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Enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for treatment of myocardial infarction.
Regenerative potential of stem cells to repair acute infarction is limited. Improved hCPC survival, proliferation and differentiation into functional myocardium will increase efficacy and advance translational implementation of cardiac regeneration.
hCPCs isolated from myocardium of heart failure patients undergoing left ventricular assist device (LVAD) implantation are engineered to express green fluorescent protein (GFP; hCPCe) or Pim-1-GFP (hCPCeP). Functional tests of hCPC regenerative potential are performed with immunocompromised mice by intramyocardial adoptive transfer injection after infarction. Myocardial structure and function is monitored by echocardiographic and hemodynamic assessment for 20 weeks following delivery. hCPCe and hCPCeP expressing luciferase are followed by bioluminesence imaging (BLI) to non-invasively track persistence.
hCPCeP exhibit augmentation of reparative potential relative to hCPCe control cells as demonstrated by significantly increased proliferation coupled with amelioration of infarction injury and increased hemodynamic performance at 20 weeks post-transplantation. Concurrent with enhanced cardiac structure and function, hCPCeP demonstrate increased cellular engraftment and differentiation with improved vasculature and reduced infarct size. Enhanced persistence of hCPCeP versus hCPCe is revealed by BLI at up to 8 weeks post delivery.
Genetic engineering of hCPCs with Pim-1 enhances repair of damaged myocardium. Ex vivo gene delivery to modify stem cells has emerged as a viable option addressing current limitations in the field. This study demonstrates that efficacy of human CPCs from the failing myocardium can be safely and significantly enhanced through expression of Pim-1 kinase, setting the stage for use of engineered cells in preclinical settings.
The human heart harbors an adult stem cell population consistent with true characteristics of stemness such as self renewal (1), clonogenicity (2) and multilineage differentiation potential (3). These ‘cardiac stem cells’ populate the heart in highly conserved atrial and ventricular niches regulating myocyte turnover (4). Recent evidence demonstrates the ability of resident human cardiac cells to differentiate into mechanically integrated cardiomyocytes (3,5) as well as vascular smooth muscle and endothelial cells, thereby supporting cardiac regeneration (6). Adoptive transfer of human cardiac stem cells results in modest repair due in part to lack of survival, proliferation and commitment of the transplanted cells after myocardial infarction. Therapeutic stem cell performance is further complicated by the elderly target population for regenerative therapy which possesses a stem cell pool adversely affected by age concomitant with upregulation of senescence markers (4), shorter telomere length (3) and decreased metabolic activity (7). These detrimental insults collectively compromise regenerative ability of stem cells in aged population, limiting their use for autologous therapy.
Modification of human cardiac progenitor cells (hCPCs) to enhance proliferation, survival and commitment increases effectiveness and buttresses use of stem cells as a viable therapeutic modality. Ex vivo genetic modification is an effective strategy to enhance stem cell function (8,9). Previously our group has shown that Pim-1 kinase, a downstream effector of AKT, enhances cell survival (10), metabolic activity (11), attenuates apoptosis (12) and maintains mitochondrial integrity (11,13). Mechanistically, apoptotic proteins such as Bad (14) and cell cycle proteins including p21 (15) have been identified as Pim-1 substrates. In the heart, Pim-1 is induced as a consequence of stress or pathological insult (10). Pim-1 also positively regulates neovasculogenesis (16) that forms an integral part of the myocardial repair response. Proof of principle studies performed with murine CPCs in a syngeneic system demonstrate that Pim-1 augments reparative processes after myocardial injury with improved cellular survival, persistence and differentiation of engrafted cells into cardiac lineages 32 weeks after transplantation (17). However, potentiation of hCPC derived from heart failure patients presents a different challenge from the healthy young CPCs used in syngeneic murine studies. Utility of hCPC as a viable therapeutic option would be further improved by interventional strategies designed to overcome inherent limitations in aged or pathologically challenged myocardial tissue.
