TY - JOUR
AU1 - Rafatian, Ghazaleh
AU2 - Davis, Darryl R.
AB - Abstract Despite progress in cardiovascular medicine, the incidence of heart failure is rising and represents a growing challenge. To address this, ex vivo proliferated heart-derived cell products have emerged as a promising investigational cell-treatment option. Despite being originally proposed as a straightforward myocyte replacement strategy, emerging evidence has shown that cell-mediated gains in cardiac function are leveraged on paracrine stimulation of endogenous repair and tissue salvage. In this concise review, we focus on the paracrine repertoire of heart-derived cells and outline strategies used to boost cell potency by targeting cytokines, metabolic preconditioning and supportive biomaterials. Mechanistic insights from these studies will shape future efforts to use defined factors and/or synthetic cell approaches to help the millions of patients worldwide suffering from heart failure. Open in new tabDownload slide Open in new tabDownload slide Adult stem cells, Cellular therapy, Insulin-like growth factors, Somatic cell, Therapy, Stromal-derived factor-1, Cardiac Significance Statement Heart failure is a rising epidemic and new strategies are needed to address this growing population. Over the past 15 years, adult heart-derived cells have garnered attention as an investigative cell-treatment option. Understanding the mechanisms underlying cell-mediated repair will help design the next generation of cardiovascular therapeutics by maximizing paracrine stimulation of natural endogenous repair mechanisms. Introduction The incidence of heart failure is rising worldwide and represents an impending epidemic. Although the number of myocardial infarctions (MIs) has declined by almost 30% over the past 10 years [1], the number of patients diagnosed with heart failure has also increased by 12% while the number of patients living with heart failure has increased by 23% [2, 3]. Once diagnosed with heart failure, these patients continue to have a dismal prognosis with a 5-year mortality approaching 50% despite innovations in device and medical therapies [4, 5]. In response, therapeutic administration of ex vivo expanded stem cells to animal models of cardiac injury or cardiomyopathic patients has been explored over the past 20 years. Early experience naturally focused on noncardiac cells sources (the blood, bone marrow, fat, and skeletal muscle) as isolation protocols already existed with an established safety record in other noncardiac diseases. While some approaches produced malignant cardiac rhythms secondary to the engraftment of electrically inhomogenous cells (i.e., skeletal myoblasts) [6], the overall experience using other noncardiac cell sources demonstrated a favorable safety profile but no consistent signal indicative of benefit [7]. The latter often being attributed to poor long-term engraftment of transplanted cells and reliance upon paracrine repair by nonspecific (noncardiac) cytokines/exosomes that target a limited number of endogenous repair mechanism(s) [8]. As these investigations unfolded, the discovery of cells expressing surface markers indicative of stem cell identity (such as abcg2, c-Kit, sca-1, and SSEA-1) within the adult heart that fulfill the classical definition for stem cells (i.e., self-renewal and multipotency) led many to explore the ability of these cells to promote myocardial healing. In comparison to other cell sources, obtaining cardiac tissue from patients themselves or unrelated donors for expansion of therapeutic active cells is not trivial as invasive biopsies (or cadaveric donors) are needed to obtain the initial cell source. In response to this challenge, we and others developed techniques to expand heart-derived cells from pieces of myocardium often smaller than the point of a pencil [9-12]. Since then, the field of adult heart-derived cell transplant has evolved to include an encouraging body of literature with promising early clinical trials [13-18] but not without controversy stemming from concerns regarding experimental methodology [19]. In this review, we discuss the experience to date using heart-derived cells with an emphasis on fundamental insights to enable means of enhancing therapeutic outcomes using biomaterials, pro-survival strategies and even synthetic cells. Heart-Derived Stem Cells Multipotent cells were first isolated from cardiac tissue using techniques perfected in the isolation of skeletal myoblasts [20]. These flow cytometry defined cells (termed side population cells) possess markers reminiscent of cardiomyocyte precursors (Nkx2.5, GATA4, and Mef2C), the ATP-binding cassette transporter Abcg2, variable surface expression of other stem cell markers (c-Kit and Sca-1) and, after inductive cell culture, the ability to adopt cardiac lineage markers (cardiac troponin T, von Willebrand factor) [21, 22]. This initial discovery spurred a flurry of research panning through digested adult heart tissue suspensions for other cell populations expressing surface markers typically found on stem cells. Of these, a body of literature emerged supporting a role for nonhematological c-Kit+ cardiac cells in the generation of cardiomyocytes during aging or after injury [23]. Based on these findings, techniques were developed to isolate c-Kit+ cells from cardiac tissue for ex vivo expansion and delivery [12]. Promising preclinical data led to c-Kit+ cardiac cells undergoing clinical testing which resulted in an encouraging Phase 1 clinical trial [24]. Despite methodological concerns arising in the trial during cell phenotype testing [19], c-Kit+ cells are still under active clinical investigation as a combinatorial cell product (Combination of Mesenchymal and C-kit+ Cardiac Stem Cells as Regenerative Therapy for Heart Failure, CONCERT-HF; ClinicalTrials.gov Identifier: NCT02501811; estimated