Indian Vet. J., December 2016, 93 (12) : 27 - 29
Isolation of Ovine Multipotent Mesenchymal Stem Cells from Umbilical Cord Tissue Wharton’s Jelly S. Eswari1, M. Monisha, K. Vijayarani and V.S. Gomathy Department of Veterinary Physiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600007, Tamil Nadu. (Received : 19-12-2015;
Accepted : 29-02-2015)
Abstract
Materials and Methods
In this study, we tried explant method and a combination of explant-enzymatic method for the extraction of Mesenchymal Stem Cells from ovine umbilical cord tissue wharton’s jelly. The isolated and culture expanded cells expressed strong alkaline phosphatase activity and they were shown to form colonies. The Mesenchymal Stem Cells showed positive for CD44 and negative for CD34 surface markers by ow cytometry. These ndings indicated that the cells with MSC phenotype can be isolated much faster and higher in mixed explant-enzymatic method from ovine UCT-WJ.
Umbilical cords were separated from the foetuses obtained from the abattoir at Perambur, Chennai and washed in sterile Phosphate buffered saline (PBS) with 200IU/ml penicillin and 200µg/ml streptomycin (Figure 1A). The WJ (Figure 1B)was cut into small pieces of 1cm3 and cultured as explant in tissue culture ask (Figure 1C). For mixed explant–enzymatic culture method, the WJ pieces weredigested in enzymatic cocktail comprising collagenase type I (4mg/ml) and hyaluronidase (1mg/ml) for 1h followed by 0.1% trypsin EDTA for 30 min at 37oC (Figure 1D). The digested cell pellet was seeded intissue culture ask in culture medium containing Dulbecco’s modied eagle’s medium (DMEM) supplemented with 10% foetal bovine serum, 2mM L- glutamine and 1% antibiotics (Figure 1E). After reaching conuence, the cells were trypsinized with 0.25% trypsin- EDTA solution. The culture dishes were observed under an inverted phase contrast microscope (TS-100 Nikon, Japan) for cell morphology and colony formation.The isolated cells were tested for the expression of alkaline phosphatase using alkaline phosphatase staining kit (Millipore, USA), cell viability by trypan blue staining (Sigma) and the presence of MSC surface markers for CD34 and CD44 (Ab Cam, USA) using a uorescence activated cell sorter(BD FACS, Jazz).
Key words : Mesenchymal stem cells, isolation, sheep, Wharton’s jelly Mesenchymal Stem Cells (MSCs) are attractive candidate for cell-based therapeutic strategies, because of their easy isolation and proliferation potential. The wharton’s jelly (WJ) is the gelatinous connective tissue from the umbilical cord tissue (UCT)and composed of myobroblast like stromal cells, collagen bres and proteoglycans (Kobayashi et al., 1998). The stem cells in WJ are expected to become seed cells for tissue engineering, cell therapy and gene therapy (Butler et al., 2000). The MSCs have been obtained from the fetal tissues (Fuchs et al., 2005), umbilical cord tissue in humans (Venugopalet al., 2011), bovine (Singh et al., 2013; Sreekumar et al., 2014) and in caprine (Moshreet al.,2010). Till date, no investigations were found about different methods for isolationof MSCs in order to obtain the cells at much faster rate. Therefore in the present experiment, the isolation of MSCs from ovine UCT-WJ by two different methods were studied. 1
Corresponding author : Email:
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Results and Discussion
Many reports have been shown for the isolation and characterization of UCT derived MSCs in humans but only a few reports were observed in animals (Zucconiet al., 2010; Moshreet al.,loccit; Sreekumar et al.,loccit). The MSCs were successfully isolated and expanded in
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Isolation of Ovine Multipotent ...
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Fig 1. (A) Umbilical cord segment separated from ovine foetus, (B) Separation of outer sheath and blood vessels from Wharton Jelly, (C) Migration of MSCs from the WJ explant tissue (200X), (D) Digestion of WJ pieces in enzyme mix, (E) Migration of MSCs from digested WJ tissue (200X).