Applicability of genetic modification to the clinical setting requires progression into an experimental model with hCPC obtained from the target population of aged patients who would be candidates for regenerative therapy: individuals undergoing left ventricular assist device (LVAD) implantation as a bridge to transplant or destination therapy. In the present study, we demonstrate that hCPCs isolated from failing myocardium and modified with Pim-1 possess enhanced reparative potential relative to control hCPC. Improvements mediated by hCPC are evident structurally and functionally, with durable human cellular persistence, engraftment, and acquisition of phenotypic characteristics consistent with differentiated myocardium. These results validate the utility of Pim-1 kinase as a molecular interventional approach to enhance hCPC-mediated regeneration, even when derived from a failing human heart.
See online supplementary section for Materials and Methods.
hCPCs are negative for hematopoietic markers CD34, CD45, CD2, CD16 and CD31 and are positive for c-kit (Supplement Fig 1A-B). hCPCe and hCPCeP are transduced with lentiviral vectors Lv-egfp and Lv-egfp+pim1 (Supplement Fig 1C). Efficiency of modification following lentiviral transduction is 74.25% and 75.95% for hCPCe and hCPCeP respectively as measured by flow cytometric analyses for eGFP (Supplement Fig 1D). Expression of eGFP and Pim-1 in hCPCe and hCPCeP is confirmed by immunoblot analysis (Supplement Fig 1E). Karyotype analyses reveals normal chromosome content in either hCPCe or hCPCeP indicating normal mitotic chromosomal segregation in the genetically engineered cells (Supplement Fig 1F).
Proliferation is increased in hCPCeP relative to hCPCs and hCPCe (P<0.001) at day 3 as measured by CyQuant (Fig 1A). Conversely, using Pim-1 pharmacologic blocker quercetagetin, proliferation is abrogated at day 1 (P <0.01) and day 3 (P<0.001), demonstrating involvement of Pim-1 in the proliferative response (Fig 1B). hCPCeP also show increased metabolic activity compared to hCPC and hCPCe at day 3 (P<0.001) measured by MTT assay (Fig 1C). Similarly, relative TERT activity measured by TRAP assay is significantly improved (P <0.05) in hCPCeP as compared to hCPCe (Fig 1D). Increased levels of phospho-p21, a Pim-1 target substrate, confirm functional activity of expressed Pim-1 protein by immunoblot analysis (Fig 1E-F). Collectively, these results indicate that Pim-1 modification of hCPC confers phenotypic properties consistent with beneficial cellular signaling.
Markers of cardiogenic lineage commitment including MEF2C, vWF and GATA-6 are upregulated in hCPCeP relative to hCPCe after dexamethasone (Dex) treatment as confirmed by quantitativeRT-PCR analysis (Fig 2A) and immunocytochemistry (Fig. 2B). MEF2C and GATA-6 signal are absent in hCPCe or hCPCeP before Dex treatment with sparse reactivity for vWF in hCPCeP (Fig 2B) and marked increases in immunolabeling for all three markers as well as morphologic remodeling of hCPCe and hCPCeP following Dex exposure (Fig. 2C). Morphological remodeling (flattening) of cells treated with Dex is consistent with previous findings (17-19). These results indicate augmentation of lineage commitment signals in hCPCeP relative to control hCPCe following rudimentary differentiation induction with Dex.
Delivery of hCPCeP, hCPCe or vehicle alone by intramyocardial injection into SCID mice concurrent with myocardial infarction was performed to determine reparative potential. Loss of function in all groups indicates comparability of infarction damage as assessed at 1 week post challenge by echocardiography (Fig 3A-B). Within four weeks after cell injection, myocardial function is significantly improved (P<0.001; supplementary Table 3) in hearts of mice receiving either hCPCe or hCPCeP compared to vehicle as measured by echocardiographic assessment of ejection fraction (EF) or fractional shortening (FS) (Fig. 3A-B). Differences in myocardial function between hCPCe and hCPCeP at 4 weeks after transplantation are not significantly different (P>0.05; supplementary table 3). However, EF and FS performance improve in the hCPCeP group from 4 to 8 weeks post-delivery, in contrast to depressed contractility for hCPCe-treated mice that is not significantly different (P>0.05) from the vehicle group (Fig 3A-B). Myocardial contractile performance of hearts receiving hCPCeP increases by 1.81 fold in EF and 1.86 fold in FS compared to hCPCe at 20 weeks after transplantation. Hemodynamic parameters are also significantly improved in hCPCeP-treated hearts compared to hCPCe (P<0.01) or vehicle (P<0.001) at 20 weeks after transplantation. hCPCeP treated hearts increase dp/dtmax and dp/dtmin by 1.29 fold and 1.23 fold, respectively (Fig 3C) together with a 1.37 fold increase in left ventricular developed pressure (LVDP) (Fig 3D) relative to hCPCe. Collectively, these results demonstrate enhanced capacity of hCPCeP to preserve and/or restore myocardial function following infarction injury.
