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Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton’s jelly

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Abstract

Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.

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Acknowledgements

This work was supported by a grant from Kerman Neuroscience Research Center, Iran. We thank Dr Alp Can and Deniz Balci from Ankara University School of Medicine for their guidance in enzymatic cell isolation from an umbilical cord.

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Authors and Affiliations

  1. Institute of Bioscience, University Putra Malaysia, Kuala Lumpur, Malaysia

    Parvin Salehinejad & Abdul Rahman Omar

  2. Faculty of Molecular and Biotechnology, University Putra Malaysia, Kuala Lumpur, Malaysia

    Noorjahan Banu Alitheen & Abdul Manaf Ali

  3. Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Avecina St., Tahmasbabad Cross, Kerman, Iran

    Seyed Noureddin Nematollahi-Mahani

  4. Department of Stem Cells, Royan Institute, Tehran, Iran

    Ehsan Janzamin & Fazel Sahraneshin Samani

  5. Department of Pathology, Kerman University of Medical Sciences, Kerman, Iran

    Maryam Mohit

  6. Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

    Zahra Torshizi

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  1. Parvin Salehinejad
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  2. Noorjahan Banu Alitheen
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  3. Abdul Manaf Ali
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  8. Zahra Torshizi
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  9. Seyed Noureddin Nematollahi-Mahani
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Correspondence to Seyed Noureddin Nematollahi-Mahani.

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Editor: T. Okamoto

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Salehinejad, P., Alitheen, N.B., Ali, A.M. et al. Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton’s jelly. In Vitro Cell.Dev.Biol.-Animal 48, 75–83 (2012). https://doi.org/10.1007/s11626-011-9480-x

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  • DOI: https://doi.org/10.1007/s11626-011-9480-x

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