N-CADHERIN+/CD168- subpopulation determines therapeutic variations of UC-MSCs for cardiac repair after myocardial infarction

doi:10.21203/rs.3.rs-4623987/v1

Download PDF

Research Article

N-CADHERIN+/CD168- subpopulation determines therapeutic variations of UC-MSCs for cardiac repair after myocardial infarction

https://doi.org/10.21203/rs.3.rs-4623987/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

The efficiency of mesenchymal stem cells (MSCs) in treating myocardial infarction (MI) remains inconsistent, which limits their therapeutic applications. Therefore, exploring the mechanism for the inconsistent efficacy of MSCs and identification the criteria for screening MSCs are important for improving the efficiency of MSCs.

Methods

Mouse model after myocardial infarction was utilized to test the role of MSCs from different donor in improving cardiac function. Heterogeneity of MSCs was identified using scRNA-seq of MSC-GY, GSEA and Scissor analyses were used to find the functional subpopulations of MSCs that promote angiogenesis. The role of functional subpopulations in promoting angiogenesis was verified by detecting the secretome proteins, the ratio of N-CADHERIN⁺/CD168⁻ subpopulations in MSCs, and the tube formation, migration, and proliferation of HUVECs after treatment with conditional medium (CM) derived from different MSCs.

Results

We found that UC-MSCs from different donors have varied therapeutic efficacy in MI mice and UC-MSCs with higher therapeutic effectiveness exhibited the most potent pro-angiogenic effects by secreting elevated levels of angiogenesis-related proteins, such as MYDGF, VEGFA, and FGF2. Single-cell RNA sequencing (scRNA-seq) of 10,463 UC-MSCs revealed that the N-cadherin⁺/CD168⁻ subpopulation was closely associated with pro-angiogenic effects, and the ratio of this cell subpopulation was positively correlated with the angiogenic potential of MSCs.

Conclusions

Our study identified that the N-CADHERIN⁺/CD168⁻ subpopulation was the functional subpopulation of MSCs in treating MI, which is essential for the development and utilization of MSCs in MI treatment.

MSCs

myocardial infarction

scRNA-seq

N-CADHERIN+/CD168-

angiogenesis

Myocardial infarction (MI) involves extensive death of cardiomyocytes due to coronary artery occlusion, leading to impaired cardiac function and progressive heart failure [1]. The transplantation of mesenchymal stem cells (MSCs) has emerged as a promising therapeutic strategy for treating MI, attributed to their low immunogenicity and paracrine effects, as demonstrated in animal studies and clinical trials [2, 3]. Lee and Amado demonstrated that MSC transplantation significantly reduced myocardial injury and improved cardiac function in the infarcted hearts of mice and pigs, respectively [4, 5]. A meta-analysis by Lalu et al. of 23 clinical trials (involving 1,148 patients) confirmed that MSC application was safe and effective for treating MI [6]. Additionally, Chullikana et al. found that MSC transplantation improved ejection fraction and reduced adverse cardiac events in MI patients [7]. However, Nowbar et al. reported the limited effects of MSCs on improving left ventricular ejection fraction based on an analysis of clinical trial data from 1252 patients [8]. Thus, achieving consistent and effective MSC treatment for MI remains a critical challenge.

Single-cell RNA sequencing (scRNA-seq) enables characterization of gene expression at the single-cell level and resolve cellular heterogeneity of MSCs. MSCs have been reported to be a heterogeneous population, with different cellular subpopulations have varying biological properties [9–12]. Sacchetti and Zhou et al. found that CD146⁺ and LepR⁺ MSCs, highly expressed bone-related genes, exhibited significant osteogenic differentiation ability [13, 14]. Similarly, Arufe and Mifune et al. identified the CD271⁺ MSCs possessed high expression of cartilage-related genes and significant chondrogenic differentiation ability [15, 16]. Additionally, the CMKLR1⁺ MSCs have been reported to possess strong immunomodulatory and osteogenic capabilities [17]. Thus, understanding the heterogeneity of MSCs would be crucial for identifying MSCs subpopulations suitable for treating specific diseases. However, it remains unclear whether specific MSC subpopulations could improve the treatment efficacy for MI.

The paracrine effects have been identified as a major mechanism of MSCs for the treatment of MI [2, 18, 19]. MSCs can secrete anti-inflammatory factors such as tumor necrosis factor stimulated gene 6 (TSG6), transforming growth factor beta 1 (TGF-β1), and interleukin 10 (IL10), which regulate macrophage polarization and inhibit T cell activation [20]. Lee et al. also found that MSCs improved cardiac function by regulating the inflammatory response, which was blocked by TSG6 knockdown [4]. Furthermore, the angiogenesis-related factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and hepatocyte growth factor (HGF) were secreted by MSCs to promote angiogenesis and cell survival [21, 22]. Lee et al. has also demonstrated that the pro-angiogenic effect is the main mechanism by which MSCs improve cardiac function and enhance therapeutic efficacy for treating MI [23]. Despite these insights, the key mechanism influencing MSC efficacy for MI treatment remains unclear.