Improvement of hemodynamic performance in hearts receiving hCPCeP (Fig 3) is accompanied by evidence of cardiogenic lineage commitment. Cardiomyocyte immunoreactivity with α-sarcomeric actin labeling in hearts receiving hCPCeP demonstrates cardiogenic commitment, together with coincident eGFP signal indicative of derivation from hCPCeP. Human origin of cells in myocardial sections from mice is evident by immunolabeling for eGFP colocalized with human-specific mitochondrial marker or by detection of characteristic repetitive Alu DNA sequence (Supplement Fig 2). hCPCeP shows a significant 2.32 fold increase (P<0.05) in telomere length following adoptive transfer to infarcted hearts relative to hCPCe at 20 weeks after transplantation consistent with a youthful cellular phenotype (Fig 4A-C). Infarction size is significantly smaller at 20 weeks in hCPCeP transplanted mice compared to hCPCe. Infarction damage involving 61.3% of the left ventricular free wall in hearts receiving hCPCeP compares favorably to 84.3% in hearts receiving hCPCe (p<0.05). Infarct size is not significantly different in hearts receiving either vehicle or hCPCe (Fig 4D-E). Presence of c-kit+/GFP+ cells derived from the adoptively transferred population increases 4.0 fold in hearts receiving hCPCeP relative to hCPCe at 12 weeks after delivery. Total c-kit+ cell number is significantly higher by 1.75 fold (P<0.05) in heart sections from hCPCeP relative to hearts receiving hCPCe at 12 weeks (Fig 4F-I). New myocyte formation in these hearts is identified by eGFP signal together with sarcomeric actin staining after 12 weeks of transplantation (Figure 5). New vessel formation is evident by coincidence of eGFP immunolabeling with smooth muscle actin (SM22) to label vascular walls as well as von Willebrand Factor (vWF) to label endothelial vessel lining (Fig 5). Myocardial sections from hearts receiving hCPCeP exhibit 28% GFP+/sm22+ cells versus 17% in hCPCe (Fig 5D). Similarly, hCPCeP-treated hearts possess 22% GFP+/vWF+ cells compared to 12% in hCPCe. Expression of Pim-1 is maintained for at least 12 weeks after delivery with increased immunolabeling for Pim-1 (Supplement Fig 3). Collectively, these results demonstrate enhance ability of hCPCeP to survive and proliferate, significantly augmenting angiogenesis and myogenesis in the infarcted heart.
hCPC persistence in vivo is longitudinally assessed over an 8 week period by BLI of luciferase signal in hearts receiving either hCPCe or hCPCeP transduced with Luc reporter construct (Fig 6A) immediately following infarction injury. Both hCPCe-Luc or hCPCeP-Luc produce robust BLI signal at day 2 in all recipient animals indicative of successful cell delivery to the heart. BLI signal remains detectable in the hCPCeP-Luc cohort throughout 56 days post-delivery (Fig 6B-C), in stark contrast to loss of signal by 14 days after delivery in the hCPCe-Luc group. These results demonstrate superior persistence of hCPCeP-Luc following delivery, particularly in the critical window of 2-4 weeks post infarction.
