Subpopulation analysis of Sca-1, Nanog, and Islet-1 positive cells in myocardial tissue

doi:10.21203/rs.3.rs-2395471/v1

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Subpopulation analysis of Sca-1, Nanog, and Islet-1 positive cells in myocardial tissue

https://doi.org/10.21203/rs.3.rs-2395471/v1

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To investigate the co-expression between different stem-cell or embryonic markers in myocardial tissue, which has rarely been discussed in previous studies, we selected three typical molecular markers, namely Sca-1, Nanog and Islet-1. In our study, we focused on the age-related changes of Sca-1, Nanog and Islet-1 expression and the co-localization levels between pin-two marker proteins to explore the heterogeneity and relevance between these three cell subpopulations. We found that Sca-1, Nanog, and Islet-1 positive cells were present in the cardiac tissue from newborn to adult mice, and their expression was negatively correlated with age. Co-localization existed between each two markers, and there were differences in co-localization levels at different stages of cardiac development. We thus hypothesize that these three positive cells are likely to be a group of cells, so that cardiac cells with myocardial differentiation potential should not be subclassified by a single cell marker, and their phenotypic changes at different stages may determine their unique roles in heart development.

Biological sciences/Cell biology

Biological sciences/Molecular biology

Biological sciences/Stem cells

Health sciences/Biomarkers

Health sciences/Cardiology

Until the beginning of this century, cardiomyocytes had been regarded as terminally differentiated cells that could not undergo endogenous repair and regeneration once damaged. The existence of stem cells with the differentiation potential within cardiac muscle tissue is a hot topic of debate. Many scholars have provided extensive studies suggesting that pluripotent stem cells with self-renewal capacity and multidirectional differentiation potential exist in the heart tissue of mature organisms, which can differentiate into cardiac structural cells such as endothelial cells, smooth muscle cells and cardiomyocytes [1–16]. However, since the findings that bone marrow-derived c-kit cells can differentiate into cardiomyocytes [17] were criticized [18], a new and intense argument [19–21] immediately arose, focusing on the fact that bone marrow-derived c-kit positive cells can neither be transformed into cardiomyocytes nor achieve therapeutic efficacy in the treatment of myocardial infarction. After a storm of controversy, it seemed that research on cardiac stem cells came to an abrupt end. The physiological and pathological characteristics of cardiac stem cells have been revisited lately by Öztürk and Elçin [22], and a number of authors have renewed new studies on c-kit cells [23, 24]. As shown by Bhartiya's more recent observation [25], the unexpected loss of stem cells during the course of performing different experiments has contributed to the misconceptions and disputes prevailing in the field of cardiac stem cells, which need to be urgently corrected. With paracrine support from mesenchymal stromal cells, VSELs/CSCs have the potential to regenerate damaged cardiac tissue.

Regardless of the presence or absence of resident stem cells within the myocardium being debated, one indisputable phenomenon is that the following types of cells expressing markers from the embryonic period are present in developing and adult myocardial tissue: Sca-1(stem cell antigen-1)-positive cells [26–30]; Nanog-positive cells [25, 31, 32]; Islet-1 positive cells [27, 33–36], previous researchers have often identified a positive expression of a marker as representing a subset of cells. However, "stem cell identity" of a cell is not solely associated with a single specific biomarker, and it is suggested that these phenotypically distinct populations of cells may be simply phenotypic variations of a unique cell type at different stages [28]. Based on our work, we found that there are not a large quantity of cells in myocardial tissue that express only one type of marker. This prompted us to hypothesize that several different stem cell markers may be co-localized on one class of cells at the same time, and therefore the classification of cardiac myocyte populations using merely one stem cell marker protein is inaccurate. Currently, there are few reports of co-expression between various marker proteins in the hearts, including limited evidence that Sca-1-positive cells could express some cardiac transcriptional regulators such as GATA-4, Mef2c and Tef1 [27, 28, 37]. c-kit was also detected to be expressed in Islet-1 positive cells in both human embryonic and fetal hearts [37]. The purpose of this study was to investigate the expression relationship between Sca-1, Nanog and Islet-1 positive cells derived from different developmental stages of the heart and to determine the heterogeneity and relevance between diverse cell subpopulations.

• Results of mouse primary cardiomyocyte culture

The isolated cells all adhere to the wall, with short, triangular or long shuttle-shaped cell morphology.On the 5th to 7th day of culture, the cells were seen to grow in both scattered and aggregated forms (Fig. 1A).

