Experimental study on co-culture of DiI-labeled rat bone marrow mesenchymal stem cells and neonatal rat cardiomyocytes to induce differentiation into cardiomyocyte-like cells

Abstract

BACKGROUND:

Myocardial infarction is a serious clinical disease with high mortality and poor prognosis. Cardiomyocytes (CMs) have limited regeneration abilities after ischemic injury. Their growth and differentiation can be enhanced by contact co-culture with stem cells.

OBJECTIVE:

The aim was to study the contact co-culture of Dil-labeled bone marrow mesenchymal stem cells (BMSCs) and CMs for inducing differentiation of CMs from stem cells for treating myocardial infarction.

METHODS:

After contact co-culture, the differentiation of BMSCs into CMs was analyzed qualitatively by detecting myocardial markers (cardiac troponin T and α-smooth muscle actin) using immunofluorescence and quantitatively using flow cytometry. To examine the mechanism, possible gap junctions between BMSCs and CMs were analyzed by detecting gap junction protein connexin 43 (C×43) expression in BMSCs using immunofluorescence. The functionality of gap junctions was analyzed using dye transfer experiments.

RESULTS:

The results revealed that BMSCs in contact with CMs exhibited myocardial markers and a significant increase in differentiation rate (P < 0.05); they also proved the existence and function of gap junctions between BMSCs and CMs.

CONCLUSIONS:

It was shown that contact co-culture can induce Dil-labeled BMSCs to differentiate into CM-like cells and examined the principle of gap junction-mediated signaling pathways involved in inducing stem cells to differentiate into cardiomyocytes.

Keywords

Stem cell therapy bone marrow mesenchymal stem cells cardiomyocytes contact co-culture DiI labeling in vitro

1. Introduction

Myocardial infarction, a common and serious disease in the clinic, has high mortality and poor prognosis. As terminally differentiated cells, cardiomyocytes (CMs) have limited regeneration abilities after ischemic injury. With breakthroughs in the field of stem cells, stem cell-based therapy has become a research hotspot in recent years [1]. Transplantation of mesenchymal stem cells (MSCs) into areas of myocardial infarction is a helpful treatment that improves cardiac function [2].

MSCs are adult stem cells that mainly exist in connective tissue, organ stroma, and bone marrow. Bone marrow mesenchymal stem cells (BMSCs) are easy to obtain and expand in vitro. Importantly, MSCs can be transplanted without immune rejection or ethical concerns. BMSCs exhibit multi-directional differentiation potential and can generate bone cells, muscle cells, chondrocytes, nerve cells, adipocytes, hepatocyte-like cells, and other cell types [3–9].

Contact co-culture is the method of putting two or more cells in direct contact within the same culture system to promote cell proliferation and/or differentiation. Contact co-culturing BMSCs with CMs promotes the proliferation and differentiation of BMSCs [10].

In this study, BMSCs were labeled with DiI dye and subsequently induced to differentiate into CM-like cells by contact co-culture with CMs to preliminarily study the mechanism and lay a theoretical foundation for further in vivo tracing experiments and clinical applications especially in the treatment of myocardial infarction.

2. Materials and methods

2.1. Materials

Clean Sprague-Dawley rats and suckling rats were purchased from SBF Biotechnology (Beijing, China). Fetal bovine serum was purchased from Gibco (Gaithersburg, MD, USA). Penicillin-streptomycin, trypsin, type II collagenase, DiI dye, and calcein were purchased from Yisheng (Shanghai, China). High-glucose Dulbecco’s Modified Eagle Medium was purchased from Hyclone (Logan, UT, USA). Flow cytometry was performed using primary antibodies against cardiac troponin T (cTnT), α-smooth muscle actin (α-SMA), connexin 43 (C×43), and secondary antibodies purchased from Abcam (Cambridge, UK).

2.2. Isolation and culture of BMSCs and identification of surface antigens

Two Sprague-Dawley rats aged 5–6 weeks were sacrificed by neck amputation. Their femur and tibia were removed, and the bone marrow cavity was washed with high-glucose Dulbecco’s Modified Eagle Medium. The flushing solution was collected, centrifuged, resuspended, and inoculated in six-well plate for culture in a 5% CO₂ incubator at 37 °C. Passage three (P3) generation BMSCs were used in subsequent experiments.