In this study, we demonstrated that variations in the angiogenic potential of MSCs affect their therapeutic efficacy in MI mice. Furthermore, we identified the N-CADHERIN⁺/CD168⁻ subpopulation as a functional MSC subpopulation in regulating angiogenesis, with the ratio of this subpopulation significantly correlating with the angiogenesis effects of MSCs. Thus, our study highlights a specific MSC subpopulation that regulate angiogenesis, which is essential for the development and utilization of MSCs in MI treatment.

Experimental animals

The C57BL/6 male mice (6–8 weeks old) used in this study were purchased from Shanghai Laboratory Animal Center (SLAC), Shanghai, China. Mice were housed in the Laboratory Animal Research Center, Tongji University, and maintained under controlled temperature (22 ℃± 1℃) and humidity conditions with a 12:12 h light: darkness cycle. Mice were euthanized by cervical dislocation and the death was confirmed by cardiorespiratory arrest. All animal experiments were conducted following the institutional animal ethics guidelines and were approved by the Animal Use Committee of Tongji University (TJAB04523102).

Cell culture

UC-MSCs were cultured in MSC medium (ScienCell #7501) supplemented with 5% FBS, 1× mesenchymal stem cell growth supplement (MSCGS), and 1× penicillin/streptomycin (PS) solution.

Construction of MI model and UC-MSCs transplantation

The MI model was established as previously describe [24]. Animals were randomly assigned to different cage locations to prevent positional effects. Adult mice were anesthetized with 2% isoflurane, and a small hole was created in the third intercostal space. The left main descending coronary artery (LCA) was sutured and ligated with a 7 − 0 silk suture, and the skin incision was closed with a 5 − 0 silk suture. For cell implantation, 2 × 10⁵ UC-MSCs suspended in 30 µL PBS were injected into the infarct border zone through three injection points immediately after ligation. Mice after MI were randomly assigned to injection of saline (Ctrl group, n = 7), MSC-CX (2×10⁵ MSC-CX group, n = 7), MSC-CJ (2×10⁵ MSC-CJ group, n = 7), MSC-GY (2×10⁵ MSC-GY group, n = 7) into the myocardium. Sham mice (Sham group, n = 7) underwent open-chest surgery but without coronary ligation. A total of 38 mice were used for MI surgery and 35 mice survived after surgery (survival rate of almost 92.1%). MI mice after 3 days with an ejection fraction lower than 40% were used for further analysis (28/28).

Assessment of cardiac function

Echocardiography was performed to assess the cardiac function of MI mice 28 days after cell transplantation (Visual Sonics Vevo 2100 system equipped with a 40-MHz 550 s probe). Left ventricular ejection fraction (EF), fractional shortening (FS), end-diastolic diameter (LVIDd), end-systolic diameter (LVIDs) was measured and calculated using cardiac echocardiography software [25].

Masson trichrome staining

Masson trichrome staining was performed as previously described [24, 26]. Briefly, mouse hearts 4 weeks after MI were fixed in 4% paraformaldehyde for 24 hours at 4°C and then dehydrated using a sucrose gradient. Hearts were sectioned from below the ligation site to the apex at 8 µm thickness, with samples taken at 500 µm intervals for staining (Yeasen). Infarct size was quantified as the average of five sections using the formula: infarct size = [infarct perimeter (infarct epicardium + infarct endocardium) × 100] / left ventricle perimeter (left ventricle epicardium + left ventricle endocardium).

Immunostaining

Immunostaining was performed as previously described [27]. Fixed slides were blocked for 1 hour at room temperature in PBS containing 10% donkey serum and 0.1% Triton X-100. Slides were incubated overnight at 4°C with primary antibodies, followed by incubation with fluorescent secondary antibodies and Hoechst33342 for nuclear staining at room temperature for 1 hour. CD31 and α-SMA positive cells were quantified from three randomly selected sections per slide.

Flow cytometry (FACS)

UC-MSCs were dissociated with TrypLE (Gibco), washed with PBS, and 3×10⁵ cells were incubated with 100 µL PBS containing 1 µL CD168 (RHAMM)-FITC antibody (bs-4736R-FITC, Bioss) and 10 µL CDH2 (N-cadherin)-PE antibody (11039-R001-PE, Sino Biological) for 30 minutes at 4°C. Cells were analyzed using a BD FACSVerse, and data were processed with FlowJo software.

Production of UC-MSCs conditioned medium

UC-MSCs (2×10⁶) from different donors were cultured in 10 cm dishes until 80–90% confluent. Cells were washed with PBS and switched to low glucose DMEM. After 5 days, supernatants were collected and stored at 4°C for further use.

