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ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 1  |  Page : 231-235

Isolation and osteogenic differentiation of umbilical cord mesenchymal stem cells


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shiben El-Kom, Egypt
2 Clinical Pathology Department, National Liver Institute, Menoufia University, Shiben El-Kom, Egypt
3 Department of Obstetrics and Gynecology, Faculty of Medicine, Menoufia University, Shiben El-Kom, Egypt

Date of Submission20-Aug-2018
Date of Decision20-Oct-2018
Date of Acceptance22-Oct-2018
Date of Web Publication25-Mar-2020

Correspondence Address:
Asmaa Z.A. Turky
Shiben El.Kom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_265_18

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  Abstract 

Objective
To investigate the isolation and in-vitro differentiation of umbilical cord mesenchymal stem cells (MSC) into osteocytes.
Background
Umbilical cord is an important source of multiple cell types such as MSCs that possess the ability of self-renewal and differentiation into several cell types including osteocytes. Bone healing is a complex process that can eventually lead to the formation of malunions, delayed unions, and osteomyelitis. Cellular therapy and bone tissue engineering are becoming a useful addition to medical therapies for bone repairing and restoring function which is a potential tool for the treatment of multiple bone diseases.
Patients and methods
The present experimental study was conducted at Clinical Pathology, Obstetrics and Gynecology Departments, Menoufia University Hospital from May 2017 to April 2018 after obtaining Menoufia Ethical Committee approval. The study was carried out on 35 umbilical cord samples. Wharton's jelly separated from the umbilical cord was divided into small pieces and cultured in tissue culture plastic flasks. MSCs were separated by plastic adherence, then subcultured in Petri dish 35 mm, treated with osteogenic differentiation media and identified by morphology and immunocytochemistry.
Results
MSCs were isolated from Wharton's jelly of the umbilical cord. Isolated MSCs were positive for the markers cluster of differentiation (CD) 44 and CD73 and negative for CD34. They were able to differentiate into osteocytes.
Conclusion
The present study showed that MSCs can be isolated from Wharton's jelly of the human umbilical cord and can be differentiated into osteocytes.

Keywords: flow cytometry, immunocytochemistry, mesenchymal stem cells, osteocytes, umbilical cord, Wharton's jelly


How to cite this article:
El-Edel RH, Hassouna MM, El-Gayed AM, Noreldin RI, Turky AZ. Isolation and osteogenic differentiation of umbilical cord mesenchymal stem cells. Menoufia Med J 2020;33:231-5

How to cite this URL:
El-Edel RH, Hassouna MM, El-Gayed AM, Noreldin RI, Turky AZ. Isolation and osteogenic differentiation of umbilical cord mesenchymal stem cells. Menoufia Med J [serial online] 2020 [cited 2020 Mar 30];33:231-5. Available from: http://www.mmj.eg.net/text.asp?2020/33/1/231/281288