study completion date May 2020) [25]. In parallel to the cardiac c-Kit+ cell experience, the literature supporting the therapeutic efficacy of cardiac explant-derived cells (EDCs) and their expanded progeny (cardiosphere-derived cells, CDCs) has grown over the past decade from data generated by over 45 independent labs worldwide. EDCs are the initial cells cultured directly from myocardial biopsies plated in adherent cell culture conditions. In a manner mirroring neurosphere techniques, adherent EDCs are collected and cultured in low attachment cell culture conditions to generate cell spheres enriched in stem cell markers and cardiomyogenic transcripts [10, 26]. Although intra-myocardial injection of cardiospheres can improve myocardial function after injury, large sphere diameter (~150–200 μm) and the risk of arteriole plugging preclude straightforward clinical intra-coronary injection. As such, Smith and colleagues explored the effect of expanding cardiospheres within monolayer conditions to generate therapeutically active CD105+/C45- single cells comprised of cardiac, endothelial and mesenchymal progenitor cells [10, 11]. CDCs have since been shown to enhance myocardial function in many models of recent and remote myocardial injury. Akin to all cell products, the acute and long-term retention of CDCs is very modest (reviewed in reference [[27]]). Thus, despite forming mature electrically coupled cardiomyocytes and endothelial cells after injection [11, 28], CDC-mediated gains in cardiac function are leveraged on paracrine stimulation of endogenous repair and tissue salvage [29]. CDCs have also been shown to be immune privileged, which might permit allogeneic transplant [30], however also limits the ability of retained cells to mature into adult cardiomyocytes as the eventual expression of foreign human leukocyte antigens leads to immune clearance. Interestingly, the phenotypic make-up of CDCs also influences cell treatment outcomes as greater numbers of CD90+ cells is associated with reduced cell potency while depleting the CD90+ cell fraction restores functional potency [31]. In contrast, depletion of c-Kit+ cells from CDCs [31] and direct head-to- head comparisons [32] suggests that c-Kit+ cells do not significantly contribute to the regenerative efficacy of CDCs. Finally, despite several positive preclinical and phase 1 studies [13-18], recent clinical trial experience has been disappointing. Notably, the ALLSTAR study (allogenic intracoronary CDCs injected <6 month after MI; ClinicalTrials.gov: NCT01458405) was abandoned as an interim analysis of data from 142 patients demonstrated a low probability (futility) of achieving a statistically significant difference in the 12-month primary efficacy endpoint (i.e., relative improvement in infarct size from baseline) [14]. Although this outcome was attributed to unforeseen improvements in the placebo arm (i.e., natural history of the disease) and technical variability seen in the scar measurements by the steering committee, these data highlight the need to maximize indirect (paracrine) repair by heart-derived cell therapies if clinical translation is to have any hope for success [33]. Engineering the Paracrine Cytokine Signature of Heart-Derived Stem Cells Given the limited number of newly formed myocytes from transplanted cells, many have concluded that secretion of cytokines and exosomes combine to stimulate resident cardiomyocyte proliferation (~13% relative contribution to the observed increases in viable myocardium), endogenous stem cells recruitment (~18% relative contribution to the observed increases in viable myocardium) and damaged tissue salvage (~68% relative contribution to the observed increases in viable myocardium) [34]. The potential cytokine mediators of these effects are numerous as the secretome from heart-derived cells contains a mixture of many cytokines implicated in angiogenesis (angiopoetin-1, angiogenin, and vascular endothelial growth factor [VEGF]), myogenesis (hepatocyte growth factor), and stem cell recruitment (stromal cell-derived factor 1α [SDF1α]) [35, 36]. Furthermore, in addition to classical cytokines, Tseliou and colleagues demonstrated that the surface marker endoglin (CD105), which is universally expressed on all heart-derived cells, is liberated in vivo and inhibits transforming growth factor beta induced fibrosis by cardiac fibroblasts (Fig. 1A) [36]. Figure 1 Open in new tabDownload slide Effects of cytokines produced by heart-derived cells. (A): The left panel demonstrates the presence of soluble endoglin found within microdissected tissue from the infarct and infarct border zone of rats 1 week after intramyocardial injection of CSp. The right panel depicts in vitro culture experiments demonstrating the effect of endoglin neutralizing Abs on dermal Fb-sourced collagen in the presence of CSp or cardiosphere CM. p values as specified on figure. Adapted with permission from Tseliou et al. [36]. (B): The left panel demonstrates the correlation between the long-term risk of coronary heart disease events (LTS score) and improvements in echocardiographic LVEF from baseline 3 weeks after injection of EDCs into an immunodeficient mouse model of myocardial injury (n = 37). The right panel shows the correlation between LTS score and SDF1α content within CM (n = 15). Adapted with permission from Mayfield et al. [37] (C): Intramyocardial injection of SDF1α or nontargeting (control) sh RNAi transduced CDCs on echocardiographic LVEF 1 day and 5 weeks after MI demonstrated that therapeutic stimulation of cardiac-repair is at least partially dependent on SDF1α secretion. *p < .05 compared to MI, #p < .05 compared to shSDF1-transduced CDCs, n = 5 mice/group. Adapted with permission from Malliaras et al. [38]. Abbreviations: Ab, antibody; CDC, cardiosphere derived cell; CM, conditioned