Tube formation assay

Tube formation assay was performed as previously described [22]. Basement membrane matrix (Matrigel®, BD Biosciences) was solidified in 24-well plates at 37°C for 30 minutes. HUVECs (8×10⁴ cells/well) were plated on Matrigel in 1640 medium with 10% FBS and 30% conditioned medium from UC-MSCs. Tube formation was imaged after 8 hours, and structures were quantified using Image J.

Cell migration assay

HUVECs (4×10⁴ cells/well) were cultured in 24-well plates for 24 hours. After creating a scratch, cells were cultured in 1640 medium with 30% UC-MSC conditioned medium for 18 hours. Images of migrated cells were taken at 0, 6, and 18 hours, and the migration rate was calculated using ImageJ.

Cell proliferation assay

HUVECs (8×10⁴ cells/well) were cultured in 12-well plates in 1640 medium with 30% UC-MSC conditioned medium for 72 hours. Cell numbers were counted at 0, 24, and 48 hours using CounterStar software.

scRNA-seq analysis

Single-cell RNA sequencing was conducted using 10× Genomics technology. Data were processed into a Seurat object, filtering out cells with fewer than 200 features or more than 5% mitochondrial gene expression. Data normalization and feature selection identified 2,000 highly variable features. Principal Component Analysis (PCA) reduced dimensionality to 30 components, which were used for clustering at a resolution of 0.25. UMAP and t-SNE techniques visualized cell populations.

Statistical analysis

All data were presented as mean ± S.E.M. of three independent experiments. Statistical significance was determined using one-way ANOVA with Bonferroni’s post hoc test for more than two groups (GraphPad Prism software). The significance levels were set at * P < 0.05, ** P < 0.01, and *** P < 0.001. The work had been reported in line with the ARRIVE guidelines 2.0.

MSCs from Different Donors Exhibit Distinct Effects on Improving Cardiac Function in Mice Post-MI

We obtained UC-MSCs from three donors, with flow cytometry results showed that these UC-MSCs were positive for CD44, CD90, CD105, and CD73, while negative for CD34 and HLA-DR (Fig. 1A). We then transplanted these three UC-MSCs into MI mice hearts via intramyocardial injection. Four weeks post-transplantation, echocardiography was performed to evaluate the cardiac function. Our results demonstrated that MSC-GY and MSC-CJ significantly improved left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). Correspondingly, we observed a decrease in left ventricular internal diameter in diastole (LVIDd) and systole (LVIDs) in the mice transplanted with MSC-GY and MSC-CJ, whereas MSC-CX transplantation had no effect on cardiac function post-MI (Fig. 1B and 1C). Additionally, the Masson's trichrome staining revealed that MSC-GY and MSC-CJ transplantation reduced the injury area in the MI mice (Fig. 1D). These results indicated that UC-MSCs derived from different donor exhibit variations in therapeutic efficacy in mice post-MI.

Variable Angiogenic Efficacies of MSCs from Different Donors

MSCs could promote angiogenesis and reduce inflammation. Vagnozzi et al. found that intramyocardial injection of both live and cryopreserved MSCs resulted in the accumulation of CCR2⁺ and CX3CR1⁺ macrophage, which alleviated inflammation in mice [28]. Based on this, we hypothesized that angiogenesis might be the primary mechanism affecting MSC efficacy in treating MI. We examined MSC-induced angiogenesis via CD31 and α-smooth muscle actin (α-SMA) immunostaining and observed that MSC-GY and MSC-CJ significantly increased α-SMA and CD31-positive cells in peri-infarct heart regions, while MSC-CX had minimal impact (Fig. 2A and 2B). To further validate the angiogenic effects of different MSCs, we treated human umbilical vein endothelial cells (HUVECs) with conditional medium (CM) derived from different MSCs. Results showed that the MSC-GY and MSC-CJ CM significantly enhanced the tube formation, migration, and proliferation of HUVECs, rather than the MSC-CX CM (Fig. 2C-2E), consistent with the results in the MI model. We then performed the secretome analysis of the three UC-MSCs CMs. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) results indicated that upregulated secretory factors in MSC-GY and MSC-CJ CMs were associated with endothelial cell proliferation, vascular development, and endothelial cell migration (Fig. 2F and fig. S1, S2). Angiogenic factors such as VEGF and FGF2 were significantly higher in the CMs of MSC-GY and MSC-CJ compared to MSC-CX (Fig. 2G). These results suggested that the angiogenic capacity of MSCs influences their efficacy in MI treatment.

