FISH) focusing on chromosomes known to be the most vulnerable to genetic changes, comparative genomic hybridization, and possibly, in the future, next-generation sequencing (Assou et al., 2018), with the caveat that highthroughput screening may still fail to pick up alterations in a minority cell population. 600 Cell Stem Cell 25, November 7, 2019

Even though cells have remained cytogenetically stable throughout the culture period, a tumor can still originate from pluripotent cells that have failed to respond to lineage-specific instructive cues and thus ‘‘contaminate’’ the final product to be delivered to the patient. However, this risk and its mitigation vary with the extent of cell differentiation. Thus, when cells are ‘‘pushed’’ toward their most terminally differentiated state, a purification step may not be required. Such is the case in spinal cord injury (Priest et al., 2015) and macular degeneration (Ben M’Barek et al., 2017; Schwartz et al., 2012) trials where ESCderived oligodendrocytes and RPE, respectively, were considered ‘‘pure’’ enough to be transplanted without an additional sorting procedure. Conversely, if cells are still at an early-differentiated progenitor state, it is mandatory, from a safety viewpoint, to eliminate those that have retained their pluripotency. This objective can be achieved by a variety of techniques including chemical inhibitors (such as etoposide), which exploit the sensitivity of PSCs to genotoxic agents (Secreto et al., 2017), small molecule-based induction of PSC apoptosis (BenDavid et al., 2013), recombinant lectin-toxin fusion proteins selectively binding to PSCs and delivering a cytotoxic cargo following their internalization (Tateno et al., 2015), or, in the case of the heart, culture under glucose depletion and lactate enrichment to enhance the exclusive survival of differentiated cardiomyocytes (Tohyama et al., 2013) (for a more extensive review of the current methods of cardiomyocyte enrichment, see Ban et al., 2017). In the clinics, however, enrichment for a pure population of progenitor cells rather relies on antibody-based sorting targeting lineage-specific surface markers such as CD142 for pancreatic endoderm cells (Kelly et al., 2011), LRTMP1 for midbrain dopaminergic neurons (Samata et al., 2016), or SSEA-1 for cardiovascular progenitors (Menasche´ et al., 2015). Of note, the unavailability of flow cytometry for clinical applications highlights the interest of immune-magnetic sorting with the caveat that it would be desirable to rather target pluripotency-specific markers such as SSEA-5 (Tang et al., 2011) or CD30 (Sougawa et al., 2018) so that the flowthrough population of interest remains free from the magnetic bead-tethered antibodies. Other GMP-compatible technologies in development might in the future increase the efficiency and scalability of purification (Rodrigues et al., 2015). At the completion of this step, the purportedly ‘‘purified,’’ genetically stable product then needs further testing, both by in vitro assays aimed at detecting residual pluripotency-associated markers (clonogenic assays, flow cytometry, qRT-PCR, or immunohistochemistry; Kuroda et al., 2013), which are useful quality controls before batch release, and in vivo by tumorigenicity/biodistribution studies (Cunningham et al., 2012; Garitaonandia et al., 2016), which are more clinically relevant than the previously mentioned surrogates and are thus pivotal in the preclinical safety screening. One important feature of these studies is to include spiking experiments in which varying ratios of pluripotent/differentiated cells are screened to determine the percentage of the former above which a tumor occurs and thus define a pre-set purity threshold for lot release (95% in our ESCORT trial). Of note, these experimental data should always be interpreted cautiously in view of their limitations, primarily the confounding effect of a usually xenogeneic setting (human cells delivered to rodents), the short lifespan of rodents precluding long term