media; CSp, cardiospheres; EDC, explant-derived cell; Fb, fibroblast; LTS, long-term stratification; MI, myocardial infarction; SDF1α, stromal cell-derived factor 1 alpha; sh, short hairpin. Figure 1 Open in new tabDownload slide Effects of cytokines produced by heart-derived cells. (A): The left panel demonstrates the presence of soluble endoglin found within microdissected tissue from the infarct and infarct border zone of rats 1 week after intramyocardial injection of CSp. The right panel depicts in vitro culture experiments demonstrating the effect of endoglin neutralizing Abs on dermal Fb-sourced collagen in the presence of CSp or cardiosphere CM. p values as specified on figure. Adapted with permission from Tseliou et al. [36]. (B): The left panel demonstrates the correlation between the long-term risk of coronary heart disease events (LTS score) and improvements in echocardiographic LVEF from baseline 3 weeks after injection of EDCs into an immunodeficient mouse model of myocardial injury (n = 37). The right panel shows the correlation between LTS score and SDF1α content within CM (n = 15). Adapted with permission from Mayfield et al. [37] (C): Intramyocardial injection of SDF1α or nontargeting (control) sh RNAi transduced CDCs on echocardiographic LVEF 1 day and 5 weeks after MI demonstrated that therapeutic stimulation of cardiac-repair is at least partially dependent on SDF1α secretion. *p < .05 compared to MI, #p < .05 compared to shSDF1-transduced CDCs, n = 5 mice/group. Adapted with permission from Malliaras et al. [38]. Abbreviations: Ab, antibody; CDC, cardiosphere derived cell; CM, conditioned media; CSp, cardiospheres; EDC, explant-derived cell; Fb, fibroblast; LTS, long-term stratification; MI, myocardial infarction; SDF1α, stromal cell-derived factor 1 alpha; sh, short hairpin. Similar to blood-derived stem cells, patient comorbidities play an important role in defining the paracrine profile of heart-derived cells [37]. Using EDCs, we recently demonstrated that increasing medical comorbidities is associated with decreased cell-mediated repair of ischemic injury and decreased production of pro-healing cytokines (Fig. 1B). Of the very few studies that dissect the contribution of cytokines to heart-derived cell outcomes, Malliaras et al., demonstrated that CDC-sourced SDF1α plays a key role in stimulating endogenous progenitors to generate new myocytes after injury (Fig. 1C); whereas, loss of VEGF had little to no effect on cell treatment outcomes [38]. These insights opened opportunities to explore whether manipulating the cytokines of heart-derived cells would improve cell-treatment outcomes. Early work in this area was discouraging as Bonios and colleagues showed that lentiviral transduction of CDCs to overexpress hypoxia-inducible factor-1α (and thus increased secretion of both erythropoietin and VEGF) reduced the beneficial effects of CDC therapy by altering the balance of paracrine factors [39]. However, given this study indiscriminately transduced all cells, we wondered whether selective transduction of a subpopulation within heart-derived cells known to contribute little to cell-mediated repair (the CD90+ population [31]) would render these cells functionally relevant. Therefore, we antigenically separated, lentiviral transduced and recombined human CD90+ cells with CD90- cells prior to intramyocardial injection into an immunodeficient mouse 1 week after left coronary artery (LCA) ligation (Fig. 2A). From the large number of cytokines involved in post infarct repair, we focused on SDF1α as decreased production by transplanted cells is associated with reduced improvements in treatment outcomes [37, 38, 42] and the SDF1α receptor (CXCR4) is robustly expressed within the mouse heart for at least 2 weeks after MI (Fig. 2B). As shown in Figure 2C, EDCs that overexpressed SDF1α improved myocardial function while reducing myocardial scarring [41]. Mechanistically, we found that increasing SDF1α reduced apoptosis while increasing the generation of new myocytes and recruitment of circulating bone marrow cells. In contrast to the prevailing literature, SDF1α overexpression failed to increase the survival and engraftment of transplanted cells (Fig. 2D). Figure 2 Open in new tabDownload slide Paracrine engineering of heart-derived cells. (A): Schema of the study design to overexpress IGF-1 or SDF1α.(B): qPCR of the IGF1R or CXCR4 after LCA ligation. *p ≤ .05 versus sham-operated mice, n = 3 mice per sampling interval. (C): Effect of SDF1α overexpression on echocardiographic left ventricular ejection fraction (left panel) and ventricular scar burden (right panel) as compared to nontransduced (NT) EDCs or vehicle. *p < .05 versus vehicle, #p < .05 versus nontransduced EDCs, n = 6 mice (left panel) and three mice (right panel) per treatment group. (D): Effect of SDF1α overexpression on the production of new myocytes (left panel) and circulation cell recruitment (right panel). As shown in the left panel, C57 mice underwent LCA ligation followed 1 week later by EDC or vehicle injection and BrdU injection (subcutaneous daily X 14 days) prior to histological analysis 1 week later for BrdU+/cardiac troponin T+ (cTNT+) cells. The right panel demonstrates qPCR for the RBMY protein within ventricular tissue from female mice after male bone marrow transplant followed by injection of female EDCs 1 week after LCA ligation. *p < .05 versus vehicle, #p < .05 versus nontransduced EDCs, n = 6. (E): Effect of lvIGF-1 transduced human EDCs on echocardiographic left ventricular ejection fraction. *p < .05 versus vehicle, #p < .05 versus lvGFP transduced EDCs 3 weeks after cell or vehicle injection, n = 8–9 mice per treatment group. (F): Single-photon emission computed tomography quantification of apoptosis within the 201Tl-defined infarct region before (D0) and 2 days after injection of lvIGF-1 or lvGFP-transduced