Single-cell Transcriptomic Analysis Reveals Four Major Subpopulations within MSCs

To explore the heterogeneity of UC-MSCs, we generated single-cell transcriptome profiles of MSC-GY, which exhibited the most significant therapeutic effects in MI. After quality control, single-cell transcriptomes of 10,463 cells were acquired for further analysis. By analyzing variably expressed genes across all cells, we identified four clusters of MSCs: Cluster 1 (35.10%), Cluster 2 (27.85%), Cluster 3 (23.06%), and Cluster 4 (13.99%) (Fig. 3A). We then performed differential gene expression analysis to find unique signatures for each cluster, presenting the top 10 significantly differentially expressed (SDE) genes of each cluster (Fig. 3B).

To reveal the biological functions of each cluster, we performed the GSEA of cluster-specific differentially expressed genes (DEGs). The results showed that cluster 1 was significantly associated with vascular development, smooth muscle cell migration and adhesion, indicating that Cluster1 might regulate angiogenesis. Cluster 2 showed enrichment in metabolic processes such as lipid metabolism and fatty acid metabolism, while Cluster 3 was associated with cell morphogenesis and cell junction assembly, and Cluster 4 was linked to cell proliferation-related biological processes such as cell division and microtubule polymerization or depolymerization. Based on their gene expression profiles and pathway enrichment analysis, we named Cluster 1 as the angiogenesis subpopulation, Cluster 2 as the metabolic regulation subpopulation, Cluster 3 as the niche support subpopulation, and Cluster 4 as the proliferation subpopulation (Fig. 3C).

Furthermore, we predicted the cell cycle phases of each cell and found that cells in the proliferation subpopulation were predominantly in the G2/M phase, confirming the strong proliferative capacity of this subpopulation (Fig. 3D). The proposed trajectory analysis can infer the evolution of cell subpopulation by their expression characteristics. We performed pseudo-time reconstruction of MSC-GY to stratify the connection of each sub-population. Pseudo-time reconstruction revealed seven cell states, starting from state 7 and ending at state 1 (Fig. 3E and 3F). The angiogenesis subpopulation served as the starting point of the trajectory, while the metabolic regulation, niche support, and proliferation subpopulation were mainly located at the terminal end (Fig. 3G). This pattern shows that the angiogenesis subpopulation also represents the stem-like cell subpopulation within UC-MSCs, which gradually transitions into other subpopulations.

The N-CADHERIN⁺/CD168⁻ Subpopulation is Associated with the Angiogenesis Capacity of MSCs

To identify the cell subpopulations associated with angiogenesis, we integrated single-cell RNA-seq data of MSC-GY with RNA-seq data from MSC-M01 and M02 in GSE13491 by Scissor analysis [29], which can identify the most phenotypically relevant cells from single-cell data. MSC-M02 in Lee et.al significantly promoted the angiogenesis in the heart of rats after MI. In this integrated analysis [23], Scissor identified 1890 Scissor⁺ cells with gene expression profiles similar to MSC-M02 (Fig. 4A), suggesting that these 1890 Scissor⁺ cells in the single-cell data were similar to MSC-M02, which could promote angiogenesis and improve heart function post- MI. Statistical analysis showed that 1,824 (96.5%) of these Scissor⁺ cells originated from the angiogenesis subpopulation (Fig. 4B). Therefore, we concluded that the angiogenesis subpopulation is the functional component of MSCs that can promote angiogenesis and improve heart function in the MI model.

Next, we performed a comprehensive analysis between the cell surface proteins and DEGs in the angiogenesis subpopulation, finding that the angiogenesis subpopulation was positive for N-CADHERIN and negative for CD168 (Fig. 4C and 4D). Flow cytometry analysis showed that N-CADHERIN⁺/CD168⁻ cells accounted for 29% of MSC-GY (Fig. 4E). Analysis of data from GSE165811 [30] revealed that CD168 was negatively correlated with the expression levels of angiogenesis-related genes such as VEGFA and FGF2, while N-CADHERIN was positively correlated with these genes (Fig. 4F). Additionally, analysis of single-cell data (GSE182158) [31] showed that genes highly expressed in the N-CADHERIN⁺/CD168⁻ cell subpopulation were associated with angiogenesis (Fig. 4G). These results indicate that N-CADHERIN⁺/CD168⁻ subpopulation is significantly associated with promoting angiogenesis of MSCs.

