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Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties

M. Ferencakova, A. Benova, I. Raska, P. Abaffy, R. Sindelka, M. Dzubanova, E. Pospisilova, K. Kolostova, T. Cajka, A. Paclik, V. Zikan, M. Tencerova

. 2023 ; 11 (-) : 1255823. [pub] 20230918

Status not-indexed Language English Country Switzerland

Document type Journal Article

Persistent link   https://www.medvik.cz/link/bmc23022850

Background: 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. Methods: 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. Results: 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. Conclusion: The type of anticoagulant in BMSC isolation did not have a significant impact on molecular characteristics and cellular composition, while in vitro cultivation caused the major change in the transcriptomics of BMSCs, which is important for future protocols using BMSCs in regenerative medicine and clinics.

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$a Background: 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. Methods: 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. Results: 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. Conclusion: The type of anticoagulant in BMSC isolation did not have a significant impact on molecular characteristics and cellular composition, while in vitro cultivation caused the major change in the transcriptomics of BMSCs, which is important for future protocols using BMSCs in regenerative medicine and clinics.
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$a Benova, Andrea $u Laboratory of Molecular Physiology of Bone, Institute of Physiology of the Czech Academy of Sciences, Prague, Czechia $u Faculty of Science, Charles University, Prague, Czechia
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$a Raska, Ivan $u Third Department of Medicine-Department of Endocrinology and Metabolism, First Faculty of Medicine, General University Hospital in Prague, Charles University, Prague, Czechia
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$a Abaffy, Pavel $u Laboratory of Gene Expression, Institute of Biotechnology of the Czech Academy of Sciences, Vestec, Czechia
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$a Sindelka, Radek $u Laboratory of Gene Expression, Institute of Biotechnology of the Czech Academy of Sciences, Vestec, Czechia
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$a Dzubanova, Martina $u Laboratory of Molecular Physiology of Bone, Institute of Physiology of the Czech Academy of Sciences, Prague, Czechia $u Faculty of Science, Charles University, Prague, Czechia
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$a Pospisilova, Eliska $u Laboratory of Personalized Medicine, Oncology Clinic, University Hospital Kralovske Vinohrady, Prague, Czechia
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$a Kolostova, Katarina $u Laboratory of Personalized Medicine, Oncology Clinic, University Hospital Kralovske Vinohrady, Prague, Czechia
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$a Cajka, Tomas $u Laboratory of Translational Metabolism, Institute of Physiology of the Czech Academy of Sciences, Prague, Czechia
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$a Paclik, Ales $u First Department of Surgery, First Faculty of Medicine, General University Hospital in Prague, Charles University, Prague, Czechia
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$a Zikan, Vit $u Third Department of Medicine-Department of Endocrinology and Metabolism, First Faculty of Medicine, General University Hospital in Prague, Charles University, Prague, Czechia
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$a Tencerova, Michaela $u Laboratory of Molecular Physiology of Bone, Institute of Physiology of the Czech Academy of Sciences, Prague, Czechia
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