  Introduction Top


Stem cells are cells found in all multicellular organisms. They are characterized by their ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialized cell types[1]. They retain their ability to divide throughout life and give rise to cells that can become highly specialized and take the place of cells that die or are lost[2]. Stem cells come from two sources: embryonic stem cells (ESCs) and adult stem cells. The stem cells that reside in a mature organism have been termed adult stem cells. ESCs originate from the inner mass of a blastocyst and are able to self-regenerate and differentiate into the three germ layers that include the endoderm, ectoderm, and the mesoderm. Adult stem cells share the same self-regenerative capability that ESCs express but have some differentiation restrictions. Their ability to give rise to only specific cell lineages makes them multipotent stem cells. Mesenchymal stem cells (MSCs) are a subset of nonhematopoietic adult stem cells that originate from the mesoderm. They possess the ability of self-renewal and multilineage differentiation into not only mesoderm lineages, such as chondrocytes, osteocytes, and adipocytes, but also ectodermic cells and endodermic cells[3],[4],[5]. MSCs can be isolated from the bone marrow, adipose tissue, and the umbilical cord and can be stored for a long time without major loss of potency and no adverse reactions to allogenic MSC transplant[6]. Bones in the body together form the skeleton. The human skeleton serves a variety of functions. The bones of the skeleton provide structural support to the rest of the body, permit movement and locomotion by providing levers for the muscles, protect vital internal organs and structures, provide maintenance of mineral homeostasis and acid–base balance, serve as a reservoir of growth factors and cytokines, and provide the environment for hematopoiesis within the marrow spaces[7]. Each bone constantly undergoes modeling during life to help it to adapt to the changing biomechanical forces, as well as remodeling to remove old and microdamaged bone and replace it with new and mechanically stronger bones to help bone strength preservation[8]. Bone defects that are due to trauma or pathological and physiological bone resorption represent a global health problem. The need for bone regeneration is one of the central issues in regenerative medicine. Cellular therapy and tissue engineering are becoming a useful addition to medical therapies for repairing and restoring function. Stem cells have the ability to become bone cells and therefore are of central importance for bone tissue engineering[9]. The potential use of stem cell-based therapies for the repair and regeneration of various tissues and organs offers a paradigm shift that may provide alternative therapeutic solutions for a number of bone diseases[10]. The aim of this study was to isolate MSCs from Wharton's jelly of the umbilical cord and evaluate the potential of their differentiation into osteocytes for further use in stem cell-based therapies and bone tissue engineering.


