Engineering HiPSC-CM And HiPSC-EC Laden 3D Nanofibrous Splenic Hydrogel For Cardiac Repair After Myocardial Infarction

Background:Cardiovascular diseases（CVD）remain the leading cause of global mortality and disease burden nowadays.The acute loss of functional myocardium after myocardial infarction（MI）and limited capability of regeneration result in irreversible cardiac damages.Treatment of myocardial infarction（MI）remains a great clinical challenge due to the limited myocardial regeneration after MI.Recently,cell therapy based on human induced pluripotent stem cell（hi PSC）derived cardiomyocytes（CMs）or endothelial cells（ECs）,has become a promising strategy to obtain a large amount of transplanted cells for cardiac repair after myocardial infarction（MI）.However,low-efficiency cell delivery caused by low retention of cell grafts and oxidative stress in the MI microenvironment,remains a significant challenge in the cell therapies.Previous studies have reported that injectable hydrogel could be used as effective carriers for delivering therapeutic cells and biomolecules to promote tissue regeneration via a minimally invasive procedure.ECM hydrogels derived from different kinds of ECMs have been reported to exhibit tissue specificity in tissue regeneration and repair.The spleen serves as a haematopoietic stem cell niche and secretes cardioprotective factors after MI,but it is unclear whether it could be used for human pluripotent stem cells（hi PSCs）cultivation and providing a proper microenvironment for cell grafts against the ischemic environment.Objectives:Here,we developed a nanofibrous porcine splenic extracellular matrix hydrogel（Sp Gel）.We hypothesized that the splenic matrix may provide a platform for the culture and differentiation of i PSCs to generate induced endothelial cells（i ECs）and cardiomyocytes（i CMs）,as well as an efficient carrier for delivering i PSC-derived cardiovascular cells to ischemic mouse hearts.Methods:1.Preparation and characterization of splenic acellular matrix hydrogels.To generate an injectable form of Sp Gel,porcine spleen tissue was decellularized,lyophilized,enzymatically digested to form splenic acellular matrix hydrogel solution.H&E and Masson staining and DNA concentration measurement were conducted to demonstrate decellularization.Picrosirius Red,Verhoeff’s and Alcian Blue stainings were conducted to confirm Sp Gel components.To identify tissue-specific protein components in the Sp Gel,label-free MS-based proteomics analysis was conducted.SEM analysis was conducted to demonstrate the microstructure of Sp Gel.The viscoelastic properties of Sp Gel were tested with a rheometer.Cytocompatibility and biodegradability of Sp Gel were also analyzed through a CCK-8 assay and subcutaneous implantation.2.Cultivation and differentiation of hi PSCs into i ECs and i CMs on splenic acellular matrix hydrogels.2.1 For i ECs differentiation,flow cytometry,immunostaining and q PCR analysis were conducted to demonstrate the differentiation of hi PSCs into i ECs on splenic acellular matrix hydrogels.SEM analysis was conducted to reveal the morphology of i ECs on the nanofibrous Sp Gel.Functional characteristics of ECs induced on Sp Gel were confirmed by tube formation capacity test.2.2 For i CMs differentiation,immunostaining and q PCR analysis were conducted to demonstrate the differentiation of hi PSCs into i CMs on splenic acellular matrix hydrogels.The morphology of i CMs on Sp Gel was shown by SEM.Contractile frequency and electrophysiological features of i CMs induced on Sp Gel were tested.The ultrastructure of i CMs generated on Sp Gel or Matrigel-covered glass was characterized by transmission electron microscopy.3.Cytoprotective effects of Sp Gel encapsulation against H₂O₂.i PSC-derived ECs and i PSC-derived CMs were harvested from Sp Gel-covered plates and encapsulated within the nanofibrous Sp Gel for 3D culture.200μM H₂O₂ was added to induce oxidative stress damage,and cytoprotective effects of Sp Gel on the survival of i ECs/i CMs were