P3 BMSCs were digested, centrifuged, counted, and adjusted to a density of 5 × 10⁴ cells/ml. Samples were aliquoted into five tubes to which monoclonal fluorescent antibodies for rat CD90, FITC-conjugated rat CD44H, APC-conjugated rat CD11b/c, and PE-cyanine7-conjugated rat CD45 were added, respectively, as well as a blank group (fifth tube) for computer detection.

2.3. DiI-labeling of BMSCs

To label P3 BMSCs, 1 mL per well of 5 μM DiI staining solution was added to the cells for 15 min. Following incubation, the cells were washed, digested, centrifuged, resuspended, counted, and adjusted to a density of 5 × 10⁴ cells/mL. Next, 2 mL of the cell suspension was added to the wells of six-well plate for culture. After 24 h, the cell morphology was observed under a fluorescence microscope.

2.4. Extraction of suckling rat CMs

Fifteen suckling rats were disinfected with 75% ethanol before opening their chest along the left side of the sternum with ophthalmic scissors. Next, the heart was removed and quickly placed in a dish containing phosphate-buffered saline (PBS). The heart tissue was cut into pieces and digested three times (20 min each at 37 °C) with 15 mL of prepared digestive solution [0.2% type II collagenase (10 mL), 0.25% trypsin (3 mL), and PBS (2 mL)]. After each digestion, the samples were centrifuged, the supernatant was aspirated, and serum was added to terminate digestion. In the third digestion cycle, the termination solution was filtered, centrifuged, resuspended, and inoculated in a culture plate. After differential adhesion for 1.5 h, the supernatant was centrifuged, resuspended, inoculated in a 6-well plate, and incubated in an incubator. After 3 days, the solution was changed and cell morphology was observed under a microscope.

2.5. Contact co-culture of DiI-labeled BMSCs and CMs

CMs on the third day of culture and DiI-labeled P3 BMSCs were used for the co-culture. The suspensions of digested CM and DiI-labeled BMSCs were mixed at a ratio of 4 CMs:1 BMSC; in total, 2.5 × 10⁴ cells/well were inoculated into the wells of 6-well plate. In the control group, the number of DiI-labeled BMSCs remained unchanged, and CMs were replaced with unlabeled BMSCs at the same cell amount. The culture medium was changed every two days, and morphological changes in the cells were observed under a microscope.

2.6. Detection of myocardial markers (cTnT and α-SMA) by immunofluorescence

After 7 days of co-culture, the expression of the myocardial markers cTnT and α-SMA was evaluated. After aspirating the culture medium, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min, and washed again three times with PBS. Next, the cells were permeabilized with 0.1% Triton X-100 for 10 min, washed three times with PBS, blocked with 5% bovine serum albumin for 30 min, and washed again three times with PBS. Three samples were selected from both the experimental and control groups. The first sample was incubated with rabbit anti-rat cTnT (1:400 in PBS), the second with rabbit anti-rat α-SMA (1:400 in PBS), and the third with rabbit anti-rat C×43 (1:200 in PBS) overnight at 4 °C. After washing three times with PBS, a goat anti-rabbit fluorescent secondary antibody was added followed by incubation in the dark at room temperature for 1 h. Following three washes with PBS for 3 times, 4′,6-diamidino-2-phenylindole (DAPI) dye was added for incubation at room temperature for 3 min, followed by three washes with PBS. Cells were observed and imaged using a fluorescence microscope.

2.7. Flow cytometric analysis of the differentiation rate of DiI-labeled BMSCs

On the first and seventh day of co-culture, two samples were selected from the experimental and control groups. After digestion, immunofluorescence staining for cTnT was performed and analyzed using flow cytometry.

2.8. Detection of C×43 by immunofluorescence and dye transfer experiment

In the co-culture group, one well was selected daily to detect C×43 expression in DiI-labeled BMSCs by immunofluorescence. In addition, CMs with good growth up to the third day were selected and labeled with 2.5 μM calcein-AM dye for 30 min. DiI-labeled BMSCs and calcein-AM-labeled CMs were mixed and co-cultured according to the proportion of cells in the contact co-culture group. The cells were observed and imaged daily using a fluorescence microscope.

2.9. Statistical analysis

Each experiment was repeated thrice, and the experimental data were statistically processed using SPSS version 20.0. Measurement data were expressed as mean ± standard deviation (x ± s). An independent sample mean t-test was used for statistical analysis. Statistical significance was set at P < 0.05.

