DOI 10.1007/s10517-017-3662-9

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Expression of Surface Molecules in Human Mesenchymal Stromal Cells Co-Cultured with Nucleated Umbilical Cord Blood Cells Yu. A. Romanov1,3, E. E. Balashova3, N. E. Volgina2, N. V. Kabaeva1, T. N. Dugina3, and G. T. Sukhikh2 Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 4, pp. 270-274, October, 2016 Original article submitted October 12, 2016 We studied the expression of different classes of surface molecules (CD13, CD29, CD40, CD44, CD54, CD71, CD73, CD80, CD86, CD90, CD105, CD106, CD146, HLA-I, and HLA-DR) in mesenchymal stromal cells from human umbilical cord and bone marrow during co-culturing with nucleated umbilical cord blood cells. Expression of the majority of surface markers in both types of mesenchymal stromal cells was stable and did not depend on the presence of the blood cells. Significant differences were found only for cell adhesion molecules CD54 (ICAM-1) and CD106 (VCAM-1) responsible for direct cell—cell contacts with leukocytes and only for bone marrow derived cells. Key Words: mesenchymal stromal cells; umbilical cord blood; co-culture; differentiation clusters; flow cytometry Over the past few decades, mesenchymal stromal cells (MSC), in particular, MSC from human umbilical cord and placental tissue are in the focus of research due to their unique biological properties and high therapeutic potential in various fields of regenerative medicine [21,24]. The therapeutic activity of MSC is determined by their capacity to differentiate into functionally active cells, paracrine regulation, and production of micro-RNA, exosomes, microvesicles, etc. [20]. Immunomodulatory, pro- and anti-inflammatory effects on different populations of blood cells (lymphocytes, neutrophils, monocytes, macrophages, and dendritic cells) are now intensively studied [4,7,8,11,17,26]. Numerous reports discussed the role of MSC in the formation of hematopoietic microenvironment, their influence on viability, proliferation, and differentiation of hematopoietic stem cells (HSC) Russian Cardiology Research-and-Production Complex, Ministry of Health of the Russian Federation; 2V. I. Kulakov Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation; 3CryoCenter Cord Blood Bank, Moscow, Russia. Address for correspondence: [email protected]. Yu. A. Romanov 1

of umbilical cord blood [5,6,10,12,18,23], and the efficacy of their transplantation [3,12]. The effects of MSC on blood cells, including HSC, are studied in detail, but little is known about inverse interactions and activation or inhibition of MSC by leukocytes [22,25]. Here we studied expression patterns of different classes of surface molecules on MSC from human umbilical cord tissue and bone marrow co-cultured with nucleated cells from umbilical cord blood (UCB).

MATERIALS AND METHODS Isolation and culturing of MSC. Detailed protocols of collection of the biological material and isolation, culturing, and cryogenic storage of MSC from human umbilical cord tissue and bone marrow were described previously [15,16]. In brief, cryopreserved passage 2 MSC were defrosted on a water bath, washed free of cryoprotectant, seeded onto 75-cm2 culture flasks, and cultured in DMEM/F12 supplemented with 100 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (Life Technologies).

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Nearly confluent cells (4-5×104 cells/cm2) were passaged into new flasks at a ratio of 1:3 and subcultured during the next two passages. Passage 4 MSC cultures were used for experiments. Isolation of nucleated UCB cells. UCB was collected into polymer bags for preservation of the blood and its components with CPDA-1 anticoagulant (Green Cross) as described elsewhere [13,14]. The fraction of nucleated cells was obtained by 2-fold centrifugation, resuspended in autologous plasma with 10% DMSO (Sigma) and 1% rheopolyglucin (Biokhimik), aliquoted in 3.6-ml cryovials (Corning; ~100×106 cells per vial), and subjected to programmed freezing to a final temperature -90oC. Frozen cells were stored in liquid nitrogen vapor phase at a temperature below -160ºC. During quarantine storage, all UCB samples were tested for blood-transmitted infection markers (HIV-1/2, hepatitis B and C, herpes simplex virus 1 and 2, HTLV-1/2, cytomegalovirus, and syphilis). Seropositive samples and cells that did not pass the tests for sterility were utilized according to the prescribed procedure. Other samples (suitable) were transferred to liquid nitrogen and stored until use. Co-culturing MSC with UCB cells. The growth medium in MSC cultures was renewed 48 h before the experiment. UCB cells were defrosted as described previously [14], washed from the cryoprotectant, resuspended in 10 ml of culture medium, and MSC were added in a concentration 5×106 cells/ml. On the next day, the medium with floating cells was replaced with a fresh portion. The culture medium was then replaced every 2-3 days (the last change was performed in 48 h before analysis). Cell growth and morphology were evaluated by phase-contrast microscopy (Axiovert-40, Nikon). Flow cytofluorometry. After co-culturing for 48  h, 1 and 2 weeks, the cultures were washed with sterile physiological saline and harvested with trypsin-EDTA (Life Technologies). Parallel MSC monocultures were harvested at the same time and served as the control. The cell suspensions were divided into 50-μl aliquots (105 MSC each) and incubated at room temperature with PE- or FITC-conjugated antibodies to the corresponding differentiation cluster (CD). Mouse monoclonal antibodies to CD13, CD29, CD40, CD44, CD54, CD71, CD73, CD80, CD86, CD90, CD105, CD106, CD146, HLA-I, and HLA-DR (Beckman Coulter or BD Pharmingen) in concentrations recommended by the manufacturer and the corresponding isotypic controls were used. To exclude blood cells and/or their aggregates with MSC from the analysis (negative gating), FITC- or PE-labeled antibodies to CD45 (Beckman Coulter) were added to each sample. The cells suspensions were analyzed on a FACSCalibur flow cytofluorom-

