AIP Conference Proceedings 2344, 040002 (2021); https://doi.org/10.1063/5.0047340 2344, 040002
© 2021 Author(s).
Characterization, differentiation, and population doubling time of Wharton’s
jelly mesenchymal stem cells (WJ-MSCs) in passage 5 and 8
Cite as: AIP Conference Proceedings 2344, 040002 (2021); https://doi.org/10.1063/5.0047340 Published Online: 23 March 2021
Rizal, Rahimi Syaidah, Ziyan Muhammad Aqsha, Adella Josephin, and Vidi Miranda Pakpahan
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Characterization, Differentiation, and Population Doubling Time of Wharton's Jelly Mesenchymal Stem Cells (WJ-
MSCs) in Passage 5 and 8
Rizal
1,a), Rahimi Syaidah
2, Ziyan Muhammad Aqsha
3, Adella Josephin
3, Vidi Miranda Pakpahan
31Biomedical Engineering program, Department of Electrical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI Depok, West Java 16424 Indonesia
2Department of Histology, Faculty of Medicine, Universitas Indonesia, Kampus UI Depok, West Java 16424 Indonesia
3Undergraduate Program in Biomedical Engineering, Department of Electrical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI Depok, West Java 16424 Indonesia
a)Corresponding author: rizal@eng.ui.ac.id
Abstract. Wharton's jelly mesenchymal stem cells (WJ-MSCs) is proposed to be the best source of mesenchymal stem cells (MSCs). It has better self-renewal and differentiation capacity compare to adult stem cells such as bone marrow- derived stem cells (BM-MSCs). However, their characteristics in different passages do not yet well described. This research aims to isolate and characterize WJ-MSCs with regard to their proliferation capacity, immunophenotypes, and differentiation capacity. The WJ-MSCs were isolated by the explant method. The cultured WJ-MSCs at P5 and P8 were then characterized using flow cytometric analysis with regard to its surface marker, which are CD44, CD90, CD105, CD73, and negative lineages. The proliferation capacity of WJ-MSCs was analyzed with the population doubling time method and the characterized cells were differentiated into three different lineages. The result revealed that the isolated cells highly express MSCs surface marker with >98% for all surface markers and 0.00% for negative lineages. Isolated WJ-MSCs also exhibited a high proliferation capacity with a range of 1.06 to 1.59 days. Both passages revealed excellent differentiation capacity into chondrocytes, adipocytes, and osteocytes.
Keywords: Mesenchymal stem cells, Wharton's jelly, proliferation, differentiation, characterization
INTRODUCTION
Mesenchymal stem cells (MSCs) are a promising candidate to treat degenerative diseases. It has been clinically applied to treat various diseases such as liver cirrhosis, Crohn disease, multiple sclerosis, myocardial infarction, acute or chronic graft versus host disease (a/cGVHD), and systemic lupus erythematosus [1,2]. Some clinical trials also have reported its promising application to treat novel diseases like COVID-19 [3]. MSCs are multipotent progenitor cells with high self-renewal capacity and hold multilineage potential to differentiate into mesodermal origin cell types such as chondrocytes, adipocytes, and osteocytes [4]. Some studies also reported its potential trans-differentiation into the endodermal and ectodermal lineage, such as neuronal cells [5], β-pancreas [6], and cardiomyocyte [7].
MSCs can be obtained from almost all tissues, including liver, fetal pancreas, lung, peripheral blood, placenta, umbilical cord, amniotic membrane, and Wharton's Jelly [8,9]. Among these sources, MSCs from natal birth byproducts pose the most promising sources [10,11]. Research reveals that MSCs derived from early phases of life, such as birth byproducts, hold greater self-renewal capacity and differentiation capacity [12,13].
Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) is one of the best sources of MSCs. It can be easily isolated through enzymatic and explant methods from the placenta[8]. WJ-MSCs have better proliferation capacity, a higher
early endodermal markers expression level in the early and late passage, and hold superior plasticity characteristics [10,11,14]. WJ-MSCs shared typical surface markers with BM-MSCs, such as immunomodulatory markers, but WJ- MSCs maintain its immunomodulatory molecules in differentiated chondrogenic, osteogenic, and adipogenic progeny [14]. WJ-MSCs and other MSCs are characterized by its surface markers such as CD73+, CD90+,CD105+,CD34–
,CD45–,CD11b–,CD14–,CD19–,CD79a–,HLA-DR–, and the differentiation capacity into adipocytes, chondrocytes, and osteocytes [4].
The biological properties of MSCs change over the passage period of the cells. Researchers recommend using early passage when it will be applied for therapy purposes. Although WJ-MSCs hold better long-term expansion capacity, several studies suggest using early passage MSCs for therapy [10,11,24]. The present study strives to reveal the characteristics of WJ-MSCs in two different early passages. It will give us information regarding the WJ-MSCs immunophenotype expression, proliferation, and differentiation capacity that can be used as a suggestion for a physician in stem cell practitioners.