3. Results

3.1. Isolation, culture, and passage of BMSCs in vitro

After 6–8 h of primary culture, the cells began to adhere to the wall and most were polygonal (Fig. 1a). After three passages, the cell volume decreased and the cells tended to be consistently polygonal or spindle-shaped.

3.2. Identification of cell phenotype by flow cytometry

CD90 was positively expressed in 99.16% of the isolated cells, while CD44H was positively expressed in 99.72% of the cells. CD11b/c, a characteristic surface antigen of leukocytes, was expressed in only 2.32% of the cells (considered negative). Similarly, CD45, a surface antigen of hematopoietic cells, was expressed in only 3.10% of the cells (considered negative). These results are consistent with the immunophenotypic characteristics of BMSCs.

3.3. Observation of DiI-labeled BMSCs by fluorescence microscopy

DiI-labeled cells emitted red light under excitation at a 550-nm wavelength. Under a fluorescence microscope, the BMSCs were polygonal and normal in shape (Fig. 1b).

3.4. Isolation and culture of neonatal rat CMs in vitro

After 3 days of culture, CMs of suckling mice were polygonal or spindle-shaped (Fig. 2), and cell pulsation was observed at a frequency of approximately 80 pulses/min.

Fig. 1.

Microscope picture of BMSCs. (a) Primary generation BMSCs under light microscope (40×); (b) DiI-labeled P3 generation BMSCs (40×).

Fig. 2.

Microscope picture of CM. Cardiomyocytes were cultured for 3 days under light microscope: (a) CMs (100×); (b) CMs (200×).

3.5. Contact co-culture of DiI-labeled BMSCs and CMs

Microscopy on the first day of co-culture revealed that the cells had adhered to the wall and started growing (Fig. 3a). Observation of the same field under a fluorescence microscope showed that DiI-labeled BMSCs emitted red light, whereas unlabeled CMs did not emit light (Fig. 3b). Compared with the pulsation of CMs, DiI-labeled BMSCs showed passive traction.

Fig. 3.

Microscope picture of contact co-culture. Comparison of co-cultured cells under light microscope and fluorescence microscope: (a) Contact co-culture underan ordinary microscope(100×); (b) Contact co-culture undera fluorescence microscope (100×).

Fig. 4.

Photos of experimental group and control group under immunofluorescence microscope. cTnT immunofluorescence staining in the experimental group: (a) the nucleus was stained blue by DAPI (200×); (b) DiI-labeled BMSCs were red (200×); (c) BMSCs and CMS expressing cTnT were green (200×); (d) Merge figure (a) (b) (c) (200×). Control group cTnT immunofluorescence staining: (e) The nucleus was stained blue by DAPI (200×); (f) DiI-labeled BMSCs were red (200×); (g) There were no green cells expressing cTnT (200×); (h) Fit figure (e) (f) (g) (200×). Experimental group α-actin immunofluorescence staining: (I) The nucleus was stained blue by DAPI (200×); (j) DiI-labeled BMSCs were red (200×); (k) Express α-BMSCsα- BMSCs and CMS of actin were green (200×); (l) Merge figure (I) (J) (k) (200×). Control group α-actin immunofluorescence staining: (m) Nuclei were stained blue by DAPI (200×); (n) DiI-labeled BMSCs were red (200×); (o) No green expression was found in α–α-Actin cells (200×); (p) Merge graph (m) (n) (o) (200×).

3.6. Detection of myocardial markers (cTnT and α-SMA) by immunofluorescence

Fluorescence microscopy revealed that on the 7th day of co-culture, in the experimental group, some DiI-labeled BMSCs in contact with CMs expressed cTnT (Fig. 4a–d) and α-actin (Fig. 4i–l). In the control group, DiI-labeled BMSCs did not significantly express cTnT (Fig. 4e–h) and α-actin (Fig. 4m–p). Among them, DiI-labeled BMSCs showed red fluorescence (Fig. 4b, f, j, n), and cells expressing myocardial markers (cTnT/α-actin) showed green fluorescence (Fig. 4c,k). In the same visual field, some DiI-labeled BMSCs in contact with CMs had both the red fluorescence of DiI-labeled and the green fluorescence of myocardial markers (Fig. 4d,l); indicating that the DiI-labeled BMSCs in that part had differentiated into cardiomyocytes. There was no obvious green fluorescence in the control group (Fig. 4g,o) indicating that the differentiation of DiI-labeled BMSCs into cardiomyocytes had not taken place.