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Review studies, the use of immune-deficient animal models, and the attendant lack of an immunosuppressive treatment that will be given to patients (for this reason, we have privileged, in our preclinical studies, the transplantation of Rhesus monkey-derived ESCs to infarcted and immunosuppressed monkeys to more closely mimic the clinically relevant scenario of an allogeneic transplantation; Blin et al., 2010). As an ultimate fail-safe system, gene-engineering technologies now allow transduction of stem cell lines with drug-inducible suicide genes linked to inducible caspase 9 (Itakura et al., 2017) or cell division genes (Liang et al., 2018) and intended to activate cell death of residual PSCs following systemic administration of a prodrug. However, one should not underscore that the clinical implementation of such a genetic engineering entails additional layers of technical, economic, and regulatory complexity. At the end, it should be recognized that tracking tiny amounts of residual undifferentiated cells in the final cell therapy product may be somewhat like looking for a needle in a haystack given the unavoidable limitations of all sensitivity assays. Therefore, the decision to move to the clinics remains ultimately based on a careful risk-tobenefit analysis, which must then be completed, once patients have received PSC-derived cell transplants, by a regular and prolonged imaging-based follow-up (Menasche´ et al., 2018). A second potential complication is the development of a clinically relevant allo-immunization. We have not seen this event in our ESCORT trial, and it has not been reported in another heart failure study of allogeneic adipose-derived stromal cells even though donor-specific antibodies could be occasionally detected during follow-up (Kastrup et al., 2017). A third safety issue specific for cardiac applications is the occurrence of ventricular arrhythmias. Not unexpectedly, the disparity in heart rates between humans and rodents has precluded to demonstrate these events in the multiple rat and mouse studies of PSC transplantation. Only large animal models with a physiology closer to that of man has unraveled arrhythmias in all PSC-treated nonhuman primates (in two out of three studies using this species; Chong et al., 2014; Shiba et al., 2016) and pigs (Romagnuolo et al., 2019). These episodes share in common to occur predominantly over the first 2 weeks after transplantation and gradually decrease thereafter. Electrophysiological studies suggest that they seem to arise through focal mechanisms at the graft/host interface (rather than by re-entry), which could be consistent with the initial presence of early-differentiated cells, the number of which subsequently declines as the graft matures. Clearly, along with the above-mentioned side effects of long-term immunosuppression, this risk of arrhythmias that can be lethal (2 out of 7 pigs in the study by Romagnuolo et al., 2019) should be a major component of the risk assessment preceding any clinical trial and needs to be weighed against a percentage of scar repopulation by the grafted cells, which seldom exceeds 15% and correspondingly translates into only modest improvements in heart function. Of note, we have not documented any arrhythmic episode in patients of the ESCORT trial (all of whom were fitted with an automatic internal defibrillator allowing repeated electrocardiographic recordings), likely because of the epicardial, as opposed to intramyocardial, location of the scaffold-embedded cardiovascular progenitors primarily intended to serve as reservoirs of paracrinally active biomolecules and not as force-generating units.

Biomaterials The recognition that cells are poorly retained and then massively die has led to the development of strategies aimed at improving their initial engraftment, discussed above, and subsequent survival whenever the target is to enhance the prolonged presence of the cells. Because of the above-mentioned caveats associated with genetic cell engineering with ‘‘pro-survival’’ genes or their preconditioning, biomaterials have emerged as a potentially more user-friendly means of addressing the retention and survival issues through their ability to act as a cell-shielding protective lubricant during injections and to provide an extracellular matrix-like tri-dimensional template featuring instructive cues to enhance cell viability, proliferation, and differentiation. Biomaterials can also serve as reservoirs controlling the release of cell-secreted paracrine factors to extend the exposure time of the target tissue to these factors and try to match their release kinetics with those of the mother cells if they were directly transplanted. The most illustrative examples of materials that have moved to the clinics pertain to diabetes (alginate capsules; Vegas et al., 2016), macular degeneration (RPE-supporting scaffold mimicking a Bruch’s membrane Kashani et al., 2018), and the heart (fibrin scaffold encaging ESC-derived cardiovascular progenitors; Menasche´ et al., 2015). Many reviews have been devoted to these biomaterials and have comprehensively discussed their respective advantages and disadvantages (O’Neill et al., 2016; Zhang et al., 2018). Only some key considerations to keep in mind will be highlighted here. First, the selection of a biomaterial regarding its chemistry, pore size, elasticity, surface topography, or sterilization technique needs to be tailored to the nature of the microenvironment in which it will be implanted (acute, highly pro-inflammatory ischemic injury or chronic scar), the phenotype of the cells intended to be seeded onto its surface or embedded within its core (Neuss et al., 2008), and its precise objective: mechanical strengthening, passive platform for delivery of cells, or entrapped cell-derived biologics or template whose features should trigger endogenous repair mechanisms. In the specific case of the heart, if the biomaterial is designed as a patch supporting cells intended to survive for a long time and integrate in the recipient myocardium, more complex engineering may be required to endow the material with angiogeneic and electro-conductive properties. The former may be achieved through fine-tuning of porosity to facilitate ingrowth of host vessels or pre-implantation microfluidics-based generation of capillary networks (Novosel et al., 2011; Redd et al., 2019), while the latter may benefit from inclusion of carbon nanotubes into elastomeric scaffolds as this has been shown to improve signal transduction through cardiomyocytes (Gorain et al., 2018). An ordered spatial patterning of the cells to be delivered according to predetermined motifs can further contribute to enhance their reparative capacity and interactions with the host extracellular matrix. Electrospinning and tri-dimensional printing now make possible to accurately control the architecture of the biomaterial, either cell or secretome laden, or free, but the availability of these technologies must be weighed against the challenges of their industryscale translatability, as discussed below. A second critical factor that influences the choice of the biomaterial is its intended route of administration. For a catheter-based delivery, the material needs to feature shear-thinning Cell Stem Cell 25, November 7, 2019 601

