Immunophenotyping of a population of cultured human umbilical cord cells from Wharton's jelly

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Currently, various methods for isolation and cultivation of mesenchymal stem cells (MSCs) and their use in clinics have been reported in the literature [1]. MSC transplantation stimulates regeneration of bone tissue, skin, myocardium, peripheral nervous system, skeletal muscle, and liver tissue, and MSCs serve as a source of growth factors and cytokines [2]. The decisive aspect that plays an important role in the selection of artificial biomedical materials is the influence of the implant on the regeneration processes of the recipient’s damaged tissues, its immunomodulatory properties [3]. Among the techniques for replacing defects in posttraumatic injuries, those involving the use of tissue engineering products and artificially cultured human cells are becoming increasingly prevalent in medical practice. In such cases, the use of the patient’s own tissues or tissues with low immunogenicity is advantageous. Several publications have reported the weak immunogenic activity of human umbilical cord tissues [5]. In this regard, human umbilical cord Wharton’s jelly stem cells (HWJSCs) are a promising source of MSCs. There are various studies on the application of HWJSCs and other umbilical cord tissues. However, there is a need for a systematic approach to organize and select the optimal variants of laboratory and technological solutions for reproducible MSC cultivation [6, 7]. Many researchers have proposed the use of human umbilical cord mucosa as an alternative source of MSCs. Given that HWJSC is the tissue of the provisional organ — the umbilical cord — procedures involving it is subject to minimal ethical restrictions. Furthermore, obtaining explants from HWJSC is performed using a noninvasive method. The process of acquiring such explants includes dissecting the surrounding tissue, including the vein and artery of the umbilical cord. This may cause explanting of tissue fragments of other types along with HWJSCs, resulting in the acquisition of cells with a different status than that of MSCs. Owing to possible errors in explant selection and cell transformation during the cultivation process, the phenotype of cells obtained as a result of tissue explantation and during their further cultivation should be determined. The International Society for Cell Therapy has defined the immunophenotype of MSCs as CD90+, CD105+, CD44+, CD29+, CD105+, CD73+, CD166+, CD45–, and CD34– [9]. The most effective method in determining characteristic cell surface proteins is the fluorescence method [10]. In the present study, the surface markers CD45, CD73, CD34, CD29, CD90, CD44, and CD105 were identified and the presence of vascular muscle tissue cells was assessed using the specific cytoskeletal marker alpha-actin in the population of cells derived from HWJSCs.

This study aimed to identify MSCs in the cell population obtained from HWJSCs using immunoenzymatic labeling.

Objectives of the study:

1. To demonstrate the absence of vascular smooth muscle cells in the obtained culture and perform immunohistochemical staining for the presence of smooth muscle actin (alpha-smooth muscle actin, α-SMA) in the cytoskeleton of the cells of the studied population
2. To examine the obtained cell population for the presence of MSCs using the surface markers CD45, CD73, CD34, CD29, CD90, CD44, and CD105 by flow cytophotometry and observe fluorescence on the cell surface during microscopy

MATERIALS AND METHODS

Obtaining a population of HWJSCs

The primary human umbilical cord material was obtained by the staff of the Department of Obstetrics and Gynecology of the Kirov Military Medical Academy and acquired in the course of planned cesarean section. The medical history of the primary material donors was recorded and stored by the staff of the department. Informed consent was obtained from the donor. The selection of primary material for further processing and harvesting of tissue fragments for explantation were performed in the delivery room under aseptic conditions. Cord material without exsanguination was placed in the transport medium immediately after separation from the placenta. Standard sterile 0.9 % sodium chloride solution in a sterile container was used as the transport medium [11]. The material was transported at ambient temperature. The umbilical cord material was transferred to the sterile room of the Cell Technology Research Laboratory of the Research Department of Biomedical Research of the Research Center, where HWJSCs were extracted and primary explants were obtained under sterile conditions.

DMEM F/12 medium, which was utilized in our previous studies to obtain cell culture from human umbilical cord tissue [11], was used to culture explants.