The Ratio of N-CADHERIN⁺/CD168⁻ Cell Subpopulation Positively Correlates with the Angiogenic Effect of MSCs

To further elucidate the relationship between the proportion of N-CADHERIN⁺/CD168⁻ cell subpopulation and the angiogenic function of UC-MSCs in MI, we detected the ratio of N-CADHERIN⁺/CD168⁻ subpopulation in three UC-MSCs. Flow cytometry analysis revealed that the ratio of the N-CADHERIN⁺/CD168⁻ cell subpopulation in MSC-CX was significantly lower than that in MSC-GY and MSC-CJ (Fig. 5A and 5B). These results indicated that the ratio of the N-CADHERIN⁺/CD168⁻ subpopulation affects the efficiency of MSCs in treating MI. To identify the key upstream regulators maintaining the N-CADHERIN⁺/CD168⁻ subpopulation, we reconstructed gene regulatory networks using SCENIC [32] and explored the unique transcriptional regulation of DEGs in distinct subpopulations. We found that the angiogenesis subpopulation might be regulated by the transcription factors CEBPB, TCF21, and TAL1 (Fig. 5C and 5D). These results suggested that CEBPB, TCF21, and TAL1 may maintain the fate of the N-CADHERIN⁺/CD168⁻ subpopulation and increase the ratio of N-CADHERIN⁺/CD168⁻ subpopulation in MSCs.

MSCs hold significant promise for MI therapy, yet growing evidence reveals inconsistent therapeutic efficacy among MSCs derived from different donors [8, 33–35]. Our study identified critical differences in the therapeutic potential of UC-MSCs from various donors, highlighting that the pro-angiogenic capacity of MSCs was a determinant of their efficacy in MI treatment. Given the variability in donor-derived MSCs used in clinical settings, our findings suggested that selecting MSCs with higher pro-angiogenic potential is essential for achieving more effective clinical outcomes.

Paracrine effects are the primary mechanism by which MSCs confer therapeutic benefits in MI. Lee et al. demonstrated that MSCs enhanced cardiac function in MI mice through the secretion of TSG6, which modulated inflammatory responses [4]. Additional studies emphasized the secretion of angiogenesis-related factors as a crucial mechanism. Hu et al. showed that pre-conditioning MSCs under hypoxic conditions (0.5% oxygen for 24 hours) significantly elevated the levels of VEGF and other angiogenic factors, thereby enhancing angiogenesis in the infarct border zone, reducing cardiac fibrosis, and improving cardiac function in MI mice [36]. Overexpressing the factors like VEGF and FGF2 had been shown to significantly boost the angiogenic potential of MSCs, thus improving their reparative capacity in damaged hearts after MI [37, 38]. Our findings corroborated that effective MSCs secrete higher levels of pro-angiogenic factors, supporting the notion that enhancing angiogenesis via paracrine effects of MSCs is the key to improve cardiac function post-MI.

Single-cell RNA sequencing (scRNA-seq) enables detailed characterization of cellular expression profiles, offering an invaluable tool for exploring MSC heterogeneity [39]. Recent studies have identified MSC transcriptome profiles based on the DEGs and differential pathways, without functional analysis of subpopulations. Some studies failed to defined the functional subpopulations because of insufficient cell numbers. Huang et al. analyzed only 361 UC-MSCs and found that the heterogeneity was primarily influenced by cell cycle differences [40], Hou et al. [41] and Wolock et al. [42] respectively identified the subpopulations in UC-MSCs and bone marrow MSCs, highlighting the differences in differentiation potentials. In this study, we expanded the analysis to 10,463 UC-MSCs, identifying four main subpopulations. Notably, the N-CADHERIN⁺/CD168⁻ subpopulation emerged as a key functional group in regulating angiogenesis. We found that the ratio of this subpopulation was positively correlated with the pro-angiogenic effects of MSCs. Together, our study demonstrated that variations in angiogenic potential influence the therapeutic efficacy of UC-MSCs in MI treatment.

This study has several limitations. First, our study found that the N-CADHERHIN⁺/CD168⁻ subpopulation was significantly associated with the proangiogenic capacity of MSCs and their efficacy in MI mice, which needs to be confirmed in a large animal model of myocardial infarction (eg, swine or nonhuman primates) and quantified to determine the relationship between the ratio of the N-CADHERHIN⁺/CD168⁻ cell subpopulation and the efficacy of MSCs, providing a basis for improving the clinical application of MSCs. Second, we found that CEBPB, TCF21, and TAL1 may maintain the fate of N-CADHERIN⁺/CD168⁻ subpopulation, but have not yet examined whether CEBPB, TCF21, and TAL1 can increase the ratio of functional cell subpopulations in MSCs. We will evaluate the role of these factors in future studies.

In summary, by exploring UC-MSC heterogeneity, we identified the N-CADHERIN⁺/CD168⁻ subpopulation as crucial for the regulation of angiogenesis. The ratio of this subpopulation determined the angiogenic efficacy of UC-MSCs, providing a basis for selecting MSCs suitable for MI therapy. This finding deepened the understanding of UC-MSC heterogeneity and established a new criteria for screening MSCs for effective MI treatment.