explored by live/dead staining following 2 h of H₂O₂treatment.The ultrastructure of cells within or without Sp Gel after treatment with H₂O₂was characterized by transmission electron microscopy.4.Cardiac repair by Sp Gel encapsulated i ECs/i CMs injection after myocardial infarction.A mouse model of acute myocardial infarction was used to investigate cell retention and therapeutic effects of i EC/i CM loaded Sp Gel after intramyocardial injection.Survival and retention analysis of cell grafts was tested by IVIS.H&E staining and immunostaining analyses were also conducted to evaluate the retention rate of PKH26-i ECs/i CMs in Sp Gel-i ECs/i CMs group and PBS-i ECs/i CMs group.To evaluate the therapeutic effects of grafts on cardiac function in MI mice,echocardiography was performed 4 weeks post injection.Masson’s stainings were conducted to estimate cardiac fibrosis in different groups.Immunostaining analyses were performed to compare neovascularization in the infarct region in different groups.5.The recruitment of regulatory T cells（Treg）by CCL22 over-expressed gene modified hi PSCs.Hi PSCs were transfected in vitro by constructing a lentiviral vector overexpressing CCL22.The expression of CCL22 in Hi PSC-CCL22 cells was analyzed by q PCR and immunofluorescence staining analyses.The secretion of CCL22 in the supernatant of hi PSC-CCL22 cells was verified by ELISA.Transwell chemotactic assay was used to detect the chemotactic effect of hi PSC-CCL22 cells culture supernatant on Treg.For the verification of multidirectional differentiation ability of hi PSC-CCL22 cells,hi PSC-CCL22 cells were induced to differentiate into cardiomyocytes and endothelial cells.Results1.We developed a thermoresponsive nanofibrous splenic matrix hydrogel（Sp Gel）successfully,which could be gelated at 37°C.H&E and Masson staining and DNA concentration measurement demonstrated effective decellularization.Picrosirius Red,Verhoeff’s and and Alcian Blue stainings confirmed the retention of collagen,elastin and glycosaminoglycans（GAGs）in Sp Gel respectively.Proteomics analysis demonstrated that1481 protein species were identified in Sp Gel,Gene Ontology（GO）enrichment analyses indicated that proteins related to the biological processes of small molecule metabolic process,extracellular structure organization,cell-substrate adhesion,cell junction organization,and cell migration were highly expressed in Sp Gel.Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analyses demonstrated that apoptosis,gap junctions,tight junctions,adherens junctions,focal adhesion,cardiac muscle contraction,adrenergic signaling in CMs and the Wnt signaling pathway related proteins were highly expressed in Sp Gel.SEM analysis demonstrated the porous nanofibrous structure of Sp Gel with an average fiber diameter of 66.67±10.60 nm.Hydrogel rheological measurements of Sp Gel showed the gelation process by rapidly increasing the temperature from 4°C to 37°C.Cytocompatibility of Sp Gel by a CCK-8 assay confirmed excellent properties of biocompatibility,and biodegradability of Sp Gel implied that Sp Gel is degradable.2.Immunostaining analysis comfirmed the pluripotency of hi PSCs（OCT4+）after seeding on Sp Gel-covered dishes,and the effective differentiation of i ECs（CD31+,v WF+）on Sp Gel.FACS analysis revealed that the proportion of CD31+/CD144+cells increased from 0.05%to 94.6%after differentiation.q PCR confirmed the low expression of pluripotent marker OCT4 in i ECs and high expression of endothelial marker CD31,CD144,v WF and CDH5.SEM images suggested the adhesion and survival of i ECs on Sp Gel,as well as cell-matrix interactions.Tube formation capacity of i ECs and HUVECs confirmed no significant variance of functional characteristics between i ECs and HUVECs.3.Immunofluorescence staining demonstrated that i CMs highly expressed cardiomyocyte-specific marker proteins,such asα-actinin,c Tn T and MLC2V.q RT-PCR also confirmed that i CMs expressed MYL3,SLC8A1 and TNNT2.SEM analysis demonstrated that the myocardial fiber