Immunophenotyping of the obtained cell population and immunohistochemical detection of SMA

Immunohistochemical staining for the presence of SMA in the cytoskeleton was performed to detect vascular smooth muscle cells in the obtained culture. Monoclonal Mouse Anti-Human Smooth Muscle Actin primary antibodies (Dako, Denmark), secondary antibodies conjugated to the fluorochrome Alexa Fluor 488 (Abcam, England), were used, and nuclei were stained with SlowFade Glass Soft-set Antifade Mountant with DAPI-Nuclear (Invitrogen, USA). The study was performed using an LSM 880 (Carl Zeiss, Germany) confocal microscope. Fluorescence was excited by a 405 nm laser with detection at 410–495 nm and an argon laser at 488 nm with detection at 495–605 nmc

The expression of stem cell surface markers in the umbilical cord MSC was investigated. Immunophenotyping was performed using a CytoFlex flow cytometer (Beckman Coulter, USA). Antibodies labeled with phycoerythrin (PE) or fluorescein (FITC) were used as labels: CD45-FITC, CD73-PE, CD34-PE, CD29-FITC, CD90-FITC, CD44-FITC, and CD105-FITC (Bio Rad, USA). Thus, cells were stripped from the surface of the culture plate by the conventional method (trypsin-serum solution at 1:3 ratio), washed three times with phosphate buffer solution (pH 7.2–7.4), and mixed with a diluted (×20) antibody dilution solution consisting of 20 μL fetal bovine serum (Gibco), 10 mg NaN₃, and 50 mL phosphate buffer. The cell suspension was injected into measuring tubes of the cytofluorimeter; then, the tubes were placed in the cytofluorimeter autosampler, and further fluorescence measurements and processing of the obtained signals were performed automatically. An Axio Imager M2 fluorescence microscope (Carl Zeiss, Germany) was used to observe the cell surface for fluorescence.

RESULTS AND DISCUSSION

Immunocytochemical examination for the presence of the specific cytoskeletal marker alpha-actin in the cytoplasm did not show autofluorescence. No specific staining of the cytoskeleton characteristic of smooth muscle cells was obtained in the cell populations studied from HWJSCs (Fig. 1B). Rat myometrium cells was stained with SMA as a positive control (Fig. 1Г).

 

Fig. 1. MSC culture: А, Б — staining with haematoxylin and eosin; В — immunocytochemical staining for the presence of smooth muscle a-actin in the cytoskeleton of cells obtained from umbilical cord, Г — stained rat myometrial cells

 

The expression of mesenchymal cell surface markers was examined by immunophenotyping with a CytoFlex flow cytometer (Beckman Coulter, USA). To check the conjugation efficiency of the antibodies, phycoerythrin-labeled CD73-PE and CD34-PE and fluorescein-labeled CD45-FITC, CD29-FITC, CD90-FITC, and CD105-FITC were used. The cells removed from the surface of the culture plastic and conjugated with antibodies were placed on slides and observed in an epifluorescence microscope using FITC light filters (green region of the spectrum for antibodies), and TRITC (red spectral region for phycoerythrin-labeled antibodies) at ×400 magnification and photofixation was performed in additive mode. The results are presented in Figure 2.

 

Fig. 2. Results of immunofluorescence labelling of surface antigens (SD) of mesenchymal cells of human umbilical cord Warton’s stool tissue. А — mesenchymal cell before labelling; Б, В, Г — images of cells labelled with different antibodies: Б — absence of conjugation with antibody (DAPI contrasting); В, Г — presence of conjugation with labelled antibody

 

Finding cells in solutions for cell removal from the culture surface, conjugating cells with labeled antibodies, and obtaining preparations for microscopy did not result in destruction of most HWJSCs studied. Figure 2A shows the original unlabeled cell in suspension and cells positively conjugated with fluorescently labeled antibodies (Fig. 2B and Г) and negatively conjugated (non-conjugated) with labeled antibodies (Fig. 2Б). The localization of the fluorescently labeled antibodies was observed on the surface of the tested cells. To obtain images of cells negatively conjugated with labeled antibodies, they were contrasted with DAPI after antibody conjugation.

Then, the suspension of labeled cells was placed into the cytofluorimeter dispenser tubes, and the distributions of surface markers in the evaluated cell population were recorded. Figure 3 presents the results of surface marker distribution of the obtained MSC culture.

