Ferencakova Michaela, Benova Andrea, Raska Ivan, Abaffy Pavel, Sindelka Radek, Dzubanova Martina, Pospisilova Eliska, Kolostova Katarina, Cajka Tomas, Paclik Ales, Zikan Vit, Tencerova Michaela
Laboratory of Molecular Physiology of Bone, Institute of Physiology of the Czech Academy of Sciences, Prague, Czechia.
Faculty of Science, Charles University, Prague, Czechia.
Front Cell Dev Biol. 2023 Sep 18;11:1255823. doi: 10.3389/fcell.2023.1255823. eCollection 2023.
Bone marrow stromal cells (BMSCs) are the source of multipotent stem cells, which are important for regenerative medicine and diagnostic purposes. The isolation of human BMSCs from the bone marrow (BM) cavity using BM aspiration applies the method with collection into tubes containing anticoagulants. Interactions with anticoagulants may affect the characteristics and composition of isolated BMSCs in the culture. Thus, we investigated how anticoagulants in isolation procedures and cultivation affect BMSC molecular characteristics. BM donors (age: 48-85 years) were recruited from the hematology clinic. BM aspirates were obtained from the iliac crest and divided into tubes coated with ethylenediaminetetraacetic acid (EDTA) or heparin anticoagulants. Isolated BMSCs were analyzed by flow cytometry and RNA-seq analysis. Further cellular and molecular characterizations of BMSCs including CFU, proliferation and differentiation assays, cytometry, bioenergetic assays, metabolomics, immunostaining, and RT-qPCR were performed. The paired samples of isolated BMSCs obtained from the same patient showed increased cellular yield in heparin vs. EDTA samples, accompanied by the increased number of CFU colonies. However, no significant changes in molecular characteristics were found between heparin- and EDTA-isolated BMSCs. On the other hand, RNA-seq analysis revealed an increased expression of genes involved in nucleotide metabolism and cellular metabolism in cultivated vs. non-cultivated BMSCs regardless of the anticoagulant, while genes involved in inflammation and chromatin remodeling were decreased in cultivated vs. non-cultivated BMSCs. The type of anticoagulant in BMSC isolation did not have a significant impact on molecular characteristics and cellular composition, while cultivation caused the major change in the transcriptomics of BMSCs, which is important for future protocols using BMSCs in regenerative medicine and clinics.
骨髓基质细胞（BMSCs）是多能干细胞的来源，对再生医学和诊断目的具有重要意义。使用骨髓穿刺术从骨髓腔中分离人BMSCs的方法是将采集的骨髓收集到含有抗凝剂的试管中。与抗凝剂的相互作用可能会影响培养中分离出的BMSCs的特性和组成。因此，我们研究了分离过程和培养中的抗凝剂如何影响BMSC的分子特性。从血液学诊所招募了骨髓供体（年龄：48 - 85岁）。从髂嵴获取骨髓抽吸物，并将其分为涂有乙二胺四乙酸（EDTA）或肝素抗凝剂的试管。通过流式细胞术和RNA测序分析对分离出的BMSCs进行分析。对BMSCs进行了进一步的细胞和分子特性分析，包括集落形成单位（CFU）、增殖和分化测定、细胞计数、生物能量测定、代谢组学、免疫染色和逆转录定量聚合酶链反应（RT-qPCR）。从同一患者获得的成对分离BMSCs样本显示，与EDTA样本相比，肝素样本中的细胞产量增加，同时CFU集落数量增加。然而，肝素分离的BMSCs和EDTA分离的BMSCs之间在分子特性上未发现显著变化。另一方面，RNA测序分析显示，无论使用何种抗凝剂，培养后的BMSCs与未培养的BMSCs相比，参与核苷酸代谢和细胞代谢的基因表达增加，而参与炎症和染色质重塑的基因表达减少。BMSC分离过程中抗凝剂的类型对分子特性和细胞组成没有显著影响，而培养导致了BMSCs转录组学的主要变化，这对未来在再生医学和临床中使用BMSCs的方案具有重要意义。