3.7. Flow cytometric analysis of the differentiation rate of DiI-labeled BMSCs

The results of the flow cytometry are shown in Fig. 5. The proportion of cTnT-positive cells in DiI stained BMSCs was used as the differentiation rate of stem cells, which was (5.59 ± 0.31)% on the first day and (21.08 ± 1.00)% on the seventh day in the experimental group; (0.54 ± 0.11)% on the first day (Fig. 5c) and (2.45 ± 1.25)% on the seventh day in the control group (Fig. 5d). In the experimental group, the differentiation rate of stem cells on the 7th day was higher than that on the 1st day (21.08% > 5.59%,p < 0.05, Fig. 5e); on the 7th day, the differentiation rate of stem cells in the experimental group was higher than that in the control group (21.08% > 2.45%, p < 0.05, Fig. 5f)

Fig. 5.

FCM of DiI-labeled BMSCs. (a) Flow analysis double staining scatter plot on the first day in the experimental group; (b) The Flow analysis double staining scatter diagram of the experimental group on the 7th day shows the group with the highest differentiation rate; (c) Flow analysis double staining scatter diagram of the control group on the 1st day; (d) Flow analysis double staining scatter diagram of the control group on the 7th day; (e) Comparison of differentiation rates of the experimental group on the 1st and 7th days; (f) Comparison of differentiation rates of the experimental group on the 7th day and the control group on the 7th day.

3.8. Detection of C×43 by immunofluorescence and dye transfer experiment

C×43 immunofluorescence staining was performed on the first two days after contact co-culture. The result was negative on the first day (Fig. 6e–h) and positive on the second day (Fig. 6a–d). DiI-labeled BMSCs emitted red fluorescence (Fig. 6b,f), and the cell membrane expressing C×43 emitted green fluorescence (Fig. 6c). On the second day, the green dot fluorescence emitted by C×43 could be seen between DiI-labeled BMSCs and CMs (Fig. 6d) indicating that they had communicated through the gap connection composed of C×43. On day 1, there was no green dot fluorescence seen between the DiI-labeled BMSCs and CMs (Fig. 6h).

Before co-culture, the calcein-stained CMs emitted green fluorescence (Fig. 6i). The dye transfer results were observed on the first, second, and third day after exposure to the co-culture. The results were negative on the first and second days, and positive on the third day (Fig. 6j–l). On the 3rd day, all cells containing calcein emitted green fluorescence (Fig. 6j), DiI-labeled BMSCs emitted red fluorescence (Fig. 6k), and cells emitting red fluorescence and green fluorescence simultaneously (Fig. 6l) could be observed in the same field of vision, that is, calcein was present in DiI-labeled BMSCs, indicating that calcein dye was transferred from CMs to DiI-labeled BMSCs.

Fig. 6.

Photo of immunofluorescence staining and dye transfer experiment under fluorescence microscope. On day 2, C×43 immunofluorescence staining: (a) The nucleus was stained blue by DAPI (400×); (b) DiI-labeled BMSCs were red (400×); (c) It can be seen that there is green dot like C×43 expression on the cell membrane of BMSCs (400×); (d) Merge figure (a) (b) (c) (400×). On day 1, C×43 immunofluorescence staining (e) The nucleus was stained blue by DAPI (200×); (f) DiI-labeled BMSCs were red (200×); (g) There was no green dot like C×43 expression on the BMSCs cell membrane (200×); (h) Merge figure (e) (f) (g) (200×). Before contact co-culture, calcein-stained CMS: (I) Calcein stained CMS was green (200×). After co-culture (j), all cells containing calcein were green (200×); (k) DiI-labeled BMSCs were red (200×); (l) Merge figure (j) (k) (200×).

4. Discussion

The efficiency of the natural differentiation of BMSCs into cardiomyocytes in vitro is very low. Various researchers have attempted to overcome this challenge. Currently, chemicals such as 5-azacytidine, salvianolic acid B, angiotensin II, and insulin-like growth factor-1 [11–13] are used for inducing differentiation. Among these, 5-azacytidine is the most popular inducer. However, even under the effect of an optimal concentration and induction time, its induced differentiation rate is still low [12,14]. Notably, most chemical inducers are cytotoxic. 5-Azacytidine induces cell wall detachment and vacuole formation, which limits its application. Simulating the myocardial microenvironment by contact co-culture of stem cells with CMs is more in line with the in vivo environment that supports cell growth and differentiation. Moreover, this approach does not elicit the toxicity or adverse reactions of traditional differentiation inducers, and thus has attracted increasing attention (Chu et al., 2020). In this experiment, by analyzing the differentiation rate of DiI-labeled BMSCs by flow cytometry, we found that contact co culture can improve the differentiation efficiency of BMSCs into CMs.