 

Fig. 3. Distribution of surface markers in the obtained population of human umbilical cord MSCs: А — CD34, CD44; Б — CD45, CD73; В — CD29, Г — CD90; Д — CD105

 

A positive reaction was detected in the markers of 5-nucleotidase CD73 (55.61 %), protein integrins CD29 (41.11 %), stem cells and axonal processes in mature neurons CD90 (6.45 %), hyaluronic acid receptor CD44 (55.62 %), and endoglin CD105 (0.52 %). A negative reaction was noted in the marker of total leukocyte antigen CD45 (24.59 %).

Studies have reported that flow cytometry, immunofluorescence, and qRT-PCR analysis show predominant expression of the surface markers CD29, CD44, CD73, CD90, CD105, and CD166 in umbilical cord MSCs and that markers of endothelial cells and hematopoietic cells are absent [9, 10]. CD29, CD90, CD105, and CD166 persist in MSCs even after adipogenic, osteogenic, and chondrogenic induction [9]. Additionally, decreased CD44 and CD73 expressions in response to induction of trilineage differentiation has been demonstrated [10]. This reveals that they are the most reliable markers of stem cells, which are observed only in undifferentiated cord MSCs [8, 9]. Positive surface markers CD73 and CD44 indicate that stem cells predominated in the culture obtained by explantation. Moreover, the expression of the marker of hematopoietic cell adhesion proteins CD34 (27.19 %) confirms the presence of hematopoietic cells in the obtained population.

FINDINGS

1. The ratio of the applied markers allows for attributing the population of cells obtained by direct explantation of HWJSC fragments to the population of MSCs.
2. Visual inspection and flow cytometry confirmed the localization of labeled antibodies on the surface of cultured cells.
3. Immunohistochemical staining for SMA did not reveal vascular smooth muscle cells in the resulting cell culture.

CONCLUSIONS

As a result, of experiments on identification of cells obtained by explantation of fragments of HWJSCs and their further cultivation, their affiliation to MSCs was established by immunofluorescence cytophotometry. No vascular muscle cells were observed in the obtained culture. The obtained population contained hematopoietic cells (cells of the hematopoietic system); however, undifferentiated stem cells predominated. Establishing a human cell culture with a preponderance of identifiable stem cells, using donor organ tissue, enables MSC extraction and allows for the use of the extracted cells in a wide range of studies of human physiology and biochemistry with minimal ethical restrictions.

ADDITIONAL INFORMATION

Funding: The search and analysis were performed within the framework of the VMA.02.06.2022/026 “Explant” research project.

Conflict of interest: The authors declare that there is no conflict of interest.

Ethical review: The conduct of the study was approved by the local ethical committee of the S.M. Kirov Military Medical Academy of the Ministry of Defense of the Russian Federation (minutes no. 230; dated December 17, 2019).

Authors’ contribution: V.E. Chernov, obtaining, maintenance of cell culture, writing the article; M.O. Sokolova, obtaining the results of experiments on the distribution of surface CD markers of cell population, writing the article; A.A. Kokorina, immunocytochemical evaluation of the presence of specific cytoskeleton marker alpha-actin in cell population by confocal microscopy; G.I. Pendinen, visual evaluation and photofixation of immunofluorescence labeling of surface antigens (SD) of mesenchymal cells of Wharton’s jelly tissue by fluorescence microscopy, editing the text of the article.

About the authors

Vladimir E. Chernov

Military Medical Academy

Author for correspondence.
Email: vmeda-nio@mail.ru
ORCID iD: 0000-0002-2440-3782
SPIN-code: 8315-1161

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Margarita O. Sokolova

Military Medical Academy

Email: vmeda-nio@mail.ru
ORCID iD: 0000-0002-3457-4788
SPIN-code: 3683-6054
Russian Federation, Saint Petersburg

Arina A. Kokorina

Military Medical Academy

Email: arina.alexandrovna.vmeda-nio@mail.ru
ORCID iD: 0000-0002-6783-3088
SPIN-code: 9371-3658
Russian Federation, Saint Petersburg

Galina I. Pendinen

N.I. Vavilov All-Russian Institute of Plant Genetic Resources

Email: pendinen@mail.ru
ORCID iD: 0000-0003-2814-7074
SPIN-code: 2120-5925

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

References

Supplementary files