Original Article Iran J Ped Hematol Oncol. 2021, Vol 11, No 3, 172-182 The Effect of Platelet Lysate on Expansion and Differentiation Megakaryocyte Progenitor Cells from Cord Blood CD34+ enriched Cells Yoda Yaghoubi MSc1,2, Ali Hassanzadeh PhD3, Adel Naimi MSc4, Sepehr Abdolahi MSc5, Mehdi Yousefi PhD2, Leili Aghebati-Maleki PhD6, Majid Zamani MSc7,* 1. Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran 2. Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 3. Department of Tissue Engineering and Applied Cell Sciences, Tehran University of Medical Sciences, Tehran, Iran 4. Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran 5. Department of Biology, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz, Iran 6. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 7. Department of Medical Laboratory Sciences, Faculty of Allied Medicine, Gonabad University of Medical Sciences, Gonabad, Iran *Corresponding author: Mr. Majid Zamani, MSc. of laboratory hematology and blood banking, Department of Medical Laboratory Sciences, Faculty of Allied Medicine, Gonabad University of Medical Sciences, Gonabad, Iran. Email: [email protected]. ORCID ID: 0000-0002-7260-2348 Received: 14 December 2020 Accepted: 09 June 2021 Abstract Background: Umbilical cord blood hematopoietic stem cells (UCB-HSCs) are an attractive source for transplantation. The generation of megakaryocyte-committed cells could lead to shorten period of thrombocytopenia after HSCs transplantation. Platelet lysate (PL) unlike fetal bovine serum (FBS) can prevent immune problems as well as avert transmission of certain diseases to the recipient. In this study, the authors aimed to assess the effect of PL on UCB CD34+ cells expansion and megakaryocyte differentiation. Materials and Methods: In this experimental study, PL prepared and the subsequent isolation of UCB CD34+ cells were done by magnetic cell sorting. The isolated cells were cultivated in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with PL or FBS. Cell expansion was evaluated using Trypan blue. Furthermore, Flow cytometry using monoclonal antibodies (CD41-FITC and CD42b-PE) and the expression of specific genes including GATA1, GATA2, FLI1, NFE2, and RUNX1 via real-time PCR were performed to evaluate the megakaryocyte differentiation. Results: The results showed that PL insignificantly enhanced UCB CD34+ cell expansion (32.83± 8.47 fold in FBS and 41.67± 10.31 fold in PL containing media). Besides, flow cytometry results showed that expression of CD41 was increased markedly (37.81± 4.78 fold in FBS and 45.78 ± 7.37 in PL containing media, P-value <0.05) but the elevation of CD42b (10.53 ± 2.13 and 13.20 ± 2.06 in FBS and PL containing media, respectively) was not significant (P-value = 0.051). The results of real-time PCR demonstrated a notable increase in GATA binding protein 1 (1.58, P-value <0.01), GATA binding protein 2 (2.45, P-value <0.001), RUNX family transcription factor 1 (1.60, P-value <0.01), Fli-1 proto-oncogene (1.87, P-value <0.001) in PL supplemented media, however, the increase of Nuclear Factor-Erythroid 2 gene expression was not significant in PL supplemented media (P- value = 0.11). Conclusion: PL improved UCB CD34+ cells expansion and megakaryocyte differentiation compared to FBS. Downloaded from ijpho.ssu.ac.ir at 3:11 IRST on Wednesday September 29th 2021 [ DOI: 10.18502/ijpho.v11i3.6563 ] Keywords: Cord Blood Stem Cell Transplantation, Hematopoietic Stem Cells, Megakaryocyte Progenitor Cells, Platelet-Derived Growth Factor Introduction HSCs can be separated from various Hematopoietic stem cells (HSCs) are a sources such as BM or mobilized peripheral small population of nucleated cells that blood (MPB) progenitor cells and umbilical have the potential to differentiate into cord blood (UCB) for the treatment of mature cells of all hematopoietic lineages hematologic disorders (3, 4). Among and self-renewal capacity for maintenance different sources of HSCs, UCB is an of HSC pool in bone marrow (BM) (1, 2). attractive source for transplantation due to superior proliferative capacity, low risk for Yaghoubi et al transmission of pathogenic agents, low zoonotic contamination (especially prions, incidence of graft versus host disease bovine diarrhea virus, mycoplasma, (GVHD), relative availability, and ease of creutzfeldt Jacob virus, viruses, and isolation (5). HSCs number in a single cord parvovirus), high cost, xenogeneic immune blood (CB) unit is acceptable for reactions, antibodies production against hematopoietic reconstruction in children. non-native proteins, inflammation, However, UCB-HSCs have some production of non-homologous immune disadvantages, for instance, the HSCs response (which leads