• Expression of Sca-1, Nanog, and Islet-1 in primary cells

Sca-1, Nanog and Islet-1 were all expressed in purified primary cells of neonatal, 1-week-old, 3-week-old and 5-week-old groups, and the expression levels of Sca-1, Nanog and Islet-1 in these cells tended to decrease as the age of mice increased. The expression sites of the three marker proteins differed, with Sca-1 mainly expressed in the cell membrane, Nanog primarily localized in the cell membrane or cytoplasm, and Islet-1 mostly present in the nucleus (Fig. 1B).

• Myocardial differentiation potential of primary Sca-1, Nanog, and Islet-1 positive cells

Sca-1, Nanog, and Islet-1 positive cells in the uninduced, normal, and induced groups all expressed cTnT, and there was cellular co-localization of cTnT with Sca-1, Nanog, and Islet-1, respectively. This co-localization relationship was significantly higher in the induced group than in the normal group and the lowest in the uninduced group. A cross-sectional comparison of the three groups indicated that Sca-1⁺ and Islet-1⁺ cells had stronger potential to express cTnT than Nanog⁺ cells (Fig. 2).

• Age-related changes in the expression of Sca-1, Nanog, and Islet-1

The results of Western blotting assays and semi-quantitative analysis showed that there was an upward and then downward trend in the expression levels of Sca-1, Nanog and Islet-1 positive cells of neonatal to 5-week-old mouse heart origin (Fig. 3).

• Co-localization analysis of Sca-1, Nanog, and Islet-1

The outcomes of immunofluorescence double-labeling staining revealed the presence of two proteins co-expressed in one cell (Fig. 4A, Fig. 4B, Fig. 4C). Statistical analysis of the immunofluorescence double-labeling images showed that the co-expression rate of Sca-1 and Islet-1 increased from birth to the third week and then gradually decreased; the co-expression rate of Sca-1 and Nanog kept increasing from birth and declined by the fifth week; the co-expression rate of Islet-1 + Nanog showed a gradual increase from one week after birth (Fig. 4D), which was consistent with the results of western blotting analysis (Fig. 3). Co-localization analysis of the immunofluorescence merge images using Image J software yielded scatter plots and Pearson correlation coefficient (PCC) metrics showing the same tendency ( Fig. 5A, Fig. 5B). The scatter plots showed that the co-localization of Sca-1 + Islet-1 double-labeled group at 3 weeks of age was closer to the diagonal than at other ages, and the slope of the scatter plots of Sca-1 + Nanog group and Nanog + Islet-1 group gradually increased and approached 1.0 with the increase of age. Moreover, the PCC value of Sca-1 + Islet-1 double-labeled group achieved its peak at 1 week of age, while that of Sca-1 + Nanog double-labeled group reached the maximum value at 3 weeks after birth.

• Cell type analysis and stem cell gene localization in juvenile mice cardiac tissue

Under the Illumina sequencing platform, an average number of 1508 genes per cell was detected using 52 million reads. In addition, the whole transcriptomes of 8918 individual cells that passed strict quality control were built, sequenced and analyzed, and finally, we identified eight major cell populations that can be categorized into five types: cardiomyocytes (1.17%), fibroblasts (76.73%), smooth muscle cells (3.20%), endothelial cells (2.11%) and immune cells (16.93%) [(B cells (0.64%), T/NK cells (4.38%), macrophages (11.00%), and granulocytes (0.91%)] (Fig. 6, Table 1). To investigate the distribution of several representative stem or progenitor cell genes (Sca-1, Nanog, Islet-1, Sox-2, NKX2.5, GATA4, Oct4) in the above-mentioned cell subpopulations, further enrichment analysis of these specific genes (Fig. 7) was done, which showed that the proportion of Sca-1 positive cells was highest, mainly enriched in the fibroblast and endothelial cells (7A); GATA4 was abundant in fibroblasts and endothelial cell populations (7B); Sox-2 was found only in the cardiomyocyte population (7C).

