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Introduction

Mesenchymal stromal cells, which also known as \'multipotent msenchyal stormal cells\' are self renewing cells which can be differentiated into multiple cells linage such as osteoblasts, chondrocyes, myocytes and adipocytes by specific growth factors that induced. For many years, bone marrow has been considered the “gold standard” for the derivation of MSCs for human stem cell engineering (Mennan, 2013) but are now described to reside in  connective tissues and notably in adipose tissue (Zuk, 2002), placenta (Fukuchi, 2004), umbilical cord (Romanov, 2003), dental pulp (Gronthos, 2000), tendon (Bi, 2007), trabecular bone (Noth, 2002) and synovium (De Bari, 2001). Bone marrow-derived and adipose tissue are the two main sources of MSCs for cell therapy through their low immunogenicity, ease accessibility, broad differentiation potential and immunomodulatory effects (Helmy, 2010). MSCs can be identified by three criteria: expression of the surface molecules CD73, CD90 and CD105 (Kariminekoo, 2016)  with a negative presence of CD34 and CD45 (Ramos, 2016) hematopoietic markers; property to adhere to plastic surface; the ability to differentiate into three mesenchlymal linages which are bone, fat, and cartilage (Maumus, 2011). As MSCs express specific set of marker proteins and by comparing the density under the flow cytometry data analysis, the cell of origin was able to be determined.

Materials and Methods

The T175 bone marrow-derived mesenchymal stromal cell (MSC) flask was washed once by adding 10 mL of Dulbecco\'s Phosphate-Buffered Saline (PBS) (no calcium, no magnesium; Invitrogen, Carlsbad, CA, USA; cat. no. 14190-144) and then the content was discarded. A volume of 2mL TrypLE™ Express, Cell Dissociation Enzyme (1X) (no phenol red; Invitrogen,; cat. no. 12604-013) was added, followed by 5 minutes incubation at 37 °C. Measured 10 mL of Dulbecco’s Modified Eagle Medium – low glucose (DMEM LG) (with GlutaMAX™ supplement and pyruvate; Invitrogen; cat. no. 10567-014) supplemented with 10 % v/v FBS and PenStrep (1X) to the flask, the mixture was suspended and the entire volume was then transferred into a 15mL centrifuge tube for centrifugation at 400xg for 3 minutes. Supernatant was removed and the cell pellet was resuspended by adding 2mL of staining buffer. The 50µL aliquot of cells was mixed with 200µL staining buffer then transferred to a 1.5ml microfuge tube for cell counting by Merck Milliopore Scepter automated cell counter. The tube was then placed in the centrifuge at 400xg for 3 minutes and the supernatant was discarded. A calculated volume staining buffer was added to achieve a concentration of 2 million cells per mL and the mixture was resuspended. Preparation of seven 1.5mL microfuge tubes by adding 50µL of cell suspension and 50µL of antibody volumes CD45-FITC, CD73-PE, CD90-FITC, CD105-FITC, IgG2a-FITC, IgG1-PE and IgG-FITC respectively followed by gentle vortex mixed. All tubes were placed in the lid covered ice container for two 10 minutes incubation and should be mixed by gentle vortex between interval. A volume of 1mL staining buffer was added to each tube, then mixed gently and centrifuged at 400xg for 3 minutes. Supernatant was discarded and the cell pellets were resuspended in 200µL of staining buffer and transferred to communal 96 well plates for the flow cytometry machine to analyze, the result then  was performed with R software statistically.

Result

The T175 flask was inspected under the microscope before the start of the experiment  and  a cell confluence of 85% was observed. Merck Millipore Scepter automated cell counter had a reading of 1.715e5 cells/mL which implied the 1.5mL microfuge tube contained 1.715e6 cells.

1.715e5 cells/mL was recorded * the dilution factor of 5 * volume of 2mL staining buffer

1.715e5 *5*2

=1.715e6 cells

The appropriate volume of staining buffer to achieve a concentration of 2 million cells per mL was calculated as 0.858mL.

1.1715e6 cells counted ÷ desired concentration of cell suspension 2e6 cells/mL

1.1715e6 ÷ 2e6

= 0.858mL

Flow cytometry analyzed density of seven surface markers under FITC and PE calibration

Fig. 1 - The density plot showed the normal distribution of the 7 antibody surface markers under the Fluorescein isothiocyanate (FITC) and Phycoerythrin (PE) channels. The density greater than 5 would be indicated as positive result and vice versa. In the FITC column,  positive results were recorded including CD105, IgG2a and IgG1 while in the PE column, only IgG1 was noted with a positive.

Discussion

The FITC and PE are two of the numerous methods for quantitative fluoresence calibration (QFC) (Wang, 2006) , FITC has one excitation and emission spectrum peak wavelengths at 530nm while PE has two different excitation wavelengths: one at around 488nm and one at around 561nm. False positive result on IgG1 under the FITC column was caused by the excitation photon of IgG1 which fluorochromes emit light over a very broad range or excited by multiple wavelengths of light and therefore both positive results were shown on the plot(Fig.1) under the QFC. In order to solve this spillover contaminated detection, compensation is necessary to obtain the true fluorescense result. Analysed plot result was not expected, bone marrow-derived MSC (BM-MSC) should have a positive density of CD73, CD90 and CD105 with a negative result of CD45; and both mouse IgG2a and IgG1 isotype control antibodies should remain negative.