EDCs (D2). Also shown are representative horizontal long-axis images from a mouse 2 days after injection of lvIGF-1 demonstrating 201Tl perfusion images (upper panel) with myocardial infarction (yellow arrow) and 99mTc-rhAnnexin V-128 images (lower panel) with uptake in the area of infarction (white arrow) and surgical site (green arrow). *p ≤ .05 D2 versus D0, n = 3 mice. (G): The left panel demonstrated histological analysis of nonobese diabetic severe combined immunodeficiency (NOD SCID) mice injected with BrdU daily for 7 days after vehicle or EDC injection. *p < .05 versus vehicle, #p < .05 versus lvGFP-EDCs, n = 5 mice. The right panel depicts qPCR of injected ventricles for retained human Alu sequences to document retention of human lvIGF-1 and lvGFP EDCs after cell transplantation. *p < .05 versus lvGFP-transduced EDCs, n = 3 mice. Adapted with permission from Jackson et al. [40] and Tilokee et al. [41]. Abbreviations: BrdU, bromodeoxyuridine; cTNT+, cardiac troponin T+; CP, crossing point; CXCR4, SDF1α receptor; EDC, explant-derived cell; IGF-1, insulin-like growth factor 1; IGF1R, IGF-1 receptor; lvGFP, lentiviral-mediated overexpression of green fluorescent protein reporter; lvIGF-1, lentiviral-mediated overexpression of mature IGF-1 with green fluorescent protein; lvSDF1α lentiviral-mediated overexpression of SDF1α; LCA, left coronary artery; LTS, long-term stratification; NT, nontransduced; SDF1α, stromal cell-derived factor 1 alpha; qPCR, quantitative reverse transcription polymerase chain; RBMY, male germ cell-specific RNA-binding protein. Figure 2 Open in new tabDownload slide Paracrine engineering of heart-derived cells. (A): Schema of the study design to overexpress IGF-1 or SDF1α.(B): qPCR of the IGF1R or CXCR4 after LCA ligation. *p ≤ .05 versus sham-operated mice, n = 3 mice per sampling interval. (C): Effect of SDF1α overexpression on echocardiographic left ventricular ejection fraction (left panel) and ventricular scar burden (right panel) as compared to nontransduced (NT) EDCs or vehicle. *p < .05 versus vehicle, #p < .05 versus nontransduced EDCs, n = 6 mice (left panel) and three mice (right panel) per treatment group. (D): Effect of SDF1α overexpression on the production of new myocytes (left panel) and circulation cell recruitment (right panel). As shown in the left panel, C57 mice underwent LCA ligation followed 1 week later by EDC or vehicle injection and BrdU injection (subcutaneous daily X 14 days) prior to histological analysis 1 week later for BrdU+/cardiac troponin T+ (cTNT+) cells. The right panel demonstrates qPCR for the RBMY protein within ventricular tissue from female mice after male bone marrow transplant followed by injection of female EDCs 1 week after LCA ligation. *p < .05 versus vehicle, #p < .05 versus nontransduced EDCs, n = 6. (E): Effect of lvIGF-1 transduced human EDCs on echocardiographic left ventricular ejection fraction. *p < .05 versus vehicle, #p < .05 versus lvGFP transduced EDCs 3 weeks after cell or vehicle injection, n = 8–9 mice per treatment group. (F): Single-photon emission computed tomography quantification of apoptosis within the 201Tl-defined infarct region before (D0) and 2 days after injection of lvIGF-1 or lvGFP-transduced EDCs (D2). Also shown are representative horizontal long-axis images from a mouse 2 days after injection of lvIGF-1 demonstrating 201Tl perfusion images (upper panel) with myocardial infarction (yellow arrow) and 99mTc-rhAnnexin V-128 images (lower panel) with uptake in the area of infarction (white arrow) and surgical site (green arrow). *p ≤ .05 D2 versus D0, n = 3 mice. (G): The left panel demonstrated histological analysis of nonobese diabetic severe combined immunodeficiency (NOD SCID) mice injected with BrdU daily for 7 days after vehicle or EDC injection. *p < .05 versus vehicle, #p < .05 versus lvGFP-EDCs, n = 5 mice. The right panel depicts qPCR of injected ventricles for retained human Alu sequences to document retention of human lvIGF-1 and lvGFP EDCs after cell transplantation. *p < .05 versus lvGFP-transduced EDCs, n = 3 mice. Adapted with permission from Jackson et al. [40] and Tilokee et al. [41]. Abbreviations: BrdU, bromodeoxyuridine; cTNT+, cardiac troponin T+; CP, crossing point; CXCR4, SDF1α receptor; EDC, explant-derived cell; IGF-1, insulin-like growth factor 1; IGF1R, IGF-1 receptor; lvGFP, lentiviral-mediated overexpression of green fluorescent protein reporter; lvIGF-1, lentiviral-mediated overexpression of mature IGF-1 with green fluorescent protein; lvSDF1α lentiviral-mediated overexpression of SDF1α; LCA, left coronary artery; LTS, long-term stratification; NT, nontransduced; SDF1α, stromal cell-derived factor 1 alpha; qPCR, quantitative reverse transcription polymerase chain; RBMY, male germ cell-specific RNA-binding protein. Based on this success, we wondered whether inducing a cytokine not otherwise found in the paracrine repertoire of heart-derived cells would improve therapeutic repair or if cytokine mediated benefits were already maximized and further stimulation of complementary pro-survival pathways would be futile. For these experiments, the pro-survival cytokine insulin-like growth factor 1 (IGF1) was chosen as it plays a vital role in early growth and development while promoting ongoing lifelong anabolic effects via the Protein kinase B and Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase (ERK/MAPK) pro-survival pathways [40]. In contrast to SDF1α-CXCR4, the IGF-1 receptor (IGF1R) is expressed for 21 days after LCA ligation (Fig. 2B). When human EDCs genetically reprogrammed to overexpress IGF1 were injected into an immunodeficient mouse model of recent myocardial injury, IGF1 overexpression resulted in better myocardial function as compared to green fluorescent protein (GFP) transduced controls (Fig. 2E). Similar to SDF1α overexpression, a portion of