Mesenchymal stem cells as a reference cell for HLA-typing

Introduction: Recognition of human leukocyte antigens (HLA) is of importance for hematopoietic stem cell transplantation. Any HLA-mismatches between the donor and recipient can cause graft rejection or other complications. In HLA-typing experiments, usage of HLA-known reference cells accompany with HLA-unknown samples is obligatory. Some international centers represent these cells with high expenses. On the other hand, transferring of these cells is problematic and in some instances is not practical. In this study, we introduced umbilical cord-derived mesenchymal stem cells (MSCs) as reference cells for HLA genotyping. These cells are national and can be prepared locally. Materials and methods: We isolated MSCs from three umbilical cord and after their growth and proliferation, these cells were characterized by flow cytometry technique using antibodies to CD29, CD34, CD44, CD45, CD73, CD90 and CD105. HLA-typing was then carried out by PCR-SSP kits for HLA-A, -B and -DRB allele’s identification. Results: Isolated MSCs were positive for MSCs markers; CD29, CD44, CD73, CD90, and CD105 and negative for hematopoietic stem cell markers; CD34 and CD45. HLA alleles were determined. One of the samples was homologous for HLA alleles and the others were heterologous. Conclusion: We can develop a reference panel for HLA-typing by obtaining MSCs from available sources like umbilical cord.


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These cells can be isolated from different sources such as, umbilical cord, Wharton's jelly, cord blood, bone marrow, and adipose tissue so they are accessible.

Materials and methods
Isolation of MSCs: Three umbilical cords were obtained from Iranian cord blood cell bank in Iran Blood Transfusion Organization (IBTO).Umbilical cord specimens were cut in to very small pieces in sterile conditions and washed with PBS.Tissue pieces were put in 1mg/ml collagenase type IV solution (GIBCO, USA) and incubated in 37°C for 3-4 h.Then the solution was filtered to isolate cell suspension from debris.Cell suspension was washed with PBS and centrifuged.The supernatant was removed and cell pellet was dissolved in DMEM medium supplemented with 10% FBS, 1% penicillin and streptomycin (prepared from a stock solution of 100X concentration contained 10,000 IU/mL penicillin and 10,000 ug/mL of streptomycin) and 1% L-gln and incubated in 37C ْ and 5% CO2 incubator.

Characterization of MSCs:
The morphology of MSCs was observed under inverted microscope.Immunophenotyping of MSCs was carried out with flow cytometry technique.Eight antibodies were used for the detection of CD markers of MSCs surface: anti CD29-FITC, anti CD34-PE, anti CD44-FITC, anti CD45-FITC, anti CD73-PE, anti CD90-PE and anti CD105-FITC (all from BD Biosciences, USA).HLA genotyping: DNA was extracted from MSCs with viability more than 95 percent.Salting out method was used for the extraction of DNA.The concentration and purity of extracted DNA was determined by NanoDrop spectrophotometer (WPA, UK).HLA genotyping and allele identification was carried out using PCR-SSP method.ABDR HLA typing kit (Olerup SSP HLA typing kit, Sweden) was used for HLA genotyping.PCR was carried out with 30 ng of the genomic DNA in a total volume of 10 µl of the reaction mixture.Addition of DNA, master mix and Taq DNA polymerase was accomplished as the manufacturer's instructions.The PCRcycling conditions were as follows: initial denaturation at 94ºC for 2 min, followed by 10 cycles of 94ºC for 10 s and 65ºC for 60 s and another 20 cycles of 94ºC for 10 s, 61ºC for 50 s and 72ºC for 30 s.PCR products were analyzed on a 1.5% agarose gel with GelRed™ (Olerup SSP HLA typing kit, Sweden).DNAs with specified alleles were used to control HLA typing results.

Results
Characterization of the isolated cells: An image for the cultured isolated cells was shown in Fig. 1.These cells were spindleshape and adherent.On the other hand, flow cytometry technique demonstrated that the surface markers of isolated cells were related to MSCs.The isolated cells were positive for MSC markers including CD29, CD44, CD73, CD90, and CD105 and negative for hematopoietic stem cell markers; CD34 and CD45 (Fig. 2).This experiment confirmed the MSCs characterize for the adherent isolated cells.HLA-genotyping results: Concentrations of extracted DNA from three umbilical cordderived MSCs (UC-MSC 1, UC-MSC 2, and UC-MSC 3) were 48 µg/ml, 75 µg/ml and 57 µg/ml, respectively.The purity of extracted DNA was determined using the ratio of OD260 to OD280 and were in an acceptable range; 1.72, 1.97 and 1.86.After PCR amplification, for identifying the HLA alleles, electrophoresis of PCR products was accomplished on a 1.5% agarose gel (Fig 3 and table 1).Each typing plate included 96 wells: 24 for HLA-A, 48 for HLA-B, 23 for HLA-DRB and 1 for negative control.The interpretation of results was accomplished according to the worksheets represented by the manufacture.

Discussion
HLA typing is essential for stem cell transplantation (SCT) because HLA compatibility between donor and recipient is important (1,4).Otherwise, the graft is rejected or GVHD is occurred (2,3).On the other hand, in HLA-typing, a suitable HLAknown control is necessary to be sure about the accuracy of HLA results (16).In this study, HLA typing was accomplished on MSCs derived from different tissues using PCR-SSP method.Since these cells were accessible and their growth and proliferation accomplished easily without need to unique medium so they could be used for preparing a reference panel for HLA genotyping.Different centers and companies such as IHWG, EBRCC-cell Culture laboratory and National Cancer Institute's Developmental Therapeutics Program (NCI-60) represented various cell lines or their related DNA and offered them as reference panel for different purposes like HLA genotyping (16,27,28).Michael Witti developed high-resolution next-generation sequencing (NGS)-based HLA-typing for cell lines from IHWG and EBRCC-cell as reference set to demonstrate the potential of NGS in HLA-typing.They used some random to fill out reference panel (29).Hemmatpour used B lymphocyte cell lines as HLA-known controls in DNA-based HLA typing.(30).NCI-60 cell lines, first, were used as screening tools to develop anti-cancer drugs, in early 90,s (31).But in 2005 Sharon Adams, performed a highresolution sequence-based HLA typing experience on this panel for using of these cells as tools in different biologic works (31).In the study of Degli-Esposti, 100 EBVtransformed cells and more than 150 cell lines were typed and were introduced as a cell panel with defined HLA alleles (32).Kim developed a Korean HLA reference panel by HLA typing of more than 400 individuals in East Asians, including Koreans (33).In the previous studies, various cell lines, virus-transformed cells or blood cells derived from healthy individuals were used to develop reference panels.But using of these panels was accompanied with difficulties such as not being national, not being easily accessible and being expensive.

Conclusion
HLA-typed MSCs could be prepared from each available MSC-containing tissue for example umbilical cord and used as reference cells in DNA-based HLA-typing techniques.These cells could be kept in liquid nitrogen and cultured periodically to obtain enough DNA as the base material in molecular studies.

Figure 1 .
Figure 1.The isolated cells (MSCs) were observed under inverted microscope and were spindle-shape and adherent (100 x).

Figure 2 .Figure 3 .
Figure 2. Flow cytometry plots.Characterization of isolated cells (A) gating of the cells.(B and C) isotype control.Immunophenotyping results showed that the isolated cells were negative for CD45 and CD34 (D and E, respectively) and positive for CD29, CD44, CD73, CD90, and CD105 (F-G, respectively).These characters were correlated with MSCs.Downloaded from jbrms.medilam.ac.ir at 15:46 IRST on Saturday December 15th 2018