DiI, a lipophilic fluorescent dye, can be used to stain cell membranes and does not usually affect the cell viability. Therefore, they are widely used for cell and tissue tracing. We used DiI dye to label BMSCs based on the traditional contact co-culture. After contact co-culture, BMSCs and CMs were distinguished by differences in fluorescence, thus laying the foundation for further tracing experiments and applications in vivo.

In this experiment, immunofluorescence staining following contact co-culture revealed that DiI-labeled BMSCs showed red fluorescence and CMs expressing myocardial markers (cTnT and α-Actin) showed green fluorescence. In the same visual field, some DiI-labeled BMSCs in contact with CMs exhibited both red fluorescence of the DiI label and green fluorescence, indicating myocardial markers; that is, a portion of DiI-labeled BMSCs differentiated into CMs. These results showed that contact co-culture can induce stem cells to differentiate into CM-like cells.

The factors induced by contact co-culture that affect the differentiation of stem cells into CMs are mainly chemical and physical. Among them, chemical factors include cytokines secreted by cells exposed to co-culture, proteins related to signaling pathways, and other molecules, whereas physical factors include signal stimulation provided by direct contact with cells, mechanical traction between cells, and myocardial electrical activity(Zhang et al., 2020). Differentiation of BMSCs in contact with CMs suggests that contact with CMs is a key factor in the differentiation of DiI-labeled BMSCs, that is, it affects the differentiation of stem cells through physical factors. However, how the contact with CMs affects stem cell differentiation remains unclear.

Gap junctions are membrane channels formed between adjacent cells that mediate signal and material transmissions between them. Gap junctions are composed of gap junction proteins, which are widely expressed in different tissues. Among these, C×43 is the most abundant subtype in heart tissue [15,16]. Accordingly, the induction of stem cell and cardiomyocyte differentiation by contact co-culture may be related to C×43-mediated gap junctions. Following contact co-culture of BMSCs and CMs in this study, we detected C×43 by immunofluorescence staining. It was found to be negative on the 1st day and positive after the 2nd day, which proved the existence of gap junctions between BMSCs and CMs. Next, we designed experiments using dye transfer, a common method to test the functionality of gap junctions [17,18], in which CMs were stained with calcein before co-culture with DiI-labeled BMSCs. Calcein was found in some DiI-labeled BMSCs after the 3rd day, but it did not appear on the 1st and 2nd days. After the gap junction was established on the 2nd day, calcein may have been transferred from cardiomyocytes to stem cells through gap junctions on the 3rd day, thus proving the function of gap junctions. Therefore, the mechanism by which contact co-culture of DiI-labeled BMSCs with CMs induces their differentiation into CM-like cells may be related to the transmission of related signaling molecules through gap junctions.

This study lays a theoretical foundation for further in vivo tracing experiments and clinical applications especially in the treatment of myocardial infarctions.

Although this experiment proved that BMSCs produce functional gap junctions after contact with CMs, we do not know which signaling pathway affects the differentiation of stem cells through gap junctions. Thus, we will examine the principle of gap junction-mediated signaling pathways involved in inducing stem cells to differentiate into cardiac myocytes. In this experiment, the observation of gap junction mainly depends on simple immunofluorescence staining. In the future experiment, I will try to use immunoelectron microscopy to further distinguish gap junction between CMs-CMs. In addition, the culture medium of this experiment contains bovine serum. Next, I will conduct a serum free experiment for further study in vivo.

5. Conclusion

Our study suggests that DiI-labeled BMSCs can be induced to differentiate into CM-like cells by contact co-culture with CMs, and the mechanism inducing this differentiation may be related to the transmission of related signaling molecules through gap junctions.

Footnotes

Acknowledgements

The authors thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), Editage () for English language editing.

Ethical approval

The study was approved by the local ethics committee (project no. GZR-3-072), as the research scheme is reasonable and standardized, the humanitarian spirit is followed in the process of animal experiment, and the relevant rights and interests of animals are fully protected, which has passed the ethical review.