eter (BD) using CellQuest Pro software; 10 4 cells were analyzed in each sample.

RESULTS The control cultures of umbilical cord MSC were characterized by expression of typical surface markers CD13, CD29, CD44, CD54, CD71, CD73, CD90, CD105, CD146, and HLA-I and weak expression of CD106 and did not express CD45, CD80, and CD86. More than a half of the cells were positively stained for CD40 (Fig. 1). No fundamental differences in the expression of CD molecules between 48-h and 1- or 2-week MSC cultures were found. Bone marrow cells expressed the same surface markers with the same fluorescence intensity except for CD106 (CD106+ cells constituted ~20-40%; Fig. 2). After addition to MSC monolayer, nucleated UCB cells adhered to MSC surface and some of them migrated beneath the monolayer. Flow cytometry after 48-h co-culturing showed the presence of all initial cell populations (lymphocytes, monocytes, granulocytes, and HSC) among adherent UCB cells and their ratio did significantly varied. At later terms (1 week or more), predominance of lymphocytic-monocytic cells was observed due to partial elimination of short-lived granulocytes and proliferation and differentiation of HSC. Similar results were obtained earlier in the study of the interaction of HSC with MSC isolated from human adipose tissue [1,9,19]. During co-culturing, the expression of studied surface markers in MSC isolated from umbilical cord tissue or from bone marrow changed insignificantly (Fig. 1). Expression of ~50% analyzed molecules (CD13, CD29, CD40, CD44, CD73, and HLA-DR) did not change at all; while for the rest molecules (CD71, CD80, CD86, CD90, CD105, and CD146), we observed moderate changes in fluorescence intensity not accompanied by changes in the proportion of positively stained cells. For instance, fluorescence intensity of CD90, CD105, CD146, and HLA-I tended to decrease; at least two of these are classic markers of MSC [2]. Significant changes in fluorescence intensity and in the number of CD54+ and CD105+ cells were found in co-cultures of bone marrow MSC (Fig. 2). Thus, the study showed that the expression of the vast majority of surface markers in MSC (from umbilical cord and bone marrow) is sufficiently constitutive and practically does not depend on the presence of blood cells. The only significant differences were found in cell adhesion molecules responsible for direct cell—cell contacts with leukocytes CD54 (ICAM1) and CD106 (VCAM-1) and only in case of bone marrow-derived cells. This can be explained by the

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Fig. 1. Expression of surface markers on MSC from human umbilical cord tissue co-cultured with nucleated UCB cells. Flow cytofluorometry. Here and in Fig. 2: black curves — negative control; green curves — control (without co-culturing); blue curves — 48 h; violet curves — 1 week; red curves — 2 weeks.

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Umbilical cord

Bone marrow

Fig. 2. Expression of surface adhesion molecules ICAM-1 (CD54) and VCAM-1 (CD106) on MSC from human umbilical cord tissue and bone marrow co-cultured with nucleated UCB cells.

fact that among MSC of different origins (umbilical cord tissue — adipose tissue — bone marrow), bone marrow-derived cells produce the maximum stimulation effect on HSC proliferation and differentiation in co-culture [6]. The study was performed within the framework of research and practical cooperation between V. I.  Kulakov Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Fe­de­ration and CryoCenter Cord Blood Bank and supported by the Russian Science Foundation (grant No. 14-25-00179).

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