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Review properties allowing it to flow through the lumen of the device before expanding as a gel once in the target tissue. Of note, while this can be easy to achieve for with a short needle, the issue may be more complex in case of a percutaneous approach to the heart because of the length of the dead space in the catheter. If the material is to be used as an epicardial patch, there is greater flexibility for fine-tuning its physico-chemical characteristics so as to make it both cell friendly and suitable for gentle grasping and intraoperative manipulations, including suturing. As an example, our experience with the fibrin patch in which the ESC-derived cardiovascular progenitor cells have been embedded has shown that a fibrinogen to thrombin ratio of 20 mg/mL-4 U was optimal for matching the above-mentioned requirements. With regard to the clinical translatability, two important issues need to be considered. The first is obviously safety and thus implies using biocompatible materials causing no or minimal inflammatory and immune responses, either by themselves or by their degradation products. The second issue pertains to manufacturing, which should be made cost effective through the use of readily available raw materials, their scalability, GMP-compliant assembly processes and quality controls, and suitability for sterilization and stability under storage. Conclusions: Considerations for Replicating Laboratory-Scale Methods The accumulated experience with cell therapy across a wide range of diseases now allows us to more clearly envision the paths worth being explored for making the use of cells or their secreted products therapeutically effective, patient friendly, and economically affordable. Given the commonality of many of the hurdles that still need to be overcome, it is always rewarding to get some insights into the experience of investigators involved in different disease areas as this cross-fertilization of disciplines can be very effective. At the end, however, a successful therapeutic outcome does not only rely on the type of cells, their mode of delivery, or their incorporation into tri-dimensional constructs. It also requires a better targeting of patients likely to benefit most from these treatments; for example, heart failure encompasses a wide variety of phenotypes (Triposkiadis et al., 2019) not all of which are likely to be responsive to cell therapy and a proper selection of the candidates is therefore critical. In this setting, it is hoped that, with the advent of big data programs, machine learning will help in discriminating responder versus nonresponder profiles, thereby allowing more accurate identification of patients most likely to demonstrate a successful outcome (Steinhoff et al., 2017). In parallel, trial designs likely need to be revisited and Bayesian-type statistical models based on predictive probabilities might be better suited than conventional randomized studies to more rapidly yield unequivocal data and hasten translatability to daily clinical practice. Given the increasing stringency of regulatory constraints, such a move requires that any developmental plan for a cell-, secretome-, or tissue-engineering-based therapy be thoughtfully designed from the onset to optimize automated manufacturing, consistency of batches, and reliability of quality controls to target cost-effectiveness, which is the prerequisite for the product to be ultimately approved by the regulators and/or reimbursed by the health stakeholders. 602 Cell Stem Cell 25, November 7, 2019