both explant culture as well as mixed explantenzymatic culture(Fig 1A-E). The cells started migrating from 24-48 h itself in mixed explant and enzymatic culture where as in explant culture it is after 8-10 days. In primary cultures, the cells grew more rapidly and reached 80-90% conuency on 6th day of culture (Fig 2B). The MSCs showed a varying morphology with spindle shaped, elongated, cuboidal and broblast like cells (Fig 2A). Conuent cells were arranged in parallel arrays. These features were observed by several studies, and they found morphology of MSCs similar to our ndings (Wang et al., 2004). The ability of the cells to form colonies is a measure of stemness of a progenitor cell activity. Presence of red reaction is an indication of strong AP activity. In the present investigation, the MSCs were isolated from WJ and examined in terms of their growth characteristics, clonogenicity and phenotypic characterization. A formal demonstration of self-renewal was provided for our isolated cells (Fig 2C). These colonies showed a strong red colour when stained with alkaline phosphatase staining kit similar to the results obtained by Moshre et al., (loc 28
cit.) in goat umbilical cord matrix. The isolated MSCs expressed strong mesenchymal marker CD44, while they are negative for the hematopoietic marker CD34 (Fig 2 D, E) suggestive of UCT-WJ derived cells were one of the MSC populations similar to those derived from bone marrow and adipose tissue. Our ndings are in consistent with other studies in human (Wang et al.,loccit). In conclusion, the WJ derived stem cells have MSC like morphology, clonogenic, alkaline phosphatase positive and expressed MSC specic surface markers similar to bone marrow and adipose derived MSCs. It could also be inferred that whenever a fast propagation of MSCs is demanded, the enzymatic - explant culture method is best method from the UCT derived WJ tissue. Summary
In this study, the cells with MSC phenotype were isolated from both explant and enzymatic explant method. Both the procedures were shown to form colonies, strong alkaline phosphatase activity and the cells appeared as homogenous spindle shaped and typical broblast like shape
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0.11%
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Fig 2.(A)Photomicrograph shows WJ derived MSCs having large nucleus and extended cytoplasm (200x),(B)Conuent culture after 6 days in in vitroculture (200x). (C) Colony formation having aggregation of cells observed in WJ derived mesenchymal stem cells culture at passage 4 (200x),(D)Flow cytometry for CD34 negative MSC marker, (E)Flow cytometry for CD44 positive MSC marker.
cells. By using ow cytometry, the MSCs showed positive for CD44 and negative for CD34 surface markers. These ndings are indicated that in mixed-enzymatic explant culture method, the MSCs can be isolated faster from the ovine umbilical cord wharton’s jelly. Acknowledgement
The authors would like to thank the Department of Biotechnology, Government of India, New Delhi for their nancial support for this project scheme No. BT/PR7142/MED/31/205/2012. References Butler, D., Goldstein, S. and Guilak, F. (2000) Functional tissue engineering: The role of biomechanics, Transac. ASME 122: 570. Fuchs, J.R., Hannouche, D., Terada, S., Vacanti, J.P. and Fauza, D.O. (2005) Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells. Stem cells, 23: 958-964. Kobayashi, K., Kubota, T. and Aso, T. (1998) Study on myobroblast differentiation in the stromal cells of wharton’s jelly expression and localizationof alpha smooth muscle actin. Early Hum. Dev.51: 223.
Moshre, M., Babaei, H and Nematollahi – Mohani, S.N. (2010) Isolation and characterization of mesenchymal cells isolated from caprine umbilical cord matrix. Anim. Reprod, 7(4):367- 372. Singh, J., Mann, A., Kumar, D., Duhan, J.S and Yadav, P.S. (2013) Cultured buffalo umbilical cord matrix cells exhibit characteristics of multipotent mesenchymal stem cells In Vitro Cellular and Developmental Biology 49:408- 417. Sreekumar, T.R., Ansari, M.M., Chandra, V and Sharma, G.T. (2014) Isolation and Characterization of buffalo Wharton’s jelly derived mesenchymal stem cells.J.Stem. Cell. Res. Ther., 4-5: 207-doi. 10.4172/2157-7633.1000207. Venugopal P, Balasubramanian S, Majumdar A.S. and Ta, M. (2011) Isolation, characterization and gene expression analysis of wharton’s jelly derived mesenchymal stem cells under xeno-free culture conditions Stem Cells and Cloning : Advances and Applications 4: 39-50. Wang, H.S., Hung, S.C., Peng, S.T., Huang, C.C., Wei, H.M., Guo, Y.J., Fu, Y.S., Lai, M.C. and Chen, C.C. (2004) Mesenchymal stem cells in the wharton’s jelly of the human umbilical cord. Stem cells, 22: 1330-1337. Zucconi, E., Vieira, N.M., Bueno, D.F., Secco, M., Jazedje, T., Ambrosio, C.E., Passos-Bueno, M.R., Miglino, M.A. and Zatz, M. (2010) Mesenchymal stem cells derived from canine umbilical cord vein- a novel source for cell therapy studies. Stem cells Dev,19: 395-402.
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