  Patients and Methods Top


The present experimental study was conducted at the Clinical Pathology Department in collaboration with the Obstetrics and Gynecology Department at Menoufia University Hospital during the period from May 2017 to April 2018. The study involved 35 umbilical cord samples collected from normal pregnant women coming for delivery after obtaining Menoufia Ethics Committee approval. Those with known history of hepatitis, infectious diseases, DM, severe hypertension, abortions, or bad obstetric history were excluded from collection. Cord samples were aseptically collected in sterile 0.9% NaCl solution and were transported immediately to a clinical pathology laboratory and were kept at room temperature. Cord blood used for serum preparation was obtained prior to the expulsion of the placenta and left at room temperature for clotting and then centrifuged for 15 min at a speed of 3000 rpm; then sera were incubated at 56°C for 20 min for heat inactivation and then liquoted and frozen for future use. Under complete aseptic conditions, umbilical cord was disinfected by 75% ethanol and divided into small segments. Each segment was opened longitudinally. Umbilical cord vessels were dissected and removed and then the cord tissues were washed with saline. Wharton's jelly was cut into small pieces of about 1.5–2.5 mm, seeded, and allowed to adhere to tissue culture plastic flasks 25 cm2 containing 5 ml of fresh complete nutrient medium composed of low-glucose DMEM, l-glutamine (2 mmol/l), penicillin–streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin) (10 μg/ml) (Lonza, Boroline Road, Allendale, NJ, USA), cord blood serum (100 μg/ml), and fungizone (0.25 μg/ml) (Gibco, Life Technologies, Grand Island, NY, USA ). The flasks were incubated in a horizontal position in an incubator with saturated humidity containing 5% CO2 at 37°C. The incubator was checked periodically for water level and CO2 pressure. The media were examined daily to look for signs of microbial contamination. Ten samples were excluded due to the appearance of contamination signs. At day 7, the tissue was removed by changing the medium and the adherent cells were fed with fresh complete nutrient media. These cells were kept until the outgrowth of fibroblast-like cells. The flasks were examined by an inverted microscope (×100–200) after removal of the tissue for assessment of cell morphology (viable cells were round, bright, and refractile). On the 14th day, when fibroblast-like cells reach 60–70% confluence, the cells were harvested by trypsinization[11]. Trypsinization was done through aseptic removal of the medium and then 2–5 ml of trypsin-EDTA detachment solution was added to the flask. The cells were examined microscopically for detachment every 2–3 min. The time required for complete detachment may vary from 5 to 15 min. Incubation at 37°C for 5 min may be required[12]. Neutralization: after complete detachment, DMEM containing 1% cord blood serum was added. The cells were transferred to 15 ml centrifuge falcon tube and centrifuged at 1800 rpm for 10 min. The cells were resuspended in complete media. Cells were examined under the microscope, counted, and the viability was assessed using the trypan blue dye. The cells were used for MSC identification by flow cytometry and osteogenic differentiation. Identification of MSCs was done by flow cytometry: on day 14, the harvested MSCs were identified by flow-cytometric analysis of surface markers cluster of differentiation (CD) 44, CD34, and CD73 using Becton Dickinson FACS Calibur (Biosciences, San Jose, California, USA)[13]. Osteogenic differentiation: 250 μl of the osteogenicsupplement (cat. no. SC006; R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN 55413, USA & Canada) was added to 5 ml of αMEM basal media for osteogenic differentiation media preparation and was mixed gently. At 50–70% confluency, osteogenic differentiation was induced by replacing the media with 4 ml of osteogenic differentiation media. The media were changed every 3–4 days and were examined daily for signs of microbial contamination (turbidity, change in the color or small fungal colonies that may be floating in the media) and evaluation of osteogenic differentiation through post-induction morphological changes followed by identification of osteocytes by fixation and immunocytochemistry. Immunocytochemistry was done by washing the cells twice with 1 ml of PBS. The cells were fixed with 0.5 ml of 4% paraformaldehyde in PBS for 20 min at room temperature. The cells were washed three times with 0.5 ml of 1% BSA in PBS for 5 min. Permeabilization and block of the cells were done with 0.5 ml of 0.3% Triton X-100 and 1% BSA at room temperature for 45 min. During the blocking, the reconstituted anti-osteocalcin antibody was diluted in PBS containing 0.3% Triton X-100, 1% BSA, and to a final concentration of 10 μg/ml. After blocking, the cells were incubated with 300 μl/well of anti-osteocalcin antibody working solution for 3 h at room temperature or overnight at 2–8°C. The cells were washed three times with 0.5 ml of 1% BSA in PBS for 5 min. The secondary antibody (e.g. NL557-conjugated donkey anti-mouse secondary antibody; R and D System) was diluted 1: 200 in 1% BSA in PBS. The cells were incubated with secondary antibody working solution in the dark for 60 min at room temperature. The cells were washed three times with 0.5 ml of 1% BSA in PBS for 5 min. The cells were covered with 1 ml of PBS and visualized with a fluorescence microscope[14].

Statistical analysis

The results were statistically analyzed by SPSS, version 22 (SPSS Inc., Chicago, Illinois, USA). Data were expressed in two phases: the first was descriptive statistics, for example, number, percentage for qualitative data, mean, range (minimum and maximum), median, and SD for quantitative data. The second was analytic statistics where Wilcoxon signed-ranked test was used for comparison of two dependent quantitative variables not normally distributed. The P value was set to a significant difference if up to 0.05 and highly significant if up to 0.01.


  Results Top


MSCs isolated from Wharton's jelly of the umbilical cord exhibit a characteristic spindle-like, fibroblastic morphology [Figure 1] and by flow cytometry the cells show positive CD44 (R = 55.0–90.0%) with a mean value of 71.2 ± 11.1 and CD73 (R = 60.0–85.0%) with a mean value of 73.8 ± 8.7 and negative CD34 (R = 0.3–3.5%) with a mean value of 2.3 ± 0.9 [Table 1].
Table 1: Descriptive analysis of the studied cases according to mesenchymal stem cell markers (n=25)

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Figure 1: (a) Inverted microscope image x100 0f adherent MSCs before removal of floating non adherent cells showed 80% fibroblasts confluence (b) Inverted microscope image x200 0f adherent MSCs after removal of floating non adherent cells showed 80-90% fibroblasts.