these benefits were attributable to increased salvage of reversibly injured myocardium (Fig. 2F). However, IGF-1 overexpression also promoted the generation of new nonmyocyte cells while increasing the survival of transplanted cells (Fig. 2G). Taken together, this data shows that increasing paracrine mediated stimulation of endogenous repair has the potential to increase cell-mediated gains in myocardial function while indiscriminately manipulating all cells may impair the production of pro-healing paracrine factors. Effects of Metabolic Preconditioning on Heart-Derived Cells Although diabetes and chronic hyperglycemia are well known to increase both cardiac and vascular disease, effects on cells grown directly from heart tissue are less well understood as ex vivo culture conditions presumably remove cells from adverse glycemic stress. To explore this issue, we cultured nondiabetic human and mice tissue in growth media containing high (25 mmol/l) or physiological (5 mmol/l) glucose conditions [43]. We found that high glucose reduced cell yields by ~50% while increasing reactive oxygen species production by ~25%. Interestingly, culture conditions had no effect on cell surface markers (CD90 or c-Kit %), susceptibility to apoptosis or cytokine production but reduced the ability of cells to improve heart function when transplanted into normoglycemic mice 1 week after LCA ligation. This observation is particularly important given that the “high glucose” conditions chosen are those found within standard Is cove's Modified Dulbecco's Medium while “physiologic glucose” conditions had to be custom made. Despite no obvious changes in cell surface markers, high glucose conditions likely alter the expressed transcriptome or proteome within genes/proteins involved in central metabolic pathways as cells adapt to a high glucose environment; highlighting the need to optimize stem cell culture conditions for their intended usage prior to implementation. To explore the preconditioning effect of donor hyperglycemia, the in vitro and in vivo behavior of heart-derived cells from patients with impaired glycemic control (HbA1c 10% ± 2%) were compared with cells from nondiabetic patients (HbA1c 6% ± 1%) [43]. Similar to the results seen in high glucose conditions, a history of poor glycemic control resulted in ~50% fewer cells cultured from myocardial biopsies, a reduced ability for cells to promote angiogenesis and a reduced ability to improve myocardial function after LCA ligation. Given these effects were partially reversed when the rate-limiting methylglyoxalase detoxification enzyme (glyoxalase-1) was overexpressed, we concluded these effects were attributable to accumulation and lasting adverse effects of reactive dicarbonyls- raising the prospect that further reversal may be possible through strategies targeting central metabolic pathways perturbed by altered substrate metabolism. Simple changes in culture oxygenation also have an impact on cell phenotype. Traditionally, cells are cultured in ~20% oxygen conditions which is much higher than the in vivo cardiac physiological microenvironment (~5% oxygen). The influence of ex vivo oxygen tension was evaluated by Li and colleagues who found that physiologic oxygen culture enhanced the genomic stability and proliferative capacity of heart-derived cells [44, 45]. Although physiological oxygenation had no effect on the cytokine production of CDCs, 5% oxygen cell culture conditions increased long-term cell retention (~2 fold) and myocardial function 3 weeks after cell injection into a mouse model of myocardial injury; an outcome that may reflect unmeasured increases in paracrine stimulation or unmeasured exosome production with altered miRNA payloads. Finally, akin to other noncardiac cell products, 24 hours of hypoxic preconditioning within 2% oxygen increases PI3-kinase/Akt signaling to improve cell-mediated repair of injured tissue [46]. Taken together, this data highlights the importance of avoiding metabolic preconditioning and the lasting impact of patient characteristics on the therapeutic potential of heart-derived cells. The Pleiotropic Cytokine Interleukin 6 Amongst the large number of cytokines produced by heart-derived cells, interleukin 6 (IL-6) is the most abundant cytokine produced with levels often 3–4 times greater than the next most abundant cytokine [35, 37, 47]. Nominally a “pro-inflammatory” cytokine, IL-6 promotes cardiomyocyte proliferation and survival while suppressing fibrosis and adverse remodeling after MI [48, 49]. However, we also noted that IL-6 production increased with accumulating medical comorbidities [37] and/or poor glycemic control [43] making the contribution of IL-6 to therapeutic repair uncertain. As shown in Figure 3A, RNA interference revealed that, instead of impairing cardiac function, increased IL-6 levels partially compensate for the loss of other cytokines and exosomes as knockdown decreased myocardial function and increased scar burden independent of donor comorbidities [47]. Mechanistically, IL-6 reduced apoptosis within injured myocardium 1 week after cell transplant (Fig. 3B) and increased the number of newly generated myocytes found within the peri-infarct region 3 weeks after cell injection. Given the IL-6 receptor is highly expressed on cardiac fibroblasts (~50%) and macrophages (~70%), fibroblast suppression and pro-healing macrophage polarization likely mediates many of these benefits (Fig. 3C). Figure 3 Open in new tabDownload slide Effect of IL-6 from explant-derived cells on ischemic-injured myocardium. (A): Comparison of the effect of IL-6 production by EDCs sourced from patients with low LTS (NT) n = 6 and IL-6 sh RNAi (shIL-6) n = 8 and high LTS (NT n = 6 and shIL-6 n = 9) scores on the echocardiographic ejection fraction of immunodeficient mice as compared to myocardial injection of the negative