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Myocardial infarction (MI) is the irreversible cardiomyocyte death resulting from prolonged oxygen deprivation due to obstructed blood supply (ischemia), leading to contractile dysfunction and cardiac remodeling. In recent decades, stem cell transplantation has been extensively investigated for the repair of injured heart in animal studies and clinical trials (Kanelidis et al., 2017; Gyongyosi et al., 2018). Among cell-based therapies in clinical development, mesenchymal progenitor cells (MPCs) are attractive candidates due to their multi-lineage potential and immunomodulatory properties (Bagno et al., 2018). However, low quality (e.g., reduced proliferative ability and increased cellular senescence at late passages) and heterogeneous cell sources, as well as poor retention and survival rate of transplanted MPCs in an in vivo niche present obstacles towards broader clinical applications (Nguyen et al., 2016; Li et al., 2020).
Forkhead box O3 (FOXO3), one of the most prominent genes related to human longevity, functions in diverse biological processes including DNA repair, oxidative stress response, cell proliferation and cellular senescence (Liu et al., 2018). We have previously reported that FOXO3 loss drives primate arterial aging and that constitutive activation of FOXO3 in human embryonic stem cell (hESC)-derived MPCs enhances their stress resistance and attenuates cellular senescence (Yan et al., 2019; Zhang et al., 2020). Here, we evaluated the cardiac repair after MI in immunodeficient mice intramyocardially transplanted with FOXO3-genetically-enhanced MPCs (FOXO3-GE-MPCs).
FOXO3-GE-MPCs were generated by directed differentiation of hESCs in which two FOXO3 phosphorylation sites were replaced with alanine (S253A, S315A) using targeted gene editing (Fig. S1A and S1B). The engineered FOXO3 could not be phosphorylated by AKT at S253 or S315 and was therefore constitutively active in the nucleus (Yan et al., 2019). Consistent with previous observations (Yan et al., 2019), FOXO3-GE-MPCs exhibited increased proliferation and decreased senescence-associated (SA)-β-gal activity relative to wildtype MPCs (WT-MPCs) (Fig. S1C–E). We next investigated whether FOXO3-GE-MPCs would be retained longer in the heart than WT-MPCs when intramyocardially delivered at the initiation of myocardial ischemia. In vivo imaging of luciferase-labelled MPCs revealed that transplanted WT-MPCs diminished within five days whereas FOXO3-GE-MPCs remained detectable until day 11 (Fig. 1A). Due to the limited resolution and sensitivity of in vivo imaging, we performed immunofluorescence staining of the human Golgi marker hTGN46 and RT-PCR of human GAPDH to further detect the transplanted cells in ischemic hearts and found that FOXO3 enhancement prolonged MPC retention up to 4 weeks after MI (Fig. 1B and 1C).
Next, we used transthoracic echocardiography to explore whether FOXO3-GE-MPCs could ameliorate cardiac dysfunction and left ventricular (LV) remodeling after MI. In control mice (MI + vehicle group), we observed decreased cardiac contractility along with enlarged LV chamber at 4 weeks after MI. These effects were partially reversed by the transplantation with FOXO3-GE-MPCs, but not WT-MPCs (Fig. 1D). Similarly, an MI-induced increase in heart to body weight ratio and a decrease in running distance (Fig. 1E), along with cardiac fibrosis, compensatory hypertrophy and cardiomyocyte apoptosis, were all ameliorated only by FOXO3-GE-MPC transplantation (Figs. 1F–H and S1F). Collectively, our data indicate that FOXO3 enhancement promotes cardiac repair by MPCs after MI, suggesting that FOXO3-GE-MPCs may provide effective biomaterials for stem cell-based therapy against ischemic heart diseases.