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Microscopic examination after differentiation showed stellate-shaped cells with cytoplasmic processes [Figure 2]. Flow-cytometric analysis of the cells was done after differentiation and showed the following results: Negative expression of CD44 (R = 0.7–6.0%) with a mean value of 3.2 ± 1.9 and negative expression of CD73 (R = 0.3–4.0%) with a mean of 1.9 ± 1.3 [Table 2]. Comparing between before and after differentiation of the umbilical cord MSCs showed highly significant difference (P < 0.001).
Table 2: Comparison between expression of cluster of differentiation 44 and cluster of differentiation 73 before and after differentiation (n=25)

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Figure 2: Stellate-shaped osteocytes with cytoplasmic processes.

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Immunocytochemical analysis showed immunoreactivity for osteocalcin in both nucleus (stained blue with donkey anti-goat IgG-conjugated antibody) and cytoplasm (stained red with DAPI; [Figure 3].
Figure 3: Immunocytochemical analysis of differentiated osteocytes stained using donkey anti-goat IgG-conjugated antibody (red) and nuclei counterstained with DAPI (blue).

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  Discussion Top


In the present study, isolation of MSCs were chosen due to their ability to differentiate, self-replicate, and secrete trophic molecules that promote cell–cell connection and reduce inflammation, apoptosis, and fibrosis of damaged tissues, while stimulating tissue regeneration. This was proved by Tang et al.[15] who studied the trophic effects of MSC implantation into the ischemic myocardium. Bone marrow had been known as the main source of MSCs, but with appearance of ethical problems and painful intervention, we had to find other sources with more little problems such as Wharton's jelly of the umbilical cord used in this study that agrees with Cheng et al.[16], who stated that for many years, bone marrow was considered the 'gold standard' for the derivation of MSCs for human stem cell engineering. However, extraction comes with ethical constraint and problems associated with painful harvesting and donor site morbidity. Umbilical cords appear to show potential as a source of MSCs for a number of reasons; they are considered medical waste; so, their use in research has little ethical concern. They proliferate rapidly in culture and are thought to be immune privileged. In the present study, MSCs were isolated successfully from Wharton's jelly of the umbilical cord. Isolated MSCs showed fibroblast-like morphology. This agrees with Haasters et al.[17], who demonstrated an elongated and spindle-shaped morphology of MSCs. Regarding the identification of MSCs by flow-cytometric analysis, this study showed that these cells were positive for human MSC markers CD44 (71.2 ± 11.1) and CD73 (73.8 ± 8.7) and negative for CD34 (2.3 ± 0.9). This agrees with Wang et al.[18], who demonstrated that Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells. Using flow-cytometric analysis, we found that the mesenchymal cells isolated from the umbilical cord, when expanded ex vivo, express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51), but not hematopoietic lineage markers (CD34, CD45). Osteogenic differentiation: as regards morphological changes, the cells were stellate-shaped cells with cellular processes and this result agrees with Heino and Hentunen[19] who demonstrated that mature osteocytes are stellate-shaped cells with long slender cytoplasmic processes which radiate in all directions. Osteocytes showed positive immunoreactivity for osteocalcin in both nucleus, stained blue with donkey anti-goat IgG-conjugated antibody, and cytoplasm, stained red with DAPI, which agrees with Yang et al.[20], who proved that positive immunoreactivity for osteocalcin is a highly sensitive and specific criterion of osteoblasts. Osteocalcin was also detected in osteocytes, and Bianco et al.[21] showed that endogenous osteocalcin was present in the cytoplasm and nucleus of osteoblasts and in the collagenous matrix.


  Conclusion Top


The present study showed that MSCs can be isolated from Wharton's jelly of the umbilical cord and were differentiated into osteocytes, which open the future for stem cell-based therapy and bone tissue engineering as alternative therapeutic solutions for a number of bone diseases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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