MSCs Mesenchymal stem cells

MI Myocardial infarction

scRNA-seq Single-cell RNA sequencing

TSG6 Tumor necrosis factor stimulated gene 6

TGF-β1 Transforming growth factor beta 1

IL10 Interleukin 10

VEGF Vascular endothelial growth factor

FGF2 Fibroblast growth factor 2

HGF Hepatocyte growth factor

LVEF Left ventricular ejection fraction

LVFS Left ventricular fractional shortening

LVIDd Left ventricular internal diameter in diastole

LVIDs Left ventricular internal diameter in systole

α-SMA α-smooth muscle actin

HUVECs Human umbilical vein endothelial cells

CM Conditional medium

GO Gene ontology

GSEA Gene set enrichment analysis

SDE Significantly differentially expressed

DEGs Differentially expressed genes

SLAC Shanghai laboratory znimal center

MSCGS Mesenchymal stem cell growth supplement

PS Penicillin/streptomycin

LCA Left main descending coronary artery

EF Ejection fraction

FS Fractional shortening

PCA Principal component analysis

Ethics approval and consent to participate

This study was approved by the Animal Experimentation Ethics Committee, Tongji University [TJABO4523102 (title: The functions and mechanisms of N-CADHERIN⁺/CD168^- umbilical cord mesenchymal stem cell subpopulation in the treatment of myocardial infarction; date:28th Feb, 2023)] and approved by the Institutional Animal Care and Use Committee of Tongji University.

Human umbilical cord mesenchymal stem cells were obtained following signed informed consent and approved for the project: The preparation and construction of clinical human umbilical cord and umbilical cord blood stem cells, the Ethics Committee of Shanghai East Hospital (No: 2017 Preliminary Review No. (001)). Oct 18th. 2019.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this manuscript and its Supplementary Information ﬁles. The scRNA-seq and proteomics data have been deposited in the Genome Sequence Archive for Human database (HRA007901). The source data presented in this study are available from the corresponding author upon request.

Competing interests

Authors declare that they have no competing interests.

Funding

This study was supported by grants from the National Key R&D Program of China (2021YFA1100400), the National Natural Science Foundation of China (82271723, 82071728 and 32170575), the Natural Science Foundation of Shanghai (23ZR1465500), and the Fundamental Research Funds for the Central Universities (22120220620).

Authors' contributions

Y.W., J. L., K.F., A.T, Y.G., W.C., W.J., X.G. and J.K. designed the overall study, including biological experiments and bioinformatics analysis. Y.W., J. L., X.G. and J.K. wrote the manuscript. Y.W. and K.F. performed the biological experiments. J.L. and Y.W. performed data analyses. A.T. carried out the random allocation process. All authors discussed the study results and worked together on preparing and editing the manuscript.

Acknowledgements

We thank Shanghai East Hospital for providing UC-MSCs. The authors declare that they have not used Artificial Intelligence in this study.