The negative CD73 and CD90 expression had clearly indicated the provided MSC cells in the T175 flask was not human MSC (hMSC), meanwhile the negative controls of IgG2 and IgG1 had  positive readings proved the above statement as these protein were not expressed on human cells or cell lines. With the positive signs of IgG2a and CD105, it was ensure that the cells in the T175 flask should be mature endothelial cells (EC) which form the inner lining of blood vessels and are therefore uniquely positioned between circulating lymphocytes and peripheral tissues (Rothermel, 2004). Flow cytometry (FCM)is suitable for study heterogeneous populations of cells rather than than homogeneous as FCM can acquire data on all subpopulations of sample with a very low error rate and alternatively ELISA is quicker and easier with a lower operating cost and for large numbers of samples running because FCM can only handle hundreds sample in a day (Martz, 2000). In order to optimised FCM , scientists suggested to reduce noise from the fluorescent signal by reagent titration, choose the right control as reference and use freshly prepared solutions to minimized the background reading. Furthermore, staining dead cells would also be useful as they bind to any antibody non-specifically which is able to be distinguished from the live cells (Moncada, 2014).

Conclusion

True MSC is able to be distinguished from the other normal cell with its specific characteristic by flow cytometry.

Reference

Bi, Y., Ehirchiou, D., Kilts, T. M., Inkson, C. A., Embree, M. C., Sonoyama, W., . . . Young, M. F. (2007). Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med, 13(10), 1219-1227. doi:10.1038/nm1630

De Bari, C., Dell\'Accio, F., Tylzanowski, P., & Luyten, F. P. (2001). Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum, 44(8), 1928-1942. doi:10.1002/1529-0131(200108)44:8<1928::aid-art331>3.0.co;2-p

Fukuchi, Y., Nakajima, H., Sugiyama, D., Hirose, I., Kitamura, T., & Tsuji, K. (2004). Human placenta-derived cells have mesenchymal s tem/progenitor cell potential. Stem Cells, 22(5), 649-658. doi:10.1634/stemcells.22-5- 649

Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., & Shi, S. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A, 97(25), 13625-13630. doi:10.1073/pnas.240309797

Helmy, K. Y., Patel, S. A., Silverio, K., Pliner, L., & Rameshwar, P. (2010). Stem cells and regenerative medicine: accomplishments to date and future promise. Ther Deliv, 1(5), 693-705. doi:10.4155/tde.10.57

Kariminekoo, S., Movassaghpour, A., Rahimzadeh, A., Talebi, M., Shamsasenjan, K., & Akbarzadeh, A. (2016). Implications of mesenchymal stem cells in regenerative medicine. Artif Cells Nanomed Biotechnol, 44(3), 749-757. doi:10.3109/21691401.2015.1129620

Martz, E (2000). Introduction to Flow Cytometry for Microbiology 542, Immunology Laboratory

Maumus, M., Guerit, D., Toupet, K., Jorgensen, C., & Noel, D. (2011). Mesenchymal stem cell-based therapies in regenerative medicine: applications in rheumatology. Stem Cell Res Ther, 2(2), 14. doi:10.1186/scrt55

Mennan, C., Wright, K., Bhattacharjee, A., Balain, B., Richardson, J., & Roberts, S. (2013). Isolation and Characterisation of Mesenchymal Stem Cells from Different Regions of the Human Umbilical Cord. BioMed Research International, 2013, 8. doi:10.1155/2013/916136

Moncada (2014). “Flow Cytometry protocol optimization, Rockland Immunochemicals

Noth, U., Osyczka, A. M., Tuli, R., Hickok, N. J., Danielson, K. G., & Tuan, R. S. (2002). Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells. J Orthop Res, 20(5), 1060-1069. doi:10.1016/s0736-0266(02)00018-9

Ramos, T. L., Sanchez-Abarca, L. I., Muntion, S., Preciado, S., Puig, N., Lopez-Ruano, G., . . . del Canizo, C. (2016). MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry. Cell Commun Signal, 14, 2. doi:10.1186/s12964-015-0124-8

Romanov, Y. A., Svintsitskaya, V. A., & Smirnov, V. N. (2003). Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells, 21(1), 105-110. doi:10.1634/stemcells.21-1-105

Rothermel, A. L., Wang, Y., Schechner, J., Mook-Kanamori, B., Aird, W. C., Pober, J. S., . . . Johnson, D. R. (2004). Endothelial cells present antigens in vivo. BMC Immunology, 5(1), 5. doi:10.1186/1471-2172-5-5

Wang, L., Abbasi, F., Gaigalas, A. K., Vogt, R. F., & Marti, G. E. (2006). Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B-cells. Cytometry B Clin Cytom, 70(6), 410-415. doi:10.1002/cyto.b.20140

Zuk, P. A., Zhu, M., Ashjian, P., De Ugarte, D. A., Huang, J. I., Mizuno, H., . . . Hedrick, M. H. (2002). Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 13(12), 4279-4295. doi:10.1091/mbc.E02-02-0105

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