METHODS WJ-MSCs Isolation
The cells were isolated by explant methods, as described in the previous study [15,16]. All Umbilical Cords (UCs) were washed with sterile phosphate-buffered saline (PBS) 1x to remove all blood cells from UCs. The UCs were placed into a transport medium containing PBS 1x (Gibco, Invitrogen, USA), 1% amphotericin-ß (v/v) (Gibco, Invitrogen, USA), 1% (v/v) penicillin-streptomycin (Gibco, Invitrogen, USA), and 20 ug/ml gentamicin (Gibco, Invitrogen, USA) and stored at 4oC for less than 24 h before tissue. Briefly, blood vessels (artery and venous) were longitudinally dissected from the UC, separating them from the WJ tissue. The WJ tissue was then cut into 1-2 cm3 explant fragments and washed with sterile PBS. The fragments were next plated on 60 mm tissue culture plates (MCD110090) and cultured in WJMSCs complete growth medium containing MEM-alpha (Gibco, Invitrogen, Canada) supplemented with 10% (v/v) fetal bovine serum (Gibco, Invitrogen Corporation, Grand Island, NY, USA), 1% (v/v) pen-strep (Gibco, Invitrogen, USA), 1% (v/v) amphotericin-ß (Gibco Invitrogen, USA), and 20 Pg/ml gentamicin (Gibco Invitrogen, USA). Cultures were incubated in an incubator (5% CO2 at 37°C). The medium was replaced every 3 days. After the cells grew, these cells were cultured and expanded in WJMSCs complete growth medium.
Population Doubling Time (PDT)
Isolated cells were detached by trypsin (Biowest, L0931-500), then incubated for 1-3 min at 37°C, completed medium consisting of 80% MEM-α, FBS 20%, 1% Antibiotic and anti-mycotic and 1% heparin were added to stop trypsin and centrifuged (MPW-2000) at 1600 rpm, 5 min at 240°C. The pellet of cells was resuspended with trypan blue solution (Sigma, 25200072) and diluted 1:1 dilution. Then, cells were counted by a hemocytometer (Neubauer,17849). Population Doubling (PD) was counted at every passage with the formula:
PD=Log10 (NH) – Log10(NI)/ Log10(2)
where NI is the inoculum cell number and NH the cell harvest number. Then, to determine cumulative PD data by added the PD at the previous passage. The PD time (PDT) was determined by the following formula:
PD time= (t (time)) ⁄ (PD (in days))
WJMSCs Characterization
The surface markers of WJ-MSCs (P5 and P8) were evaluated by flow cytometry (Macsquant, Analyzer 10, Beach Road, Singapore). WJ-MSCs at 80% confluence were harvested and dissociated with trypsin-EDTA and centrifuged at 5000 rpm for 5 minutes. The pellet was resuspended with PBS 1x, FBS, and cells were counted with a hemocytometer. Between 1x105 – 2x105 cells in 500 μL PBS cells were stained with appropriate positive surface antibodies as of CD90-FITC, CD105-PerCP-Cy5, CD73-APC, CD44-PE and negative surface antibodies of CD34- PE, CD116-PE, CD19-PE, CD45-PE, HLA-DR-PE according to manufacturer's protocol (BD stem flowKit, Becton, USA). Isotype controls were used as background staining. All experiments were performed in triplicate.
Differentiation Capacities
Differentiation into adipocytes was conducted through the following method. Cells (P5 and P8) were plated at density 1.1x104 cell/cm2 in four-well plates (Nunc, Thermo Scientific, Massachusetts, USA), grown to confluence, and incubated around two days in growth medium at 37°C, 5% CO2. The cells were then exposed to StemPro Adipogenesis Differentiation Medium (Gibco, Invitrogen, USA) for approximately 21 days. After three weeks of culture with fresh medium replacement every three days, the cells were fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature and stained using Oil Red O (Sigma, Missouri, USA) to examine intracellular lipid droplets[15, 17]. Osteogenic differentiation was conducted using StemPro Osteogenesis Differentiation Kit (Gibco A10072-01) under manufacturer protocol. 5x103 cells hWJ-MSCs were seeded in 24 well plates (Nunc, 72296-18) and were exposed by the StemPro Osteogenesis Differentiation medium for three weeks. Calcium deposits were stained using Alizarin red S (Sigma, A5533). For chondrogenic differentiation, 5x103 hWJ-MSCs were seeded in 4 well plates (Nunc, 176740) and were exposed with StemPro Chondrogenesis Differentiation Kit (Gibco, A10071-01) for three weeks. Chondrocytes were stained using Alcian blue (Sigma, A5268).
RESULTS
Morphological Appearance
Isolated MSCs exhibited spindle-shaped and fibroblast-like MSCs, as shown in Fig. 1. The cells that are attached to the plastic flask exhibited monolayer homogenous MSCs. This revealed that the explant method is one of the best methods of WJ-MSCs isolation. The isolated cells then cultured until passage 8.
