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RESEARCH ARTICLEDOI: 10.4274/tjh.2016.0498Turk J Hematol 2018;35:42-48The Effect of Bone Marrow Mesenchymal Stem Cells on theGranulocytic Differentiation of HL-60 CellsKemik İliği Mezankimal Hücrelerinin HL-60 Hücrelerindeki Granülositik FarklılaşmasıÜzerine EtkisiHossein Nikkhah 1 , Elham Safarzadeh 2,3 , Karim Shamsasenjan 1 , Mehdi Yousefi 2,3 , Parisa Lotfinejad 1,3 , Mehdi Talebi 1 ,Mozhde Mohammadian 4 , Farhoud Golafshan 5 , Aliakbar Movassaghpour 11Tabriz University Faculty of Medicine, Hematology and Oncology Research Center, Tabriz, Iran2Tabriz University Faculty of Medicine, Drug Applied Research Center, Tabriz, Iran3Tabriz University Faculty of Medicine, Department of Immunology, Tabriz, Iran4Mazandaran University Faculty of Medicine, Amol Faculty of Paramedical Sciences, Sari, Iran5Hamline University Faculty of Medicine, Department of Biology, Minnesota, USAAbstractObjective: Mesenchymal stem cells (MSCs) are multipotent stromalcells that can differentiate into a variety of cell types. They control theprocess of hematopoiesis by secreting regulatory cytokines and growthfactors and by the expression of important cell adhesion molecules forcell-to-cell interactions. This investigation was intended to examinethe effect of bone marrow (BM)-derived MSCs on the differentiationof HL-60 cells according to morphological evaluation, flow cytometryanalysis, and gene expression profile.Materials and Methods: The BM-MSCs were cultured in Dulbecco’smodified Eagle’s medium supplemented with 10% fetal bovine serum(FBS). After the third passage, the BM-MSCs were irradiated at 30Gy. To compare how the HL-60 cells differentiated in groups treateddifferently, HL-60 cells were cultured in RPMI-1640 and supplementedwith 10% FBS. The HL-60 cells were seeded into six-well cultureplates and treated with all-trans-retinoic acid (ATRA), BM-MSCs, orBM-MSCs in combination with ATRA, while one well remained asuntreated HL-60 cells. The expression levels of the granulocyte subsetspecificgenes in the HL-60 cells were assayed by real-time polymerasechain reaction.Results: Our results revealed that BM-MSCs support the granulocyticdifferentiation of the human promyelocytic leukemia cell line HL-60.Conclusion: Based on the results of this study, we concluded thatBM-MSCs may be an effective resource in reducing or even preventingATRA’s side effects and may promote differentiation for shortmedication periods. Though BM-MSCs are effective resources, morecomplementary studies are necessary to improve this differentiationmechanism in clinical cases.Keywords: Mesenchymal stem cells, HL-60 cells, Differentiation, Alltrans-retinoicacidÖzAmaç: Mezankimal kök hücreler (MKH) birçok hücre tipine görefarklılaşabilen multipotent stromal hücrelerdir. Hematopoez sürecinidüzenleyici sitokinler ve büyüme faktörleri salınımı ile ve hücreler arasıetkileşim için önemli hücresel adezyon moleküllerinin ifadesi yoluylakontrol ederler. Bu çalışma da kemik iliği (Kİ) kaynaklı MKH HL-60hücrelerinin farklılaşması üzerine etkisini morfolojik değerlendirme,akım sitometri ve gen ifade analizi yöntemleriyle araştırılmasıamaçlanmıştır.Gereç ve Yöntemler: Kİ-MKH %10 fetal sığır serumu (FSS) içerenDulbecco’nun modifiye Eagle ortamında kültür edildi. Üçüncüpasaj sonrası, Kİ-MKH 30 Gy ile ışınlandı. HL-60 hücrelerinin farklışartlarda nasıl farklılaştığını karşılaştırmak için HL-60 hücreler %10FSS eklenmiş RPMI-1640 ortamında kültür edildi. HL-60 hücreleri altıkuyucuklu plaklarda all-trans retinoik asit (ATRA), Kİ-MKH ve ATRAile birlikte Kİ-MKH ile muamele edilirken, bir kuyucuğa sadece HL-60 hücreleri kondu. HL-60 hücrelerinde granülosit alt gruplarına özgügenlerin ifade düzeyleri gerçek zamanlı polimeraz zincir reaksiyonu iledeğerlendirildi.Bulgular: Sonuçlarımız Kİ-MKH’nin insan promiyelositik lösemi hücredizisi HL-60’ın granülositik farklılaşmasını desteklediğini gösterdi.Sonuç: Bu çalışmanın bulgularına göre, Kİ-MKH’nin ATRA yan etkileriniazaltıcı ve hatta önleyici etkili bir kaynak olduğu ve kısa ilaç kullanımısüreçlerinde farklılaşmayı uyarabileceği sonucunu çıkarttık. Kİ-MKHetkili bir kaynak olsa da, klinik olgularda bu farklılaşma mekanizmasınıiyileştirmek için destekleyici ek çalışmalara ihtiyaç vardır.Anahtar Sözcükler: Mezankimal kök hücreler, HL-60 hücreleri,Farklılaşma, All-trans retinoik asit©Copyright 2018 by Turkish Society of HematologyTurkish Journal of Hematology, Published by Galenos Publishing HouseAddress for Correspondence/Yazışma Adresi: Ali Akbar MOVASSAGHPOUR, M.D.,Tabriz University Faculty of Medicine, Hematology and Oncology Research Center, Tabriz, IranPhone : +984 133 343 888E-mail : movassaghpour@tbzmed.ac.ir ORCID-ID: orcid.org/0000-0002-6990-9260Received/Geliş tarihi: December 27, 2016Accepted/Kabul tarihi: June 12, 201742
