Isolation of human BM-MSCs
BM-MSCs obtained from healthy 22- and 25-year-old male and 80- and 86-year-old female volunteers (young KNT cells: KNT_D_170714, KNT_L_170714; elderly KNT cells: KNT_80_170825 and KNT_86_170825, Kintaro Cells Power Co., Tokyo, Japan) were used in this study (Table 1). These cells were collected under the control of the Federal Research and Clinical Center of the Federal Medical Biological Agency of Russia (FRCC FMBA, Moscow, Russia) after approval from the institutional ethical committee (approval 5-11-03-2013 of March 03, 2013).
We seeded BM-MSCs in αMEM (Gibco, Carlsbad, CA, US) supplemented with 5% PLTMax Human Platelet Lysate (Millipore, Temecula, CA, US) and 2 U/ml Heparin (Sigma, St. Louis, MO, US) at 37 °C in a humidified atmosphere containing 5% CO2. Adherent cells were harvested by trypsinization and either passaged (passages 1–4) for expansion or subjected to analyses for subsequent cytogenetic analysis. The following cell lines derived from hematologic malignancies were purchased from the Health Science Research Resource Bank, JCRB Cell Bank, and ATCC: three lymphoma cell lines (SUDHL4, CRL-2957; DL40, JCRB1334; Pfeiffer, CRL-2632), three lines with multiple myeloma (RPMI8226, JCRB0034; KMS-11, JCRB1179; U266, TIB-196), and three lines with leukemia (K562, JCRB0019; HL-60, IFO50022; U937, JCRB9021). These cells were cultured in RPMI1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT, US) at 37 °C in a humidified atmosphere containing 5% CO2.
Flow cytometry and cytogenetic analysis
To characterize KNT cells using their surface antigen, the following monoclonal antibodies were used: CD90-FITC (Cat. No. 555595), CD73-APC (Cat. No. 560847), CD105-PerCPCy5.5 (Cat. No. 560819), CD34-FITC (Cat. No. 555821), CD45-PE (Cat. No. 555483), HLA-DR-PE (Cat. No. 555812), and CD271-PE (Cat. No. 557196) (BD Biosciences, San Jose, CA, US). The KNT cells were incubated with these antibodies and analyzed by an Accuri C6 cytometer (BD Biosciences), according to the manufacturer’s instructions. Chromosome analysis was performed with a conventional Giemsa-banding method after colcemid exposure. A further cytogenetic study was performed using commercially available spectral karyotyping (SKY) from SRL Inc. (Hachioji, Hino, Tokyo, Japan).
Osteoblast and adipocyte differentiation
To confirm the adipogenic potential of young and elderly KNT cells, these cells were incubated in αMEM with 5% PLTMax until cells were confluent. Thereafter, KNT cells were cultured with adipogenic induction medium (Lonza, Basel, Switzerland). After 3 days, maintenance medium was added to cells, and three cycles of induction and maintenance media were completed. Cells were fixed with 10% formalin (Sigma-Aldrich) and stained with 0.5% Oil Red O (Sigma-Aldrich) in methanol (Sigma-Aldrich). To confirm the osteogenic potential of KNT cells, they were incubated in αMEM with 5% PLTMax until a confluent layer was achieved. Thereafter, osteogenic differentiation medium (Lonza) was added. Medium was changed every 3–4 days. After 17 days, cells were fixed in 10% formalin and stained with 10% alizarin red (Sigma-Aldrich).
Collection of conditioned medium (CM) and EV
Young and elderly KNT cells were cultured in maintenance medium (αMEM with 5% PLTMax) which was replaced with serum-free MSCBM basal medium (Lonza). The KNT cell-derived conditioned medium (KNT cell-CM) was collected after 48 h. The collected CM was centrifuged for 15 min at 2500 rpm to remove cell and cell debris, and then concentrated (approximately tenfold concentration) using Amicon Ultra-15 3 kDa MW cut-off filter units (MilliporeSigma, Burlington, MA, US) to exclude possible contamination from lipids and metabolites. KNT cells (4 × 104 cells/cm2) were cultured in 5 ml of MSCBM basal medium (Lonza) in a T-25 flask. The culture supernatants were harvested after 48 h of incubation, and the EV fraction was purified with Exoquick-TC reagent (System Biosciences, Palo Alto, CA, US) according to the manufacturer’s instructions. EV pellets were resuspended in 500 µl of MSCBM basal medium.
Evaluation of the anti-neoplastic activity of various hematologic malignant cells
To evaluate the proliferative effect of KNT cells, lymphoma cells (SUDHL4, DL40, and Pfeiffer), multiple myeloma cells (RPMI8226, KMS-11, and U266), or myeloid leukemia cells (K562, HL-60, and U937) were used. Neoplastic cells (2 × 105 cells/mL in maintenance medium) were cultured with or without concentrated CM or EV from KNT cells. Thirty microliters of concentrated CM or EV fractions from the KNT cells were then added to the cultured hematological malignant cell lines. Cell proliferation rates were measured using the IncuCyte Zoom live-cell analysis system (Essen Biosciences, Ann Arbor, MI, US).
Effect on neoplastic cell-induced tube formation
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and cultured in maintenance medium [EBM-2 medium supplemented with EGM-2 bulletKit (Lonza)]. HUVECs (300 µL of 4 × 105 cells/mL in EBM-2 basal medium) were placed on a 260-µl Matrigel (BD Biosciences) in a 24-well plate and incubated overnight at 37 °C. Neoplastic cell (SUDHL4, RPMI8226, or K562)-derived concentrated CM (20 µL/well) with or without KNT cell-derived CM (20 µL/well) or KNT cell-derived EV was subsequently added. KNT cells used were KNT_D_176714 (as young KNT cells) or KNT_86_170825 (elderly KNT). After 24 h, cells were stained with calcein AM (BD Biosciences) and visualized under a fluorescence microscope. Total tube area was quantified as the mean pixel density obtained from image analysis of five random microscopic fields using Image-J software .
Data are expressed as mean ± SD. Two treatment groups were compared using Student’s t test. Multiple group comparisons were performed by ANOVA. GraphPad Prism version 5c for Macintosh (GraphPad Inc., La Jolla, CA, US) was used for statistical analyses. Results were considered statistically significant when P was < 0.05.