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  1. Effect of culture media on expansion properties of human umbilical cord matrix-derived mesenchymal cells
    Salehinejad P, Alitheen NB, Nematollahi-Mahani SN, Ali AM, Omar AR, Janzamin E, et al.
    Cytotherapy, 2012 Sep;14(8):948-53.
    PMID: 22587592 DOI: 10.3109/14653249.2012.684377
    BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have been isolated from a number of different tissues, including umbilical cord. Because of the lack of a uniform approach to human umbilical cord matrix-derived mesenchymal (hUCM) cell expansion, we attempted to identify the optimum conditions for the production of a high quantity of hUCM cells by comparing two media.

    METHODS: We compared the ability of Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) and Alpha Minimum Essential Medium (α-MEM) with Glutamax (GL) (α-MEM/GL) to expand hUCM cells. For this purpose, hUCM cells were cultured in plates containing different culture media supplemented with 10% fetal bovine serum (FBS). Culture dishes were left undisturbed for 10-14 days to allow propagation of the newly formed hUCM cells. The expansion properties, CD marker expression, differentiation potential, population doubling time (PDT) and cell activity were compared between the two groups.

    RESULTS: The hUCM cells harvested from each group were positive for MSC markers, including CD44, CD90 and CD105, while they were negative for the hematopoietic cell surface marker CD34. Differentiation into adipogenic and osteogenic lineages was confirmed for both treatments. Cell activity was higher in the α-MEM/GL group than the DMEM/F12 group. PDT was calculated to be 60 h for the DMEM/F12 group, while for the α-MEM/GL group it was 47 h.

    CONCLUSIONS: Our data reveal that α-MEM/GL with 10% FBS supports hUCM cell growth more strongly than DMEM/F12 with 10% FBS.

  2. Effect of EGF and FGF on the expansion properties of human umbilical cord mesenchymal cells
    Salehinejad P, Alitheen NB, Mandegary A, Nematollahi-Mahani SN, Janzamin E
    In Vitro Cell Dev Biol Anim, 2013 Aug;49(7):515-23.
    PMID: 23708920 DOI: 10.1007/s11626-013-9631-3
    Mesenchymal stem cells have been increasingly introduced to have great potential in regenerative medicine, immunotherapy, and gene therapy due to their unique properties of self-renewal and differentiation into multiple cell lineages. Studies have shown that these properties may be limited and changed by senescence-associated growth arrest under different culture conditions. This study aimed to present the ability of some growth factors on human umbilical cord mesenchymal (hUCM) cells expansion and telomerase activity. To optimize hUCM cell growth, epidermal growth factor (EGF) and fibroblast growth factor (FGF) were utilized in culture media, and the ability of these growth factors on the expression of the telomerase reverse transcriptase (TERT) gene and cell cycle phases was investigated. TERT mRNA expression increased in the hUCM cells treated by EGF and FGF. So, the untreated hUCM cells expressed 30.49 ± 7.15% of TERT, while EGF-treated cells expressed 51.82 ± 12.96% and FGF-treated cells expressed 33.77 ± 11.55% of TERT. Exposure of hUCM cells to EGF or FGF also promoted the progression of cells from G1 to S phase of the cell cycle and induced them to decrease the number of cells entering the G2/M phase. Our study showed that EGF and, to a lesser extent, FGF amplify the proliferation and expansion of hUCM cells.
  3. Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton's jelly
    Salehinejad P, Alitheen NB, Ali AM, Omar AR, Mohit M, Janzamin E, et al.
    In Vitro Cell Dev Biol Anim, 2012 Feb;48(2):75-83.
    PMID: 22274909 DOI: 10.1007/s11626-011-9480-x
    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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