Table 1

Proportion of each cell subset of t-SNE clustering analysis
Cell type	Number	Frequence/%
B cell	50	0.643
T/NK cell	340	4.375
Macrophages	855	11.002
Granulocytes	71	0.914
Endothelial cell/endocardial cell	164	2.110
Cardiac muscle cell	79	1.017
Smooth muscle cell	249	3.204
Fibroblast	5963	76.734
Total	7771	100

The existence of stem cells in the heart has become a hot topic in the field of cardiac research in recent years. Cardiac biology and heart regeneration have been intensively investigated and debated in the last 15 years. Nowadays, the well-established and old dogma that the adult heart lacks of any myocyte-regenerative capacity has been firmly overturned by the evidence of cardiomyocyte renewal throughout the mammalian life as part of normal organ cell homeostasis, which is increased in response to injury. Concurrently, there is research evidence that the adult heart possesses a pool of multipotent cardiac stem/progenitor cells capable of sustaining cardiomyocyte and vascular tissue refreshment after injury [38]. At present, the commonly used stem cell labeling method is that a marker represents a class of cells. This method is used to detect myocardial stem cells in situ have created the paradox of the adult heart harboring at least seven different cardiac progenitor or stem populations. We found that the density of positive cells expressing stem cell markers within myocardial tissue was not significant. On the basis of these contradictory findings, we re-identified the myocardial stem cell marker-positive cells by cross co-localization of two proteins. The results showed that Sca-1 and Islet-1, Sca-1 and Nanog, and Nanog and Islet-1 were co-expressed in the separate cell subsets. It is suggested that the cellular taxa detected with a single marker do not reflect the true picture of cell classification.

It is generally accepted that stem cells are undifferentiated, immature, self-replicating cells that express two or more marker proteins in the embryonic stage and have the potential to regenerate tissues and organs. This definition is the basic yardstick for judging the presence or absence of stem cells in myocardial tissue. The cells isolated and purified in this study have a good ability to divide and proliferate. Sca-1, Nanog and Islet-1 expressed by these cells are commonly used stem cell marker proteins. At the same time, they can be induced to express the cardiac-specific protein cTnT and differentiate into cardiomyocyte-like cells. The above three aspects are in line with the basic characteristics of stem cells, so we believe that the cells isolated in this study should belong to stem cells. Lin et al. argue that there are stem cells in the myocardium, and these cells can differentiate into one or more of three cell lineages—endothelial cells, smooth muscle cells, and cardiomyocytes in vitro and in vivo, however, the amount of stem cells will gradually decrease as the myocardial tissue matures [2]. A recent study has proven that 80% of Sca-1 positive cells can differentiate into osteoblasts, adipocytes and chondrocytes [26]. Other research evidence shows that a considerable number of stem cells are still present in adult heart tissue, although their stemness and ability to repair myocardial damage are greatly reduced [4, 5]. The discrepancy in conclusions obtained from in vitro experiments to determine the presence or absence of stem cells in adult hearts may be associated with the different methods of isolation and culture in vitro. Previously, most studies have adopted the tissue block applanation culture method [38], which is simple and operational, but has the disadvantage of poor purity of the harvested cells and long culture period. More importantly, the number of stem cells in the primary cell population obtained by this method was low, and its abundance could not meet the requirements of subsequent experiments. In this study, the density gradient centrifugation method [39]-using 0.2% type II collagenase and the cell separation solution poly sucrose-sodium pantothenate solution (1.077 g/mL Ficoll solution)-was used to separate cardiac cells of different densities, with the stem cell population being of medium density and present in the intermediate layer after centrifugation, the "buffy coat". In this way, the number and purity of stem cells we get are greatly improved.

The diversity and heterogeneity of stem cells in myocardial tissue remains controversial. Past works usually assumed that the positive expression of a certain marker means that the positive cells are a cell subset. In this regard, at least subsets of cells such as Sca-1⁺ cells, lateral population (SP) cells, cardiomyocyte-derived cells (CDCs), Islet-1⁺cells, and epicardium-derived stem cells (EPDCs) have been identified. If these cells are independent, the true functional relationship between these cell populations is still unclear. We hypothesized that if two or more markers were co-expressed on a single cell, the actual quantity of cell subpopulations would be much smaller than the previously delineated. For this reason, two-two cross co-expression was performed on the isolated cells in this study.