Myocardial contractile performance impairment is initially comparable shortly following cardiomyopathic challenge in cohorts of hCPCe-Luc or hCPCeP-Luc indicative of similar infarction injury by echocardiographic assessment. Subsequently, mice receiving hCPCeP-Luc exhibit improvement in FS, left ventricular end diastolic dimension (LVEDd), left ventricular end systolic dimension (LVESd), and anterior wall thickness (AW) at 8 weeks post-injury (Fig 7A-D; P<0.05). Magnetic resonance imaging (MRI) at 1 and 8 weeks post-infarction substantiate echocardiographic results, revealing improved hemodynamic parameters at 8 weeks in mice receiving hCPCeP Luc compared to hCPCe Luc (Fig 7E-H) with respect to left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV), and EF (P<0.05). P values for all parameters by MRI or echocardiography are provided (supplement Table 4). hCPCeP-Luc treated hearts exhibit 22.5% increase in anterior wall dimension with 10% decrease in LVEDd and 20.6% decrease in LVESd by echocardiography 8 weeks after transplantation relative to hCPCe-Luc.
Discovery of hCPC contributing to cardiomyogenesis within the heart and supporting myocardial repair has revolutionized the conceptual view of treatment for heart disease, as supported by the capacity of hCPCs to form functionally integrated cardiomyocytes and vasculature (20). However, survival and persistence of adoptively transferred hCPC used for therapeutic purposes remains a major concern, particularly when the donor cell population used for autologous therapy is derived from pathologically stressed myocardium. Regenerative capabilities of adult hCPCs are likely to be impaired by age (21) and disease (22) limiting the reparative and regenerative potential of these autologously-derived cells. Ex vivo modification or preconditioning has been shown to prime adoptively transferred cells for myocardial repair (23,24). Genetic modification to augment cellular survival and proliferation is a viable molecular interventional strategy as previously published by our group for syngeneic murine CPCs (17,25). This study addresses a critical issue by demonstrating that Pim-1 modification augments regenerative and reparative potential of hCPC derived from heart failure patients, bringing this conceptual approach another step closer to therapeutic implementation.
hCPC isolated from heart failure patients amenable to modification with Pim-1 also display phenotypic characteristics consistent with enhanced survival, proliferation, and reversal of senescent characteristics. hCPCeP exhibit high proliferation and metabolic activity in vitro (Fig 1A-C). Telomere lengths of hCPCeP are also preserved, suggesting an important role of Pim-1 in maintaining telomere length (Fig 1D) to antagonize cellular senescence that is currently being investigated by our group. Pim-1 also increases phosphorylation of p21 (Fig 1E-F) a cyclin dependent kinase inhibitor, as well as stabilizing c-Myc and the nuclear mitotic apparatus (26). Increased survival of Pim-1 engineered cells is likely due to ability of the kinase to promote proliferation and attenuate apoptotic signaling (10,27), moderating enhanced proliferation and persistence of the transplanted cells to augment the reparative process.
Potentiation of hCPC from heart failure patients undergoing LVAD implantation reported in our study addresses the heretofore critical unanswered issue of whether aged hCPC from pathologically damaged myocardium would retain capacity to benefit from genetic engineering. Indeed, heart failure associated with aging has been proposed to be a “stem cell disease” characterized by impaired functional reserve of the endogenous stem cell pool due to exhaustion, senescence, depletion, or inability to cope with the environmental stressors (28). Recent clinical results using autologous hCPC to restore myocardial performance in the SCIPIO trial show that the c-kit+ cell population is capable of mediating improvement in both EF as well as reduction in infarct size (29). With unequivocal evidence of clinical relevance for treatment of heart failure using c-kit+ hCPC, the future of hCPC therapy will inevitably turn toward assessment of approaches to enhance the regenerative process.