Dreyer RP, Zheng X, Xu X et al. Sex differences in health outcomes at one year following acute myocardial infarction: A report from the China Patient-Centered Evaluative Assessment of Cardiac Events prospective acute myocardial infarction study [in English]. Eur Heart J-Acute Ca 2019;8(3):273-282.
Bagno L, Hatzistergos KE, Balkan W et al. Mesenchymal Stem Cell-Based Therapy for Cardiovascular Disease: Progress and Challenges. Mol Ther 2018;26(7):1610-1623.
Williams AR, Hare JM. Mesenchymal stem cells: biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease. Circ Res 2011;109(8):923-940.
Lee RH, Pulin AA, Seo MJ et al. Intravenous hMSCs Improve Myocardial Infarction in Mice because Cells Embolized in Lung Are Activated to Secrete the Anti-inflammatory Protein TSG-6 [in English]. Cell Stem Cell 2009;5(1):54-63.
Amado LC, Saliaris AP, Schuleri KH et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A 2005;102(32):11474-11479.
Lalu MM, Mazzarello S, Zlepnig J et al. Safety and Efficacy of Adult Stem Cell Therapy for Acute Myocardial Infarction and Ischemic Heart Failure (SafeCell Heart): A Systematic Review and Meta-Analysis [in English]. Stem Cell Transl Med 2018;7(12):857-866.
Chullikana A, Sen Majumdar A, Gottipamula S et al. Randomized, double-blind, phase I/II study of intravenous allogeneic mesenchymal stromal cells in acute myocardial infarction [in English]. Cytotherapy 2015;17(3):250-261.
Nowbar AN, Mielewczik M, Karavassilis M et al. Discrepancies in autologous bone marrow stem cell trials and enhancement of ejection fraction (DAMASCENE): weighted regression and meta-analysis [in English]. Bmj-Brit Med J 2014;348.
Zhang C, Han X, Liu J et al. Single-cell Transcriptomic Analysis Reveals the Cellular Heterogeneity of Mesenchymal Stem Cells. Genomics Proteomics Bioinformatics 2022;20(1):70-86.
Zhang P, Dong J, Fan X et al. Characterization of mesenchymal stem cells in human fetal bone marrow by single-cell transcriptomic and functional analysis. Signal Transduct Target Ther 2023;8(1):126.
Zhang YY, Li F, Zeng XK et al. Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development. World J Stem Cells 2023;15(6):589-606.
Miura T, Kouno T, Takano M et al. Single-Cell RNA-Seq Reveals LRRC75A-Expressing Cell Population Involved in VEGF Secretion of Multipotent Mesenchymal Stromal/Stem Cells Under Ischemia. Stem Cells Transl Med 2023;12(6):379-390.
Sacchetti B, Funari A, Michienzi S et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment [in English]. Cell 2007;131(2):324-336.
Zhou BO, Yue R, Murphy MM et al. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell 2014;15(2):154-168.
Arufe MC, De la Fuente A, Fuentes I et al. Chondrogenic potential of subpopulations of cells expressing mesenchymal stem cell markers derived from human synovial membranes. J Cell Biochem 2010;111(4):834-845.
Mifune Y, Matsumoto T, Murasawa S et al. Therapeutic superiority for cartilage repair by CD271-positive marrow stromal cell transplantation. Cell Transplant 2013;22(7):1201-1211.
Xie Z, Yu W, Ye G et al. Single-cell RNA sequencing analysis of human bone-marrow-derived mesenchymal stem cells and functional subpopulation identification. Exp Mol Med 2022;54(4):483-492.
Shi W, Xin Q, Yuan R et al. Neovascularization: The Main Mechanism of MSCs in Ischemic Heart Disease Therapy. Front Cardiovasc Med 2021;8:633300.
Sun SJ, Wei R, Li F et al. Mesenchymal stromal cell-derived exosomes in cardiac regeneration and repair. Stem Cell Reports 2021;16(7):1662-1673.
Shi Y, Wang Y, Li Q et al. Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases. Nat Rev Nephrol 2018;14(8):493-507.
Kwon HM, Hur SM, Park KY et al. Multiple paracrine factors secreted by mesenchymal stem cells contribute to angiogenesis. Vascul Pharmacol 2014;63(1):19-28.
Park SJ, Kim RY, Park BW et al. Dual stem cell therapy synergistically improves cardiac function and vascular regeneration following myocardial infarction. Nat Commun 2019;10(1):3123.
Lee EJ, Choi EK, Kang SK et al. N-cadherin determines individual variations in the therapeutic efficacy of human umbilical cord blood-derived mesenchymal stem cells in a rat model of myocardial infarction. Mol Ther 2012;20(1):155-167.
Wu Y, Guo X, Han T et al. Cmarr/miR-540-3p axis promotes cardiomyocyte maturation transition by orchestrating Dtna expression. Mol Ther Nucleic Acids 2022;29:481-497.
Guo X, Xu Y, Wang Z et al. A Linc1405/Eomes Complex Promotes Cardiac Mesoderm Specification and Cardiogenesis. Cell Stem Cell 2018;22(6):893-908 e896.
Feng K, Wu Y, Li J et al. Critical Role of miR-130b-5p in Cardiomyocyte Proliferation and Cardiac Repair in Mice After Myocardial Infarction. Stem Cells 2024;42(1):29-41.
Xi J, Wu Y, Li G et al. Mir-29b Mediates the Neural Tube versus Neural Crest Fate Decision during Embryonic Stem Cell Neural Differentiation. Stem Cell Reports 2017;9(2):571-586.
Vagnozzi RJ, Maillet M, Sargent MA et al. An acute immune response underlies the benefit of cardiac stem cell therapy. Nature 2020;577(7790):405-409.
Sun D, Guan X, Moran AE et al. Identifying phenotype-associated subpopulations by integrating bulk and single-cell sequencing data. Nat Biotechnol 2022;40(4):527-538.
Zhang C, Zhou L, Wang Z et al. Eradication of specific donor-dependent variations of mesenchymal stem cells in immunomodulation to enhance therapeutic values. Cell Death Dis 2021;12(4):357.
Wang Z, Chai C, Wang R et al. Single-cell transcriptome atlas of human mesenchymal stem cells exploring cellular heterogeneity. Clin Transl Med 2021;11(12):e650.
Aibar S, Gonzalez-Blas CB, Moerman T et al. SCENIC: single-cell regulatory network inference and clustering. Nat Methods 2017;14(11):1083-1086.
Cho HM, Lee KH, Shen YM et al. Transplantation of hMSCs Genome Edited with LEF1 Improves Cardio-Protective Effects in Myocardial Infarction. Mol Ther Nucleic Acids 2020;19:1186-1197.
Yan W, Lin C, Guo Y et al. N-Cadherin Overexpression Mobilizes the Protective Effects of Mesenchymal Stromal Cells Against Ischemic Heart Injury Through a beta-Catenin-Dependent Manner. Circ Res 2020;126(7):857-874.
Lei J, Wang S, Kang W et al. FOXO3-engineered human mesenchymal progenitor cells efficiently promote cardiac repair after myocardial infarction. Protein Cell 2021;12(2):145-151.
Hu X, Xu Y, Zhong Z et al. A Large-Scale Investigation of Hypoxia-Preconditioned Allogeneic Mesenchymal Stem Cells for Myocardial Repair in Nonhuman Primates: Paracrine Activity Without Remuscularization. Circ Res 2016;118(6):970-983.
Karpov AA, Udalova DV, Pliss MG et al. Can the outcomes of mesenchymal stem cell-based therapy for myocardial infarction be improved? Providing weapons and armour to cells [in English]. Cell Proliferat 2017;50(2).
Bagno LL, Carvalho D, Mesquita F et al. Sustained IGF-1 Secretion by Adipose-Derived Stem Cells Improves Infarcted Heart Function [in English]. Cell Transplant 2016;25(9):1609-1622.
Chen P, Tang S, Li M et al. Single-Cell and Spatial Transcriptomics Decodes Wharton's Jelly-Derived Mesenchymal Stem Cells Heterogeneity and a Subpopulation with Wound Repair Signatures. Adv Sci (Weinh) 2023;10(4):e2204786.
Huang Y, Li Q, Zhang K et al. Single cell transcriptomic analysis of human mesenchymal stem cells reveals limited heterogeneity. Cell Death Dis 2019;10(5):368.
Hou WH, Duan L, Huang CY et al. Cross-Tissue Characterization of Heterogeneities of Mesenchymal Stem Cells and Their Differentiation Potentials [in English]. Frontiers in Cell and Developmental Biology 2021;9.
Wolock SL, Krishnan I, Tenen DE et al. Mapping Distinct Bone Marrow Niche Populations and Their Differentiation Paths [in English]. Cell Reports 2019;28(2):302-+.