In our study, normal BALB/c mice were grouped into four periods, namely the newborn group, the 1-week-old group, the 3-week-old group, and the 5-week-old group to demonstrate the age-related changes in the quantity and differentiation potential of stem cells in myocardial tissue. Previous studies have rarely performed such a long continuous follow-up. The results of this study exhibited a concurrent change in the co-expression levels of each two markers as time increased. The co-localization level of Sca-1 + Islet-1 gradually decreased from newborn to 5 weeks postnatal heart; the co-localization level of Sca-1 + Nanog first increased and then decreased; the co-localization level of Islet-1 + Nanog gradually increased. Comparing the above findings with the results of single marker labeling showed a high degree of consistency. The expression of Sca-1 reached the maximum at around one week after birth, while that of Nanog and Islet-1 culminated at the 3rd week. Combining the results of single protein expression and co-localization in this study, we found the following regularities: Sca-1⁺ cell population was more functionally active before 3 weeks of life, and co-expression with Islet-1 mainly occurred within 1 week after birth (probably contributes to cardiac development) and with Nanog predominantly at the period from 1 to 3 weeks of life; Nanog⁺ cells were dominant between 3 and 5 weeks after birth, and then the co-expression of Islet-1 and Nanog was enhanced, with a gradual synergistic effect on the heart.

To further clarify the relationship between the proportion of Sca-1, Nanog and Islet-1 positive cells in myocardial tissue, we selected several cardiac-specific stem cell genes (including Sca-1, Nanog, Isl-1, Sox-2, NKX2.5, GATA4, Oct4) from the numerous results obtained by scRNA-seq for enrichment analysis. The results showed a strong positive expression of Sca-1, while Nanog and Islet-1 genes were less expressed. This result is consistent with the single-labeling and co-localization results, indicating that both gene and protein expression of Sca-1 is stronger than that of Nanog and Islet-1, which may be attributed to the fact that Nanog and Islet-1 are mainly present in naive cardiomyocyte populations and their expression intensity gradually decreases after birth as cells mature, and furthermore, Nanog has been lately reported to be a marker of muscle senescence [40].

As shown by single-cell gene sequencing and stem cell gene enrichment (Fig. 6,7), Sca-1, GATA4 and Sox2 genes were mainly enriched in the fibroblast cluster, indicating that stem cells may have some kind of functional relationship with fibroblasts. A number of findings have shown that fibroblasts have the features of stem cells and can differentiate in multiple directions [41–43]. Does this suggest that the so-called cardiac stem cells are part of the fibroblast population? It is well worth further exploration.

In conclusion, our study demonstrated that one cell co-expresses two or more proteins through the pairwise co-localization of three stem cell marker proteins, Sca-1, Nanog, and Islet-1. Therefore, one stem cell marker alone cannot be used to classify subpopulations of cardiac stem cells. There are dynamic changes in Sca-1, Nanog, and Islet-1 positive cells at different cardiac developmental stages, which may lead to differences in their naïveté and differentiation potential. Furthermore, what functional relationship exists between cells expressing stem cell markers and fibroblasts needs to be further investigated.

Experimental animals

One hundred and forty healthy male BALB/c mice, including 60 newborn mice within 2 days, 40 at 1 week of age, 20 at 3 weeks of age, and 20 at 5 weeks of age, weighing (2.0 ± 0.5) g, (6.5 ± 0.5) g, (12.0 ± 0.5) g, and (20.0 ± 0.5) g were purchased from the Experimental Animal Center of Xinxiang Medical College [Physical Certificate: SCXK (Yu) 2017-0001].

Ethics approval

This study was performed in line with the principles of “Guiding Opinions on Treating Experimental Animals” issued by the Ministry of Science and Technology of the People's Republic of China. Approval was granted by the Animal Care and Use Committee of Xinxiang Medical University (Xinxiang China). The authors complied with the ARRIVE guidelines.

Cell isolation and culture

Twenty newborn mice within 2 days old, ten 1-week-old mice, five 3-week-old and 5-week-old mice were anesthetized using sodium pentobarbital via intraperitoneal injection, and the hearts were removed by rapid thoracotomy after disinfection in 75% alcohol. All hearts from the same age group were placed in the same sterile centrifuge tube (containing 10 mL of pre-cooled Hank's solution), and the tubes were transferred to the ultraclean bench. The hearts were washed again with Hank's solution and cut into small pieces of 2–4 mm³. The tissue pieces were resuspended with 0.2% type II collagenase solution, mixed thoroughly, and digested at 75×g for 1h at 37°C on a thermostatic shaker. Add complete medium (volume of medium = 2–3 times the volume of the tissue mixture). Filter the cell suspension sequentially through 100 µm and 40 µm cell strainers. The cells were separated by density gradient centrifugation: firstly, the filtrate was gently overlay with an equal volume of the Ficoll 1.077 solution and the gradient mixture was centrifuged at 500×g for 20 min at room temperature. And then remove the middle layer "buffy coat" from the centrifuged stratified solution. Lastly, Add complete medium to the cell suspension, and centrifuge for 5 min at 500×g, 4°C twice to remove the residual Ficoll solution. The cell precipitate containing purified stem cell population was resuspended in complete medium. The cell suspension was inoculated in 25 cm² culture flasks and incubated at 37℃ and 5% CO2. The medium was changed daily for the first 3 days and every 2–3 days thereafter.