Can Pim-1 be considered an appropriate molecular interventional strategy for enhancing cardiogenesis? Pim-1 induces proliferation of endothelial (16) and vascular smooth muscle (30) cells as well as promoting lineage commitment as evidenced by increased expression of cardiogenic transcripts in Pim-1 engineered CPC (Fig 2 and (17)). hCPCeP express vWF transcript before and after differentiation in vitro and after transplantation into damaged myocardium. Moreover, clear evidence of myocytes derived from adoptively transferred hCPCeP is revealed after 20 weeks following delivery by immunohistochemistry (Fig 5 and Supplement Fig 2). Persistence, expansion, and integration of the hCPCeP into myocardial tissue translates into progressive improvement in myocardial structure and function evident up to 20 weeks post-delivery relative to hCPCe (Fig. 3 and and6).6). The durability of repair together with the superior improvement of functional parameters of myocardial hemodynamic performance support the use of Pim-1 as a plausible molecular strategy to enhance myocardial regeneration with modified hCPC.
Despite pro-proliferative effects mediated by Pim-1, oncogenic transformation has never been observed in any of our human samples and all engineered hCPCeP were amenable to differentiation in vitro that resulted in acquisition of post-mitotic characteristics (Fig 2). In vivo studies show cardiogenic commitment of hCPCeP to all three essential cell lineages for reconstitution of myocardial tissue: cardiomyocytes, vasculature, and endothelium (Fig 5). Furthermore, karyotypic analyses show normal chromosome content in hCPCeP (Supplement Fig 1F). Although oncogenic risk needs to be carefully evaluated when genetic engineering is proposed, it is important to consider that lentiviral vectors have also made their way to clinics for therapies (31) including advanced forms of HIV infections(32), Parkinson’s disease (33) and inherited disorders affecting hematopoietic cells (34). Also, lentiviral vectors have integration sites away from transcriptional regulatory sites making them a safe therapeutic option (35). These findings are in stark contrast to published literature showing chromosomal abnormalities in certain ES and iPS cells where oncogenic transformation remains a significant barrier to therapeutic implementation (36,37).
Clinical trials using bone marrow-derived stem cells (TAC-HFT) (38) and hCPC (29) effectively demonstrate improved cardiac function after transplantation of stem cells. Narrow inclusion criteria for these clinical trials leave open the issue as to whether initially promising findings will be broadly applicable to the much greater segment of patients suffering from debilitating consequences of aging and multiple concurrent cardiac problems. Nevertheless, despite severe deterioration of myocardium necessitating surgical intervention and mechanical assist device implantation in the 68 year old source of our hCPC, Pim-1 expression effectively increased myocardial repair in immunosuppressed murine recipients, whereas hCPCe without Pim-1 expression are ineffective. Persistence of BLI signal of hCPCeP until approximately two months after delivery (Fig 6) reinforces earlier findings with syngeneic CPCeP in mice (17) and supports the notion that hCPCeP become permanently integrated into myocardium unlike control hCPCe undetectable after two weeks following delivery. Moreover, notably enhanced signal from hCPCeP at 2-4 weeks following transfer coincides with timing for recruitment of endogenous repair in the infarcted heart (25). Increased presence of total c-kit+ cells in the myocardium of hearts receiving hCPCeP (Fig 4F-I) likely reflects augmentation of endogenous repair previously postulated to play a critical role in mediating myocardial repair (39). The ensuing progressive loss of BLI signal in hearts of recipient mice receiving hCPCeP over two months could be caused by ongoing molecular and cellular processes such as promoter silencing (29,40) or rejection of the allogenic human cells from remnants of non-T, non-B cell immunity in the NOD/SCID mice (41). It is reasonable to posit that persistence of hCPCeP could be further improved with autologous transfer as well as by using humanized expression vectors such as minicircles that can persist for months in non-dividing cells without integrating into chromatin, thereby minimizing concerns of insertional mutagenesis (42). Ongoing studies are evaluating minicircle technology and other protocol modifications to further refine the safety and efficacy of Pim-1 genetic engineering to enhance myocardial regeneration.
We thank all members of the Sussman laboratory for their helpful discussions and technical support.
GRANTS: M.A.S was supported by National Institutes of Health grants R21HL102714, R01HL067245, R37HL091102, P01HL085577, RC1HL100891, R21HL102613, R21 HL104544, and R01HL105759. J.C.W was supported by RC1HL100891 and R01EB009689.
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