To dissect the underlying mechanisms of cardiac repair by FOXO3-GE-MPCs, we performed RNA-seq analysis of heart tissues of the infarct border zone (Figs. 2A, 2B and S1G–J) and identified a panel of MI-upregulated genes that were reversed by the transplantation of FOXO3-GE-MPCs, including those involved in inflammatory response (Fig. 2B). In addition, RelA (p65) that is a subunit of the NF-κB transcription complex (Wang et al., 2018) was upregulated in ischemic hearts and its upregulation was attenuated only by the transplantation with FOXO3-GE-MPCs (Figs. 2C and S1K). Some of the MI-upregulated genes rescued upon FOXO3-GE-MPC transplantation were enriched in NF-κB pathway (Fig. S1L). Furthermore, several NF-κB target genes including Cxcl13, Mmp9, Itgam, Tlr2 and Hmox1 were upregulated after MI and attenuated by FOXO3-GE-MPC delivery as verified by RT-qPCR (Fig. S2A). Likewise, ORF1p, which is encoded by the autonomous non-LTR retrotransposon LINE-1 and involved in the induction of IFN-I and other pro-inflammatory cytokines (De Cecco et al., 2019), was increased upon MI and decreased by the transplantation of FOXO3-GE-MPCs (Figs. 2C and S2B). The serum levels of pro-inflammatory factors including TNF-α, IL-1β and IFN-γ were also increased after MI and reversed only by FOXO3-GE-MPCs (Fig. 2D). Altogether, these findings suggest that the transplantation of FOXO3-GE-MPCs attenuates inflammatory response after MI, which may partially explain the cardioprotective effects of FOXO3-GE-MPCs.
Given that neovascularization is essential for the repair of heart damage, we investigated whether it might underlie the cardioprotective effects of FOXO3-GE-MPCs in ischemic hearts. Indeed, we found more α-SMA-positive cells and CD31-positive cells in the infarct border zone of mouse hearts transplanted with FOXO3-GE-MPCs at 4 weeks after MI as compared to those treated with vehicle or WT-MPCs (Figs. 2E, S2C and S2D), suggesting that neovascularization may occur in the infarct border zone upon the transplantation with FOXO3-GE-MPCs and facilitate cardiac repair. To further dissect how neovascularization could be induced by the transplanted FOXO3-GE-MPCs, we evaluated the possible paracrine effects of FOXO3-GE-MPCs by culturing human aortic endothelial cells (HAECs) in different media. Improved migration and tube formation capacities along with an increasing trend in proliferative ability were observed in HAECs cultured in conditioned medium harvested from FOXO3-GE-MPCs relative to HAECs cultured in fresh MPC medium or conditioned medium harvested from WT-MPCs (Figs. 2F and S2E–G). Consistently, RNA-seq analysis of FOXO3-GE-MPCs revealed the upregulation of a list of angiogenic genes as compared to those in their wildtype counterparts, including secretory factors ESM1, IGF2 and FGF16 that were verified by RT-qPCR (Fig. 2G and 2H). Altogether, these results suggest that FOXO3-GE-MPCs promote cardiac repair after MI at least partially in a paracrine angiogenic manner.
In this study, we demonstrated that intramyocardial transplantation of FOXO3-GE-MPCs exhibited enhanced cardioprotective effects than WT-MPCs did against myocardial infarction in mice. MPCs have been used in preclinical and clinical settings against ischemic cardiac diseases (Kanelidis et al., 2017; Bagno et al., 2018; Gyongyosi et al., 2018). However, primary MPCs isolated from various human tissues, including bone marrow, adipose tissue and umbilical cord, possess diverse characteristics that aggravate cellular heterogeneity, likely resulting in batch-dependent effects (Le Blanc and Davies, 2018). The age of the donor may also affect the quality of primary MPCs as it has been reported that MPCs from older donors proliferate slowly and partially lose their stem cell characteristics (Block et al., 2017). In addition, upon serial passaging, primary MPCs exhibit the onset of senescence phenotypes and progressive loss of self-renewal and differentiation abilities, further compromising the quality and quantity of MPCs for cell therapy. By comparison, directed differentiation from pluripotent stem cells has emerged as a new strategy to generate a large number of high-quality MPCs, providing unprecedented and valuable cell resources for preclinical and clinical applications. Here, our study indicates that genetic activation of FOXO3 represents a novel strategy to generate even superior and safer cell materials for MPC-based therapies against ischemic cardiac diseases (Fig. 2I).
A major challenge of MPC-based therapy resides in the massive loss of transplanted cells upon delivery, which is a yet-to-be-resolved issue that significantly compromises the therapeutic benefits. The poor retention may be attributed to various intrinsic and extrinsic factors such as pro-apoptotic stressors, inflammation, hypoxia, and oxidative stress (Salazar-Noratto et al., 2020). Genetic activation of FOXO3 has been demonstrated to increase the resistance of MPCs to oxidative stress-induced apoptosis (Yan et al., 2019). Here, we explored whether constitutive activation of FOXO3 in grafted MPCs may be beneficial for counteracting the hostile microenvironment in an ischemic context and found that FOXO3-GE-MPCs were retained longer than WT-MPCs, along with improved therapeutic effects in ischemic heart. Likewise, upregulation and activation of FOXO3 have been reported in renal tubular cells under hypoxic conditions to protect kidney from ischemic injury (Li et al., 2019). Mechanistically, growing evidence indicates that the protective role of FOXO3 against tissue damage may be associated with the expression of antioxidant enzymes and the activation of other pro-survival pathways (Lim et al., 2017; Yan et al., 2019). Therefore, FOXO3-GE-MPCs may provide a feasible option for more effective preclinical and clinical applications targeting ischemic diseases.