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. stem.2019.10.001. ACKNOWLEDGMENTS We thank the LabEx REVIVE (ANR-10-LABX-73), the Fondation de France (FDF/2014 00047970), the Fondation pour la Recherche Me´dicale (Prix Victor et Erminia Mescle, PME20180639496), and the Fondation de l’Avenir (AP-RM18-017) for their funding support. MD is supported by a grant from the Fondation Marion Elisabeth Brancher. DECLARATION OF INTERESTS P.M. acts as a consultant for Fujifilm-Cellular Dynamics, Inc.; M.D. has no conflicts of interest. REFERENCES Agarwal, U., George, A., Bhutani, S., Ghosh-Choudhary, S., Maxwell, J.T., Brown, M.E., Mehta, Y., Platt, M.O., Liang, Y., Sahoo, S., and Davis, M.E. (2017). Experimental, Systems, and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients. Circ. Res. 120, 701–712. Aguado, B.A., Mulyasasmita, W., Su, J., Lampe, K.J., and Heilshorn, S.C. (2012). Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers. Tissue Eng. Part A 18, 806–815. Assou, S., Bouckenheimer, J., and De Vos, J. (2018). Concise Review: Assessing the Genome Integrity of Human Induced Pluripotent Stem Cells: What Quality Control Metrics? Stem Cells 36, 814–821. Ban, K., Bae, S., and Yoon, Y.-S. (2017). Current Strategies and Challenges for Purification of Cardiomyocytes Derived from Human Pluripotent Stem Cells. Theranostics 7, 2067–2077. Barile, L., Cervio, E., Lionetti, V., Milano, G., Ciullo, A., Biemmi, V., Bolis, S., Altomare, C., Matteucci, M., Di Silvestre, D., et al. (2018). Cardioprotection by cardiac progenitor cell-secreted exosomes: role of pregnancy-associated plasma protein-A. Cardiovasc. Res. 114, 992–1005. Bartolucci, J., Verdugo, F.J., Gonza´lez, P.L., Larrea, R.E., Abarzua, E., Goset, C., Rojo, P., Palma, I., Lamich, R., Pedreros, P.A., et al. (2017). Safety and Efficacy of the Intravenous Infusion of Umbilical Cord Mesenchymal Stem Cells in Patients With Heart Failure: A Phase 1/2 Randomized Controlled Trial (RIMECARD Trial [Randomized Clinical Trial of Intravenous Infusion Umbilical Cord Mesenchymal Stem Cells on Cardiopathy]). Circ. Res. 121, 1192–1204. Bartunek, J., Terzic, A., Davison, B.A., Filippatos, G.S., Radovanovic, S., Beleslin, B., Merkely, B., Musialek, P., Wojakowski, W., Andreka, P., et al.; CHART Program (2017). Cardiopoietic cell therapy for advanced ischaemic heart failure: results at 39 weeks of the prospective, randomized, double blind, sham-controlled CHART-1 clinical trial. Eur. Heart J. 38, 648–660. Bellamy, V., Vanneaux, V., Bel, A., Nemetalla, H., Emmanuelle Boitard, S., Farouz, Y., Joanne, P., Perier, M.-C., Robidel, E., Mandet, C., et al. (2015). Longterm functional benefits of human embryonic stem cell-derived cardiac progenitors embedded into a fibrin scaffold. J. Heart Lung Transplant. 34, 1198–1207. Ben-David, U., and Benvenisty, N. (2011). The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat. Rev. Cancer 11, 268–277. Ben-David, U., Gan, Q.-F., Golan-Lev, T., Arora, P., Yanuka, O., Oren, Y.S., Leikin-Frenkel, A., Graf, M., Garippa, R., Boehringer, M., et al. (2013). Selective elimination of human pluripotent stem cells by an oleate synthesis inhibitor discovered in a high-throughput screen. Cell Stem Cell 12, 167–179. Ben M’Barek, K., Habeler, W., Plancheron, A., Jarraya, M., Regent, F., Terray, A., Yang, Y., Chatrousse, L., Domingues, S., Masson, Y., et al. (2017). Human ESC-derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration. Sci. Transl. Med. Published online December 20, 2017. https://doi.org/10.1126/scitranslmed.aai7471. Blin, G., Nury, D., Stefanovic, S., Neri, T., Guillevic, O., Brinon, B., Bellamy, V., €cker-Martin, C., Barbry, P., Bel, A., et al. (2010). A purified population of Ru

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Review Huang, N.F., Niiyama, H., Peter, C., De, A., Natkunam, Y., Fleissner, F., Li, Z., Rollins, M.D., Wu, J.C., Gambhir, S.S., and Cooke, J.P. (2010). Embryonic stem cell-derived endothelial cells engraft into the ischemic hindlimb and restore perfusion. Arterioscler. Thromb. Vasc. Biol. 30, 984–991.

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