FIGURE 1. The morphological appearances of isolated WJMSCs in 40x magnification (A) and 100x magnification (B).
Surface Marker Characterizations
MSCs in both passages (P5 and P8) exhibited the same expression levels of MSCs surface markers. The dot blot visualization of flow cytometric analysis were depicted in Fig. 2. At a glance, there are no significant differences in the expression level of CD90, CD44, CD105, CD73, and negative lineage. The lowest expression was CD44. The expression for Passage 5 was 89,34±1,08, and for P8 was 91,19±0,82. The highest expression were CD105 (99,97±0,06 for P5 and 99,99±0,03 for P8).
FIGURE 2. The immunophenotypes expression of WJ-MSCs in two different early passages. A) dot blot visualization of flow cytometric analysis of WJ-MSCs in passage 5. B) dot blot visualization of flow cytometric analysis of WJMSCs in passage 8. a)
gating strategy, b)CD90, c)CD44, d)CD73, e)CD105, f) negative lineage.
FIGURE 3.The percentage of surface markers (CD90, CD44, CD105, CD73 and negative lineages) expressions
Population Doubling Time (PDT)
The PDT was analyzed to evaluate the self-renewal capacity of WJ-MSCs. Population doubling time is the time required for the population to double its population size. The result revealed that the PDT from P5 to P8 was approximately ranging from 1.06±0.04 in P8 to 1.59±0.03 in P6. The lowest PDT was in P8, and the highest PDT was in P5. The PDT of isolated MSCs can be seen in Fig. 4.
FIGURE 4. Population doubling time (PDT) of WJ-MSCs from P5 up to P8.
Cells Differentiation
The differentiation capacity was evaluated and the isolated WJ-MSCs can be differentiated into three different lineages (adipocytes, chondrocytes, and osteocytes), as shown in Fig. 5.
0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00
CD90 CD44 CD105 CD73 Negative
Lineage
Surface Markers Expression (%)
Surface Markers Passage 5 Passage 8
b
c
b
a
0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60 1,80
P5 P6 P7 P8
Population Doubling Time (PDT) (Days)
Passage
FIGURE 5. Differentiation capacity f WJ-MSCs into adipocytes, chondrocytes, and osteocytes.
DISCUSSION
WJ-MSCs have been known as one of the best sources for cell transplantation therapy. Compare to BM-MSCs, WJ-MSCs has better proliferation capacity and differentiation capacity. The use of MSCs should follow many qualifications before it can be transplanted into the human body [11]. Several parameters to assess the quality of mesenchymal stem cells are proliferation capacity, differentiation capacity, senescence, and expressions of MSCs surface markers [18].
Isolated MSCs, as shown in Fig. 1 revealed the same morphological characteristics as the common MSCs, which are spindle-shape and fibroblast-like cells [1]. The present study revealed that in the early passage, the isolated WJ- MSCs have high proliferation capacity. The PDT decreases significantly in P5 and P8. This means that in those passages, the isolated WJ-MSCs still have excellent proliferation capacity. This result similar to previous research conducted by Antonius et al. [19] and Widowati et al. [8]. Nekanti et al. [11] stated that the highest proliferation capacity of MSCs was in early passages (passages 5-10).
Almost all populations of isolated WJ-MSCs in P5 and P8 express CD90, CD73, CD105, and CD44 in high expression. These surface markers are likely to have a function in the cell cycle, proliferation, survival, migration, adhesion, and differentiation [20,21,22]. Passage number and cell manipulation techniques have a significant role in surface marker expression [10,23]. Because P5 and P8 are considered as an early passage, at least from this research, there are no significant differences in the expression of all markers. The isolated cells in both passages also measly express the negative CD34, CD11b, CD19, CD45, and HLA-DR. Based on the surface markers expression levels, the method of isolation and culture of MSCs in this research generates high-quality stem cells that can be used as cell transplantation therapy [24].
The result revealed that the isolated WJ-MSCs in both passages could be differentiated into adipocyte, chondrocyte, and osteocyte cells. Several findings revealed that serial passages affect the differentiation capacity of MSCs [16,25,26]. Moreover, Rizal et al. [16] exhibited that serial passages decrease the adipogenic differentiation capacity of WJ-MSCs. In this study, we did not quantify the differentiation performance of isolated MSCs in both passages. However, due to this study measuring the differentiation capacity in an early passage, there are no significant differences in differentiation capacity in both passages.
CONCLUSION
The entire data revealed that the isolation and culture method used in this study generates high-quality stem cells with high proliferation capacity and can be differentiated into adipocyte, chondrocyte, and osteocyte and express a high level of surface marker proteins. There are no significant differences in the biological properties of isolated WJ- MSCs in P5 and P8.
ACKNOWLEDGEMENT
This study was supported by a grant from Universitas Indonesia, PUTI Prosiding 2020, contract no. NKB- 912/UN.RST/HKP.05.00/2020
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