Notably, vascular reconstruction has been identified as a key event for the repair of ischemic heart and we have reported downregulation of FOXO3 as a key driver for primate vascular endothelial aging and activation of FOXO3 conferring vascular protection (Yan et al., 2019; Zhang et al., 2020). In this study, we showed that FOXO3-GE-MPCs promoted angiogenesis in the ischemic border zone at least partially via the secretion of pro-angiogenic factors, as evidenced by the higher expression levels of ESM1, FGF16 and IGF2 in FOXO3-GE-MPCs compared with those in WT-MPCs. Likewise, enhanced migration, clonal expansion and tube formation capacities of HAECs were observed upon the incubation with conditioned medium from FOXO3-GE-MPCs when compared with those in conditioned medium from WT-MPCs, further supporting the notion of possible paracrine effects by FOXO3-GE-MPCs in promoting angiogenesis and cardiac repair against ischemic injury.
In conclusion, genetic activation of FOXO3 extends the retention time of hESC-derived MPCs upon transplantation and confers better therapeutic effects in a mouse model of myocardial infarction. This new finding, together with the previous observations that FOXO3-enhanced hMPCs are resistant to oncogenic transformation (Yan et al., 2019), may support for the safety and effectiveness of using these cells to treat human myocardial infarction diseases in the future.
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FOOTNOTES
The authors acknowledge Bai L, Chu Q, Bai R, Lu J, Yang Y and Ma S for administrative assistance, Jia J (IBP, CAS) for his help with FACS experiments, Xu Y (IBP, CAS) for her help in optical in vivo tracing, Hao J (IBP, CAS) for pathological analysis, Li W, Jia J (Xuanwu Hospital Capital Medical University), Jiao J (IOZ, CAS), Shi X, Wu X, Wang M, Yang S, Duo S, Du K, and Zhou L (IBP, CAS) for the management of laboratory animals. This work was supported by the National Key Research and Development Program of China (2018YFC2000100), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010100), the National Key Research and Development Program of China (2017YFA0102802, 2017YFA0103304, 2018YFA0107203), the National Natural Science Foundation of China (Grant Nos. 81801370, 81625009, 91749202, 81861168034, 81921006, 31671429, 91949209, 91749123, 81671377, 81822018, 81870228, 81922027, 81701388, 81901433, 91849132), the Program of the Beijing Municipal Science and Technology Commission (Z191100001519005), Beijing Natural Science Foundation (Z190019), Beijing Municipal Commission of Health and Family Planning (PXM2018_026283_000002), Advanced Innovation Center for Human Brain Protection (3500-1192012), the Key Research Program of the Chinese Academy of Sciences (KFZD-SW-221), K.C. Wong Education Foundation (GJTD-2019-06, GJTD-2019-08), Youth Innovation Promotion Association of CAS, China Postdoctoral Science Foundation (2018M640154), CAMS Innovation Fund for Medical Sciences (2018-I2M-1-002), the State Key Laboratory of Stem Cell and Reproductive Biology and the State Key Laboratory of Membrane Biology. Work in the laboratory of J.C.I.B. was supported by The Moxie Foundation.
Jinghui Lei, Si Wang, Wang Kang, Qun Chu, Zunpeng Liu, Liang Sun, Yun Ji, Concepcion Rodriguez Esteban Esteban, Yan Yao, Juan Carlos Izpisua Belmonte, Piu Chan, Guang-Hui Liu, Weiqi Zhang, Moshi Song and Jing Qu declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
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Lei, J., Wang, S., Kang, W. et al. FOXO3-engineered human mesenchymal progenitor cells efficiently promote cardiac repair after myocardial infarction. Protein Cell 12, 145–151 (2021). https://doi.org/10.1007/s13238-020-00779-7
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DOI: https://doi.org/10.1007/s13238-020-00779-7