to the transplant number in a single CB unit is insufficient rejection), batch-to-batch variation, fetus for an adult patient so this can lead to a scarification during sample collection, and delayed or failed reconstruction of the ethical issues regarding the animal welfare hematopoietic system (5, 6). UCB-HSCs (15-19). Due to the drawbacks of serum- are immature, therefore, more cell divisions free culture medium as well as animal are needed before differentiation. serum, it is imperative to search for an Following UCB transplantation, it could alternative human resource. These delay platelet recoveries (6). Allogenic alternatives are autologous and allogeneic platelet transplantation after HSCs serum albumin, human serum, umbilical transplantation has some disadvantages cord blood serum, plasma derivative including bacteremia, alloimmunization, material, platelet derivatives, and acellular high-cost, and febrile reactions. Using Wharton’s jelly (15, 20). A high megakaryocyte progenitor along with the concentration of growth factors available in immature hematopoietic stem/progenitor platelet lysate (PL) makes it a proper cells (HSPCs) during the first few months replacement for animal serum in cell after transplantation can support short-term culture (21). PL granules, specially α- platelet production (7, 8). Cultivation of granules, release various cytokines and HSCs and generation of megakaryocyte- growth factors including chemokine ligand committed cells could lead to rapid cell 2(C¬XCL2), CXCL4 (PF4), CXCL10, reconstitution and shorten the period of CXCL12 (SDF-1), CXCL1, CXCL3, thrombocytopenia after HSCs CXCL5, granulocyte-macrophage colony- transplantation (6, 7). Differentiation of stimulating factor (GM-CSF), granulocyte- cells toward the megakaryocyte-platelet colony stimulating factor (G-CSF), linage requires the expression of specific connective tissue growth factor (CTGF), glycoproteins such as CD41 (glycoprotein hepatocyte growth factor (HGF), platelet- IIb), CD42b (glycoprotein Ib), and CD61 derived growth factor (PDGF), epidermal (glycoprotein IIIa) (9-12). These growth factor (EGF), insulin-like growth glycoproteins are involved in platelet factor-I (IGF-I), and basic fibroblast- aggregation/function and facilitate the derived growth factor (b-FGF). Compared identification and isolation of to FBS, the promotion of proliferation and megakaryocyte cells (13). Cell culture is differentiation of cells observed in PL may Downloaded from ijpho.ssu.ac.ir at 3:11 IRST on Wednesday September 29th 2021 [ DOI: 10.18502/ijpho.v11i3.6563 ] required for cell proliferation in vitro. Some be due to the abundance of growth factors mediums are serum-free which have some and cytokines with synergic effect (21-24). disadvantages including high cost and need In the current study, the effect of PL as a a protein/growth factor supplement, and substitute for PBS on the HSCs culture and some others, need supplementation with megakaryocyte differentiation has been animal or human serum (14, 15). In cell investigated. culture, fetal bovine serum (FBS) supplement has been considered as the gold Materials and Methods standard media, however, clinical use of PL procurement FBS has some limitations including In this experimental study, PL preparation transmission possibility of pathogens, was performed via freeze/thaw protocol Iran J Ped Hematol Oncol. 2021, Vol 11, No 3, 172-182 173 The Effect of Platelet Lysate on Expansion and Differentiation Megakaryocyte Progenitor Cells from Cord Blood CD34+ enriched Cells from platelet bags obtained from the Iranian Cell expansion assay Blood Transfusion Organization. Briefly, To evaluate the UCB CD34+ cells 10 bags of platelet (isolated from peripheral expansion, the culture was collected and the blood) were collected, and PL was prepared cells counted using the Trypan Blue (Gibco, by freeze/thaw protocol. The pooled USA) on day 8. To determine expansion platelet bags were frozen at -80°C/24 hour fold, counted viable cells on day 8 were and subsequently thawed three times at 37 divided by the cells seeded on the first day. °C to release PDGFs available in platelets. 2.5 Immunophenotypic marker analysis Then, bags were centrifuged at 4000 g/15 Megakaryocyte lineage markers were min at 4 °C, and to isolate leukocytes along evaluated by the flow cytometry with platelet fragments, the supernatant was (FACSCalibur, Becton Dickinson, US) on filtered by 0.2μm filter. For future use, PL day 8 in both culture conditions. Cells were was stored at -80°C (25). incubated with monoclonal antibodies including CD41-FITC, and CD42b-PE UCB CD34+ cell selection (both from Beckman Coulter, France) for After receiving informed consent,