Immunocytochemical staining

The cells obtained above were inoculated into 24-well plates, and once the cells reached the optimal fusion level (50–70%), the medium was aspirated and washed thrice with PBS. The cells were fixed with 4% paraformaldehyde for 20 min and washed thrice with PBS for 5 min each time. Next the cells were subjected to 0.2% Triton X-100 solution for 10 min and washed thrice with PBS. Incubated with 0.3% H2O2 for 10 min and washed thrice with PBS. Goat serum blocking ensued for for 1h. monoclonal Primary antibodies: rat anti-mouse Sca-1 antibody (1:100), mouse anti-mouse Nanog antibody (1:500) and rabbit anti-mouse Islet-1 antibody (1:250) were added dropwise overnight at 4°C. The secondary antibodies were added to each well: goat anti-rat IgG, goat anti-mouse IgG and goat anti-rabbit IgG, respectively. The next staining procedure was conducted with the commercially available IHC assay kits. The coverslips were taken out from the plate and attached to the slides with Clear-Mount. The images were obtained by the light microscope.

Identification of myocardial differentiation potential

The primary cells from 5-week-old myocardial tissue were seeded in 48-well plates with 18 wells (A1-A6, B1-B6, C1-C6) and divided into four groups: A1, B1 and C1 wells were blank control group; A2, B2 and C2 were uninduced group; A3, B3 and C3 were normal group; the rest of wells were induced group. The uninduced group was added with low-glucose complete medium containing 12% fetal bovine serum and the cells were fixed for immunofluorescence staining on the 2nd day after inoculation. The normal group was also cultured with complete medium, and the induction group was added with myocardial induction solution (IMEM, 15% fetal bovine serum, 2 mM glutamine, 40 ng/ml bFGF, 0.01 mM β-mercaptoethanol, 100ug/ml penicillin, 100ug/ml streptomycin, 10uM CHIR99021, 1uM Dorsomorphin, 5uM SB431542, 500uM VPA, 10uM Trichothecene). Then put the 48-well plate at cell incubator, and the medium was changed every 2–3 days. The normal and induced groups were fixed and immunofluorescently stained on the 10th day after inoculation. Then the expression of cardiomyocyte-specific protein cardiac troponin T(cTnT) and three marker proteins (Sca-1, Nanog, Islet-1). Blank control group (wells A1, B1, C1) without primary antibody, wells A2-A6, B2-B6 and C2-C6 were double-stained to compare the expression differences between three groups. A2-A6 with two antibodies added in sequence: rat anti-mouse Sca-1 antibody (1:100) and mouse anti-mouse cTnT antibody (1:200). B2-B6 were add with mouse anti-mouse Nanog antibody (1:500) and rabbit anti-mouse cTnT antibody (1:100). Rabbit anti-mouse Islet-1 antibody (1:100) and mouse anti-mouse cTnT antibody (1:200) were added sequentially in C2-C6.

Western blotting and detection of proteins

Sample preparation for Western blotting was performed with the third generation of the cultured cells from different age groups(newborn, 1 week, 3 weeks, 5 weeks). The lysis mixture containing RIPA solution and protease inhibitor cocktail in the proportion 100:1. The lysed cell mix was centrifuged at 12,000 r/min for 5 min and the supernatant was the whole cell proteins we needed. The samples were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were then incubated with primary antibodies and peroxidase-conjugated secondary antibodies. Chemiluminescence quantification was performed using Chemi-Lumi HRP Substrate, followed by image acquisition on ChemiDoc XRS + System(Bio-Rad). The primary antibodies were Rat anti-Sca-1 (1:500), Mouse anti-Nanog (1:1000) and Rabbit anti-Islet-1 antibody (1:10000).