vehicle control (n = 8) or EDCs transduced with lentiviral SCR (n = 8). *p < .05 vs. NT EDCs at the same LTS score, # versus SCR EDCs. (B): Effects of IL-6 knockdown (shIL-6 EDCs) on the expression of anti (Bcl-2) and pro (Bax, P53) apoptotic transcripts within microdissected tissue from the infarct, infarct zone and posterior wall of SCID mice 2 weeks after left coronary artery ligation and 1 week after intra-myocardial injection of EDCs. *p < .05 versus NT EDCs, n = 4/group. (C): Effects of IL-6 on the number of newly generated myocytes (cTnT+/BrdU+ cells) within the infarct border zone using equivalent histological sections taken from immunodeficient mice treated with SCR or shIL-6 transduced high LTS score EDCs. *p < .05 versus SCR EDCs, n = 4/group. Adapted with permission from Mayfield et al. [47]. Abbreviations: BrdU, bromodeoxyuridine; cTNT+, cardiac troponin T+; EDC, explant-derived cell; IL-6, interleukin 6; LTS, long-term stratification; NT, nontransduced; SCR, scramble control; sh, short hairpin. Figure 3 Open in new tabDownload slide Effect of IL-6 from explant-derived cells on ischemic-injured myocardium. (A): Comparison of the effect of IL-6 production by EDCs sourced from patients with low LTS (NT) n = 6 and IL-6 sh RNAi (shIL-6) n = 8 and high LTS (NT n = 6 and shIL-6 n = 9) scores on the echocardiographic ejection fraction of immunodeficient mice as compared to myocardial injection of the negative vehicle control (n = 8) or EDCs transduced with lentiviral SCR (n = 8). *p < .05 vs. NT EDCs at the same LTS score, # versus SCR EDCs. (B): Effects of IL-6 knockdown (shIL-6 EDCs) on the expression of anti (Bcl-2) and pro (Bax, P53) apoptotic transcripts within microdissected tissue from the infarct, infarct zone and posterior wall of SCID mice 2 weeks after left coronary artery ligation and 1 week after intra-myocardial injection of EDCs. *p < .05 versus NT EDCs, n = 4/group. (C): Effects of IL-6 on the number of newly generated myocytes (cTnT+/BrdU+ cells) within the infarct border zone using equivalent histological sections taken from immunodeficient mice treated with SCR or shIL-6 transduced high LTS score EDCs. *p < .05 versus SCR EDCs, n = 4/group. Adapted with permission from Mayfield et al. [47]. Abbreviations: BrdU, bromodeoxyuridine; cTNT+, cardiac troponin T+; EDC, explant-derived cell; IL-6, interleukin 6; LTS, long-term stratification; NT, nontransduced; SCR, scramble control; sh, short hairpin. Collectively, this data suggests that numerous cytokines and exosomes (outlined below) produced by heart-derived cells overlap to stimulate multiple cardio-protective/pro-survival pathways simultaneously to synergistically improve cardiac function after myocardial injury. Exosomes and Heart-Derived Cells With the recent explosion of knowledge highlighting the importance of exosome transfer in autocrine and paracrine signaling, it seems inevitable that a portion of the benefits conferred by heart-derived cells are mediated through exosomes. Recently, two labs independently reported the importance of exosome-mediated protein and RNA transfer in therapeutic repair of injured myocardium by CDCs [50, 51]. Since then, a number of reports have emerged confirming injection of heart-derived exosomes replicates many of the benefits seen after cell transplant in both small [52, 53] and large [54] animal models. The mechanisms underlying exosome-mediated effects are complex as these packed nanovesicles contain numerous candidate proteins and RNAs capable of influencing postinfarct repair [51, 55]. Within the population of miRNAs found within CDCs, mir-146a is the most highly enriched transcript and replicates many of the antiapoptotic and cardiomyogenic effects of CDC exosomes without improving function or scar mass [50]. These findings highlight one of the important limitations of using a single miRNA as therapy given that not all the salutary benefits of cell therapy may be faithfully recapitulated while underscoring the complexity of exosomes that contain over 20,000 molecules needed in different combinations or at different times after injury to provide maximal benefit. Intriguingly, CDC exosomes also contain a substantial population of small noncoding yRNAs (~20% of total RNA) that dwarfs the miRNA population (~5% of total RNA) [52]. Application of the most abundant yRNA (EV-YF1, RNA central access: URS000072DA11) has been shown to reprogram host macrophages to increase IL-10 production which, in turn, modulates cardiac repair through apoptosis inhibition and suppression of inflammation. CDC-derived exosomes have also been shown to recruit host cardiac fibroblasts to boost local production of pro-healing cytokines (SDF1α and VEGF) and shift the miRNA signature of fibroblast exosomes to express transcripts implicated in reducing fibrosis and apoptosis [53]. Although a relation between exosome dose and cardiac function has yet to be established, the inverse relationship between exosome production and accumulating medical comorbidities hints that cell exosome production is intimately tied to treatment outcomes [37]. The Engraftment Hypothesis: Does Cell Dose Matter for Adult Cell Therapies? The heart is a formidable target to establish successful long-term engraftment of any transplanted cell [27]. Extracellular matrix remodeling, harsh inflammatory conditions, and nutrient/oxygen starvation conspire to limit cell retention after injection into a moving, strongly contracting, and inherently vascular organ. Remarkably, despite their ephemeral nature, heart-derived cells are able to provide paracrine pulses to injured heart tissue that stimulate endogenous repair mechanisms. Specifically, early work showed that acute engraftment of saline suspended heart-derived cells into coronary ligation models approaches 15% of the initial injectate while only 1%–2% of transplanted cells persist weeks