bundles were arranged tightly and regularly.i CMs induced on Sp Gel revealed an increased contraction amplitude and a higher beating frequency（approximately60 beats/min）than i CMs induced on Matrigel-covered dishes（approximately 10 beats/min）.MEA analyses revealed that i CMs induced on Sp Gel may promote functional improvement during CM differentiation.Transmission electron microscopy analyses demonstrated that sarcomeres,Z-line structures and gap junctions were clearly observed in the Sp Gel group,while in the Matrigel-covered glass group,sarcomeric disarray without Z-line structures was observed.4.Pseudocolor SEM and H&E staining images revealed the morphology and distribution of i ECs/i CMs within Sp Gel.Confocal microscopic examination demonstrated cell membrane interactions between i ECs（red,hu CD31）and i CMs（green,α-actinin）.Live/dead staining analysis showed that viability was significantly improved in the presence of Sp Gel compared with the absence of Sp Gel（p<0.0001）following 2 h of H₂O₂treatment.TEM graphs demonstrated disrupted cytoarchitecture,cell vacuolation and necrosis in cells without Sp Gel after treatment with H₂O₂.However,cells on Sp Gel appeared to be free of serious structural deformity,and autophagic vacuoles in the cytoplasm were observed.5.IVIS fluorescent imaging revealed that Sp Gel encapsulation significantly improved cell retention in the ischemic heart seven days after injection（p=0.0014）.H&E staining and immunostaining analyses of sectioned heart tissues indicated that the retention rate of Sp Gel-i ECs/i CMs was approximately 7-fold and 3-fold higher than that of PBS-i ECs/i CMs on day 1（p<0.0001）and day 7（p=0.0376）,respectively,which suggested that encapsulation in Sp Gel significantly increased the retention of the cell grafts in the mouse hearts.Echocardiographic measurement of cardiac function demonstrated that injection of Sp Gel-i ECs/i CMs led to the highest LVEF measurements（54.53±2.09%）at 4 weeks,while the PBS-i EC/i CM group（46.88±1.73%）and Sp Gel group（39.91±1.34%）showed an increase in LVEF compared to the PBS group（31.68±2.17%）.Statistical analysis of the infarct size based on Masson’s staining revealed that treatment of MI with Sp Gel-i ECs/i CMs（16.64±4.59%）significantly decreased the extent of infarct fibrosis compared to treatment with PBS-i ECs/i CMs,Sp Gel and PBS（33.04±7.13%vs 44.59±3.80%and 60.40±4.30%,respectively）.Immunostaining analyses showed that the arterial（α-SMA+）and capillary（CD31+）density in Sp Gel-i EC/i CM-treated group was significantly higher than that in the PBS group,indicating that treatment with Sp Gel-i ECs/i CMs promoted neovascularization in the infarct region.6.A lentiviral vector overexpressing CCL22 was successfully constructed and transfected into hi PSC in vitro to achieve CCL22 gene overexpression.q PCR analysis indicated that CCL22 was highly expressed at m RNA level.Immunofluorescence stainings confirmed the expression of CCL22 at the cellular level.ELISA confirmed that CCL22 could be expressed in the form of exocrine protein by hi PSC-CCL22 cells.Transwell chemotactic assay was used to detect the chemotactic effect of hi PSC-CCL22 cell culture supernatant on Treg.Immunofluorescence staining confirmed the efficient differentiation of hi PSC-CCL22into cardiomyocytes and endothelial cells,respectively.Conclusion:We developed a thermoresponsive nanofibrous splenic matrix hydrogel（Sp Gel）to maintain the culture and promote the differentiation of hi PSCs.In vivo study indicated that application of Sp Gel-encapsulated i EC/i CMs effectively improved cell grafts retention,accelerated cardiac function recovery,inhibited fibrosis and promoted revascularization of ischemic tissue.What’s more,Sp Gel could also be used as a biocompatible bioink for 3D printing cardiac organoids in the future.This study revealed that Sp Gel could be used as a platform for culture and differentiation of hi PSCs,and an injectable cell transplantation carrier for regenerative medicine research and clinical utility.