Conflict of interest

No potential conflict of interest was reported by the authors.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

This study was partially supported by the National Natural Science Foundation of China (nos 81870181 and 82270255).

Author contributions

ZZ carried out the molecular genetic studies, participated in the immunoassays and drafted the manuscript. BP carried out the flow cytometric. ZJ participated in the co-culture. YB participated in the design of the study and performed the statistical analysis. MJ and ZF conceived the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

References

Parizadeh

S.M.

Jafarzadeh Esfehani

Ghandehari

, Stem cell therapy: A novel approach for myocardial infarction, Journal of Cellular Physiology 234(10) (2019), 16904–16912.

Kim

S.H.

Cho

J.H.

Lee

Y.H.

, Improvement in left ventricular function with intracoronary mesenchymal stem cell therapy in a patient with anterior wall ST-segment elevation myocardial infarction, Cardiovascular Drugs and Therapy 32(4) (2018), 329–338.

Wang

Y.H.

Wang

D.R.

Guo

Y.C.

, The application of bone marrow mesenchymal stem cells and biomaterials in skeletal muscle regeneration, Regen Ther 15(2020) (2020), 285–294.

Zakirova

E.Y.

Valeeva

A.N.

Aimaletdinov

A.M.

, Potential therapeutic application of mesenchymal stem cells in ophthalmology, Exp Eye Res 189 (2019), 107863.

Guo

Bai

Zhang

, Cardiomyocyte differentiation of mesenchymal stem cells from bone marrow: New regulators and its implications, Stem Cell Research & Therapy 9(1) (2018), 44.

Wang

Jing

, Bone marrow- and adipose tissue-derived mesenchymal stem cells: characterization, differentiation, and applications in cartilage tissue engineering, Crit Rev Eukaryot Gene Expr 28(4) (2018), 285–310.

Wang

Meng

Wang

, Differentiation of bone marrow mesenchymal stem cells in osteoblasts and adipocytes and its role in treatment of osteoporosis, Medical Science Monitor 22 (2016), 226–233.

Liu

W.H.

Song

F.Q.

Ren

L.N.

, The multiple functional roles of mesenchymal stem cells in participating in treating liver diseases, Journal of Cellular and Molecular Medicine 19(3) (2015), 511–520.

Defeng

Z.Y.C.Y.

, Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells, Neural Regeneration Research 9(12) (2014), 1241–1248.

10.

Yang

Tian

Feng

, Trichostatin a promotes cardiomyocyte differentiation of rat mesenchymal stem cells after 5-azacytidine induction or during coculture with neonatal cardiomyocytes via a mechanism independent of histone deacetylase inhibition, Cell Transplant 21(5) (2012), 985–996.

11.

Gong

Wang

, Inhibition of IGF-1 receptor kinase blocks the differentiation into cardiomyocyte-like cells of BMSCs induced by IGF-1, Mol Med Rep 16(1) (2017), 787–793.

12.

Hou

A.L.

Liu

B.W.

, Combination of BMP-2 and 5-AZA is advantageous in rat bone marrow-derived mesenchymal stem cells differentiation into cardiomyocytes, Cell Biol Int 37(12) (2013), 1291–1299.

13.

Fan

Wang

, Angiotensin II increases periostin expression via Ras/p38 MAPK/CREB and ERK1/2/TGF-beta1 pathways in cardiac fibroblasts, Cardiovasc Res 91(1) (2011), 80–89.

14.

Wakitani

Saito

and Caplan

A.I.

, Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine, Muscle Nerve 18(12) (1995), 1417–1426.

15.

Epifantseva

and Shaw

R.M.

, Intracellular trafficking pathways of C×43 gap junction channels, Biochim Biophys Acta Biomembr 1860(1) (2018), 40–47.

16.

Ribeiro-Rodrigues

T.M.

Martins-Marques

Morel

, Role of connexin 43 in different forms of intercellular communication - gap junctions, extracellular vesicles and tunnelling nanotubes, J Cell Sci 130(21) (2017), 3619–3630.

17.

Abbaci

Barberi-Heyob

Blondel

, Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication, Biotechniques 45(1) (2008), 33–52, 56-62.

18.

Fonseca

P.C.

Nihei

O.K.

Savino

, Flow cytometry analysis of gap junction-mediated cell-cell communication: Advantages and pitfalls, Cytometry A 69(6) (2006), 487–493.