Turk J Hematol 2018;35:42-48Nikkhah H, et al: BM-MSCs’ Effects on HL-60 Cell DifferentiationIntroductionThere are different cell types of the osteoblast lineage in boneand the bone marrow, the most primitive of them being themesenchymal stem cells (MSCs) [1,2]. MSCs can differentiate intoseveral types of cells and produce important growth factors andcytokines [3,4]. MSCs are defined by the International Societyof Cellular Therapy based on three properties: the adherence toplastic in standard culture; the expression of CD105, CD73, andCD90 and lack of expression of CD45, CD34, CD14 or CD11b,CD79α or CD19, and HLA class II; and differentiation potentialinto osteocytes, adipocytes, and chondrocytes [5,6]. These cellsare involved in the regulation of hematopoietic precursor cellproliferation and differentiation [7,8].All-trans-retinoic acid (ATRA) has a potential role in treatingacute myeloid leukemia (AML) and some hematologicaldisorders [9]. It has been recognized that ATRA induces thedifferentiation of myeloid leukemic cells through growthinhibition [10]. Many studies have reported severe adverseeffects of ATRA. Therefore, novel therapeutic strategies needto be developed to decrease ATRA’s potential side effects andenhance the efficacy of this drug. One possible approach is theuse of ATRA-based combinations that are more efficient thanthe single components [11,12,13]. The roles of the various cellsin the bone marrow niche are unclear in the differentiation ofhematopoietic stem cells, and MSCs, as the precursors of thecellular components, are important cells of the bone marrowniche [14]. To understand the precise interaction between MSCsand leukemic cells, in the current study we investigated whetherMSCs affect the differentiation of HL-60 cells.Materials and MethodsCell CultureHuman promyelocytic leukemia cell line HL-60 (a kind giftfrom Dr. Abroun, Tarbiat Modares University, Tehran, Iran)was cultured in RPMI-1640 medium (Sigma-Aldrich, St.Louis, MO, USA) supplemented with 10% fetal bovine serum(HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 µg/mLstreptomycin (Sigma, St. Louis, MO, USA). The BM-MSCs (StemCell Technology, Tehran, Iran) were cultured in low-glucoseDulbecco’s modified Eagle medium (GIBCO BRL, GaithersburgMD, USA) containing 10% fetal bovine serum.Co-culture ExperimentsHL-60 cells (10 5 cells/mL) were seeded onto plates and treatedwith ATRA at a concentration of 5x10 -7 M (Sigma-Aldrich) for48 h. The co-culture experiments were performed in six-wellplates including the HL-60 cells treated with BM-MSCs or thosetreated with BM-MSCs and 5x10 -7 M ATRA together. Before coculturingwith cancer cells, the BM-MSCs were irradiated at 30Gy when they reached 60% confluence. The HL-60 cells cameinto direct contact with the BM-MSCs.Morphological Study of Differentiated Granulocyte CellsTo study the morphological changes, the HL-60 cells weretreated with ATRA, BM-MSCs, or a combination of ATRA andBM-MSCs. After 48 h of incubation, the cells were stained withWright-Giemsa stain and studied by light microscope.Flow Cytometric Assessment of Granulocytic Markers forDifferentiationThe HL-60 cells (1x10 6 ) of the different groups, the co-cultureof the HL-60 cells with BM-MSCs, the HL-60 cells with BM-MSCs and ATRA in combination, the HL-60 cells with ATRA asa positive control, and the HL-60 cells without additions as anegative control were harvested and incubated with FITClabeledanti-CD11b (Becton Dickinson, San Jose, CA, USA) for 30min at 4 °C. The cells were then analyzed for the evaluation ofCD11b expression (a myeloid differentiation marker) with a flowcytometer (Becton Dickinson).Real-Time Polymerase Chain ReactionThe expression of the granulocyte subset-specific genes in thetreated HL-60 cells was investigated by real-time polymerasechain reaction (RT-PCR) after an incubation period of 48 h.Total RNA was extracted using the QIAzol lysis reagent (QIAGEN,Germantown, MD, USA) according to the manufacturer’sinstructions. The cDNA was prepared according to theinstructions of the Revert Aid Single Strand Kit (Fermentas,Burlington, ON, Canada). The mRNA levels of PU.1, CD11b,lysozyme, C/EBP-ALPHA, C/EBP-BETA, C/EBP E, MPO, CD64,CD16, GCSFR, and cathepsin G were analyzed using qRT-PCR.The GAPDH gene was used as an internal control (Table 1).Statistical AnalysisData were reported as mean ± standard deviation and wereanalyzed using Graph Pad Prism v 5.00 (Graph Pad Software, Inc.,La Jolla, CA, USA). Student’s t-test for single comparisons and twowayANOVA for multigroup comparisons were used for analysisand p<0.01 was regarded as denoting statistical significance.ResultsFlow Cytometry Confirmation of the Nature of the BM-MSCsTo verify the mesenchymal nature of the BM-MSCs, the surfaceantigens were assessed by flow cytometry, including CD14,CD19, CD34, CD45 CD90, CD105, and CD73. The characterizationexperiments performed in our study demonstrated that theBM-MSCs were negative in the expression of the hematopoieticmarkers for CD14, CD19, CD34, and CD45, and they had positiveexpression for CD90, CD105, and CD73 markers (Figure 1).43
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