after injection [56]. Given the dose–response relationships established between retained cells and subsequent myocardial function shown using cardiac [44, 57] and noncardiac [58] cells, early attention shifted towards using inert [56, 59] and functional [60, 61] biomaterials or magnetic cell trapping [62] as a means of boosting cell retention. All of these approaches prolong the residency of transplanted cells within the injured heart and, in the case of biomaterials, limit detachment induced cell loss secondary to enzymatic dissociation prior to intramyocardial injection. However, the number of transplanted cells that finally engraft even using biomaterials is small (~1–2,000) and unlikely to account for the cell-mediated benefits seen in myocardial function and scar size. As such, we explored the possibility that cell dose has finite effects on treatment outcomes by broadening the paracrine output of individual cells without altering cell retention [63]. This was accomplished by varying the density of an inert nanoporous gel (NPG) cocoon surrounding transplanted cells (Fig. 4A). Previously, we demonstrated that encapsulating EDCs within 3.5% NPG cocoons increases acute cell retention, decreases detachment induced cell loss and boosts cell treatment outcomes [59]. As shown in Figures 4B and 4C, we found that reducing NPG density from 3.5% to 2.5% decreased cocoon stiffness which, in turn, reduced the migratory capacity of encapsulated cells. While cocooning cells increased acute and long-term retention compared to injection of suspended cells, reducing NPG density had no effect on cell retention. Despite equivalent numbers of retained cells, reducing cocoon density provided cell treatment effects equivalent to intra-myocardial injection of suspended cells. In contrast, encapsulating within 3.5% cocoons markedly boosted myocardial function (Fig. 4D). We found that the cell-matrix-biomaterial interactions within 3.5% NPG cocoons prompted EDCs to produce greater amounts of cytokines, exosomes and miRNA (Fig. 4E) to increase the generation of new blood vessels and myocytes while limiting scar expansion. This data suggests that, while EDC dose plays a role in transplant outcomes, these benefits have limits and, once a critical dose is reached, further increases may not be possible until a more potent paracrine cell product is applied. Figure 4 Open in new tabDownload slide Biomaterials increase the paracrine potency of cells while changes in cell retention represent an epiphenomenon. (A): In the left panels, cocooned EDCs are shown after staining with actin filaments using phalloidin (green) with the dashed area indicating the area of increased magnification displayed in the bottom panel. The right panels are representative transmission electron microscopy images showing cocooned EDCs with annotations depicting the nucleus (N), cell, capsule and a membrane protrusion (arrow, scale bar 20 μm). (B): Atomic force microscopy measures showing the force-displacement curves for NPG capsules of varying density. (C): The left panels are representative displacement plots showing the path of cell migration when cocooned cells are placed in hypoxic low serum conditions. Shown in the right panel is grouped data summarizing the rate of 2D-cell migration as EDCs emerge from capsules of different NPG concentrations. (D): Echo analysis showing left ventricle ejection fraction of immunodeficient mice after cell or vehicle injection. *p < .05 versus vehicle, #p < .05 for 3.5% versus 2% NPG cocooned or suspended EDCs, n = 10–9 mice/group. (E): The left panels demonstrate the effect of increasing capsule density on exosome miRNAs. The right panel summarizes the effect of cocoon density on hierarchical clustering of differentially expressed miRNA. Adapted with permission from Kanda et al. [63]. Abbreviations: ADH, adherent EDC; EDC, explant-derived cell; LCA, left coronary artery; NPG, nanoporous gel. Figure 4 Open in new tabDownload slide Biomaterials increase the paracrine potency of cells while changes in cell retention represent an epiphenomenon. (A): In the left panels, cocooned EDCs are shown after staining with actin filaments using phalloidin (green) with the dashed area indicating the area of increased magnification displayed in the bottom panel. The right panels are representative transmission electron microscopy images showing cocooned EDCs with annotations depicting the nucleus (N), cell, capsule and a membrane protrusion (arrow, scale bar 20 μm). (B): Atomic force microscopy measures showing the force-displacement curves for NPG capsules of varying density. (C): The left panels are representative displacement plots showing the path of cell migration when cocooned cells are placed in hypoxic low serum conditions. Shown in the right panel is grouped data summarizing the rate of 2D-cell migration as EDCs emerge from capsules of different NPG concentrations. (D): Echo analysis showing left ventricle ejection fraction of immunodeficient mice after cell or vehicle injection. *p < .05 versus vehicle, #p < .05 for 3.5% versus 2% NPG cocooned or suspended EDCs, n = 10–9 mice/group. (E): The left panels demonstrate the effect of increasing capsule density on exosome miRNAs. The right panel summarizes the effect of cocoon density on hierarchical clustering of differentially expressed miRNA. Adapted with permission from Kanda et al. [63]. Abbreviations: ADH, adherent EDC; EDC, explant-derived cell; LCA, left coronary artery; NPG, nanoporous gel. It follows that, amongst the many strategies used to enhance adult cell potency dogmatic “cell dose” groupthink has distracted attention from the true paracrine mechanism(s) driving treatment outcomes. It is very likely that many of these cell rejuvenation papers increased indirect repair of injured myocardium alone without any meaningful contribution from retained