AuthorChecklistFull.pdf
SupplementalInformation.docx
Supplementary Information The supplementary information contains results of the supplementary experiment (Figure S1-2) and antibodies information (Table S1-3).

Download PDF

Reviewers agreed at journal
22 Jul, 2024
Reviewers invited by journal
22 Jul, 2024
Editor assigned by journal
16 Jul, 2024
First submitted to journal
16 Jul, 2024
Editorial decision: Major Revision
02 Jul, 2024

You are reading this latest preprint version

N-CADHERIN+/CD168- subpopulation determines therapeutic variations of UC-MSCs for cardiac repair after myocardial infarction

Status:

Version 1

Abstract

Figures

Introduction

Methods

Experimental animals

Cell culture

Construction of MI model and UC-MSCs transplantation

Assessment of cardiac function

Masson trichrome staining

Immunostaining

Flow cytometry (FACS)

Production of UC-MSCs conditioned medium

Tube formation assay

Cell migration assay

Cell proliferation assay

scRNA-seq analysis

Statistical analysis

Results

MSCs from Different Donors Exhibit Distinct Effects on Improving Cardiac Function in Mice Post-MI

Variable Angiogenic Efficacies of MSCs from Different Donors

Single-cell Transcriptomic Analysis Reveals Four Major Subpopulations within MSCs

The N-CADHERIN⁺/CD168⁻ Subpopulation is Associated with the Angiogenesis Capacity of MSCs

The Ratio of N-CADHERIN⁺/CD168⁻ Cell Subpopulation Positively Correlates with the Angiogenic Effect of MSCs

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

N-CADHERIN+/CD168- subpopulation determines therapeutic variations of UC-MSCs for cardiac repair after myocardial infarction

Status:

Version 1

Abstract

Figures

Introduction

Methods

Experimental animals

Cell culture

Construction of MI model and UC-MSCs transplantation

Assessment of cardiac function

Masson trichrome staining

Immunostaining

Flow cytometry (FACS)

Production of UC-MSCs conditioned medium

Tube formation assay

Cell migration assay

Cell proliferation assay

scRNA-seq analysis

Statistical analysis

Results

MSCs from Different Donors Exhibit Distinct Effects on Improving Cardiac Function in Mice Post-MI

Variable Angiogenic Efficacies of MSCs from Different Donors

Single-cell Transcriptomic Analysis Reveals Four Major Subpopulations within MSCs

The N-CADHERIN+/CD168− Subpopulation is Associated with the Angiogenesis Capacity of MSCs

The Ratio of N-CADHERIN+/CD168− Cell Subpopulation Positively Correlates with the Angiogenic Effect of MSCs

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

The N-CADHERIN⁺/CD168⁻ Subpopulation is Associated with the Angiogenesis Capacity of MSCs

The Ratio of N-CADHERIN⁺/CD168⁻ Cell Subpopulation Positively Correlates with the Angiogenic Effect of MSCs