Double-labelled immunofluorescence

The primary cells were seeded in 48-well plates. When the cells grew and fused to 70%-80%, perform the same dyeing procedure as described above. After the two rounds of staining, the nuclei were stained with DAPI solution and immunofluorescence images were obtained under an inverted fluorescence microscope. The blank control was PBS instead of the primary antibody, and the rest of the operations were the same as the experimental group. Primary antibodies used: rat anti-Sca-1 antibody (1:100), mouse anti-Nanog (1:250) and rabbit anti-Islet-1 antibody (1:100). Secondary antibodies used: Alexa Fluor 488-labeled goat anti-rat secondary antibody (1:50), Cy3-labeled goat anti-rabbit secondary antibody (1:500), Cy3-labeled goat anti-mouse secondary antibody (1:500) and FITC-labeled goat anti-mouse secondary antibody (1:500).

IF co-expression analysis

The above immunofluorescence images were merged and divided into 3 major groups: (1) Sca-1 + Islet-1 double-labeled group; (2) Sca-1 + Nanog double-labeled group; (3) Nanog + Islet-1 double-labeled group, and each major group was divided into 4 subgroups according to the age of mice: newborn, 1 week, 3 weeks and 5 weeks. 20 fields were randomly selected in each group, and the co-expression rate was calculated (co-expression rate = number of co-expressed cells/total number of nucleated cells) to compare the co-expression between the 3 different cardiac stemness markers and the expression differences in different cardiac developmental stages.

IF co-localization analysis qualitatively and quantitatively

The above superimposed IF images were analyzed by Image J software to depict the scatter plot and analyze the relationship between the scatter plot and the diagonal line, which can qualitatively analyze the co-localization relationship between the two labeled proteins represented by the two colors. The ROI was manually boxed and Pearson's Correlation Coefficient (PCC) was calculated for each group to quantitatively analyze the co-localization level. The above scatter plot depiction and PCC calculation can be implemented in Coloc 2 or JACoP plug-in of Image J software.

Single-Cell RNA Sequencing analysis

Freshly isolated hearts were placed in MACS tissue storage solution (Mitenyi Biotec) and quickly sent to Mingma Technologies for scRNA-seq analysis of the tissue samples. Data analysis was performed using Seurat 3.2.3 software, including: t-SNE clustering analysis, cell type identification, searching for class-specific expressed genes (differential genes), specific marker protein enrichment analysis and gene ontology (GO) analysis.

Statistical methods

Data were processed using SPSS 19.0 and Graphpad Prism 6.0 software, and all data were expressed as standard deviation of the mean(x̅ ± S). T-test was used for comparison between two samples, and one-way ANOVA was used for comparison of multiple sample means, P < 0.05 was considered statistically significant.

Data Availability The datasets generated and/or analysed during the current study are available in the “Figshare” repository at https://doi.org/10.6084/m9.figshare.21775001.v1.

Acknowledgements This research is supported by the National Natural Science Foundation of China. We would like to thank Henan Key Laboratory of Cardiac Reconstruction and Transplantation for providing important help for this study.

Author contributions Conception and design: Z.G.,X.L.; literature search and collection of the scientific information: X.L.; material preparation, data collection and analysis: W.G.,J.D.,X.L.; writing and revision of the manuscript: Y.X.,X.L.,Z.G. All authors reviewed the manuscript.

Additional Information

Competing interests The authors declare no competing interests.

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Subpopulation analysis of Sca-1, Nanog, and Islet-1 positive cells in myocardial tissue

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Abstract

Figures

Introduction

Results

• Results of mouse primary cardiomyocyte culture

• Expression of Sca-1, Nanog, and Islet-1 in primary cells

• Myocardial differentiation potential of primary Sca-1, Nanog, and Islet-1 positive cells

• Age-related changes in the expression of Sca-1, Nanog, and Islet-1

• Co-localization analysis of Sca-1, Nanog, and Islet-1

• Cell type analysis and stem cell gene localization in juvenile mice cardiac tissue

Discussion

Methods

Experimental animals

Ethics approval

Cell isolation and culture

Immunocytochemical staining

Identification of myocardial differentiation potential

Western blotting and detection of proteins

Double-labelled immunofluorescence

IF co-expression analysis

IF co-localization analysis qualitatively and quantitatively

Single-Cell RNA Sequencing analysis

Statistical methods

Declarations

References

Additional Declarations

Supplementary Files

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