cells. In the absence of quantitative cell tracking, comprehensive paracrine screening and targeted ablation transplanted cells, interpretation of these studies is difficult and unlikely to identify new avenues to advance the field. Synthetic Cell Strategies While soluble factors account for a sizable portion of the benefits seen after CDC injection, recent work has shown that direct cell-to-cell contact plays an important role in potentiating cell treatment effects as β1 integrin blockade reduces the ability of CDCs to promote myocyte cycling [64]. These insights prompted Tang and colleagues to explore the utility of dispensing with cells altogether by injecting synthetically engineered cell-mimicking microparticles (CMMPs) into injured hearts [65]. In this report, spheres (~20 μm diameter) made from Poly Lactic-co-Glycolic Acid were soaked in CDC conditioned media prior to being cloaked with sonicated CDC membrane particles to provide a synthetic reservoir of cytokines expressing a surface antigenic profile similar to native CDCs. When injected at the time of LCA ligation, CMMPs improved myocardial function to a degree indistinguishable from injection of suspended CDCs; thus, providing a synthetic off-the-shelf product that reflects the ex vivo expanded cell products without in culture time constraints. Interestingly, complementary work to take cells out of cell therapy has shown that the exosome-based paracrine effects of other cell types, such as induced pluripotent stem cells or induced pluripotent stem cell-derived cardiomyocytes, promote cardiac recovery through the distribution of cardio-protective pro-healing miRNAs within injured tissue [66, 67]. Despite arising from noncardiac cell types, this data will likely inform and guide additional research focusing on promoting endogenous cardiac repair. Ultimately, a noninvasive strategy targeting exosomes to areas of cardiac injury would be ideal [68] but if this novel intravenous method will supplant direct intra-myocardial injection of paracrine factories (i.e., cells) remains to be shown. Translational Considerations Small and large animal studies have shown heart-derived cells improve cardiac function by stimulating new vessel growth, the production of new cardiomyocytes and salvage of reversibly damaged myocardium [27, 69]. Invariably, these proof-of-concept models focus on the defining allogeneic or autologous cell effects using healthy animals soon after cardiac injury (<1 week). Evidence supporting the use of heart-derived cells even in the late phases after infarction (~4 weeks) is limited and these studies have shown the effect of delayed (+35 days post MI) delivery is reduced as compared to early (+7 days post MI) administration [36, 70, 71]. Early clinical trials prudently reflect the timing of these models as patient were received cells soon (1–6 months) after their first large infarct [13, 72]. With the advent of modern reperfusion therapies, large first infarcts that moderately reduce function to ejection fractions less than 45% (with normal being 50% and above) are increasingly rare. As such, the large number of contemporary patients potentially in need of cell therapy have suffered multiple infarcts and already undergone progressive adverse neurohormonal/structural remodeling. These patients also suffer from multiple comorbidities and advanced age further depleting the responsiveness of endogenous repair mechanisms. Thus, the effect of adding more cytokines or exosomes within this depleted/remodeled milieu deserves more attention prior to clinical translation once a promising strategy has been identified using reductionist uncomplicated models of recent cardiac injury. The early stuttering clinical progress of noncardiac cell therapies bear evidence for this need to use clinically realistic models and valid experimental methodology to identify promising strategies that enhance endogenous repair to ensure success in the clinic. Conclusion The past two decades have taught much about the potential benefits and limitations to cardiac cell therapy. Although adult cell products can differentiate and integrate into damaged heart tissue, with current application techniques, this is rare, and it is widely recognized that the current benefits seen after cell injection are largely attributable to transplanted cell sourced cytokines and exosomes. Thus, to realize their full potential, adult cell products should logically focus on fully exploiting these paracrine mediators of benefit. It is noteworthy that direct injection of cell sourced factors may replicate many of the benefits seen after cell injection. However, these factors only briefly pulse the injured myocardium and are soon cleared. In contrast, transplanted cells promise to deliver sustained doses of pro-healing factors in areas local to the injury which may be needed to reverse long-standing remodeling in response to injury. While the work outlined above has laid a promising foundation to enable cardiac cells to enter the clinic, the coming years will need to define the role of cells or defined factors to help treat the millions of patients worldwide with heart failure. Acknowledgments This work was supported by the Canadian Institutes of Health Research Clinician Scientist Award (MC2-121291) and the Heart and Stroke Foundation of Canada (NA-7346). 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Google Scholar Crossref Search ADS WorldCat © AlphaMed Press 2018 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Concise Review: Heart-Derived Cell Therapy 2.0: Paracrine Strategies to Increase Therapeutic Repair of Injured Myocardium
JF - Stem Cells
DO - 10.1002/stem.2910
DA - 2018-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/concise-review-heart-derived-cell-therapy-2-0-paracrine-strategies-to-5q2m0PbqD4
SP - 1794
EP - 1803
VL - 36
IS - 12
DP - DeepDyve
ER -