Eleni Gounari1, Angelos Daniilidis2, Nikolaos Tsagias3, Anna Michopoulou4, Kokkona Kouzi5, George Koliakos4. 1. Biohellenika Biotechnology Company, Thessaloniki, Greece; Department of Biological Chemistry, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece. Electronic address: egounari@biohellenika.gr. 2. 2nd Department of Obstetrics and Gynecology, Hippokratio General Hospital, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece. 3. Biohellenika Biotechnology Company, Thessaloniki, Greece. 4. Biohellenika Biotechnology Company, Thessaloniki, Greece; Department of Biological Chemistry, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece. 5. Biohellenika Biotechnology Company, Thessaloniki, Greece; Department of Histology Embryology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece.
Abstract
BACKGROUND: Recent studies highlight the existence of a population of cord blood (CB)-derived stem cells that bare embryonic features (very small embryonic-like stem cells [VSELs]) as the most primitive CB-stem cell population. In the present study, we present for the first time a novel and high purity isolation method of VSELs with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). METHODS: The experimental procedure includes isolation upon gradually increased centrifugation spins and chemotaxis to Stromal cell-derived factor 1a (SDF-1a). Τhis cell population is characterized with flow cytometry, alkaline phosphatase (ALP) staining and qRT-PCR. The functional role of the isolated VSELs is assayed following co-culture with WJ-MSCs or bone marrow-derived mesenchymal stromal cells (BM-MSCs), whereas the stimulation of the quiescent VSEL population is verified via cell cycle analysis. The in vitro hematopoietic capacity is evaluated in methylcellulose cultures and also through induction of erythroid differentiation. RESULTS: The final isolated subpopulation is characterized as a small-sized CD45/Lineage-/CXCR4+/CD133+/SSEA-4+cell population, positive in ALP staining and overexpressing the Oct3/4, Nanog and Sox-2 transcription factors. Upon the co-culture with MSCs, a stimulation of the quiescent VSEL population is observed. An impressive increase in the co-expression of the CD34+/CD45+ markers is observed following the co-culture with the WJ-MSCs, which is confirmed by the intense clonogenic ability suggesting in vitro differentiation toward all of the hematopoietic cell lineages and successful differentiation toward erythrocytes. DISCUSSION: Conclusively, we propose a novel, rapid and rather simplified isolation method of CB-VSELs, capable of in vitro hematopoiesis.
BACKGROUND: Recent studies highlight the existence of a population of cord blood (CB)-derived stem cells that bare embryonic features (very small embryonic-like stem cells [VSELs]) as the most primitive CB-stem cell population. In the present study, we present for the first time a novel and high purity isolation method of VSELs with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). METHODS: The experimental procedure includes isolation upon gradually increased centrifugation spins and chemotaxis to Stromal cell-derived factor 1a (SDF-1a). Τhis cell population is characterized with flow cytometry, alkaline phosphatase (ALP) staining and qRT-PCR. The functional role of the isolated VSELs is assayed following co-culture with WJ-MSCs or bone marrow-derived mesenchymal stromal cells (BM-MSCs), whereas the stimulation of the quiescent VSEL population is verified via cell cycle analysis. The in vitro hematopoietic capacity is evaluated in methylcellulose cultures and also through induction of erythroid differentiation. RESULTS: The final isolated subpopulation is characterized as a small-sized CD45/Lineage-/CXCR4+/CD133+/SSEA-4+cell population, positive in ALP staining and overexpressing the Oct3/4, Nanog and Sox-2 transcription factors. Upon the co-culture with MSCs, a stimulation of the quiescent VSEL population is observed. An impressive increase in the co-expression of the CD34+/CD45+ markers is observed following the co-culture with the WJ-MSCs, which is confirmed by the intense clonogenic ability suggesting in vitro differentiation toward all of the hematopoietic cell lineages and successful differentiation toward erythrocytes. DISCUSSION: Conclusively, we propose a novel, rapid and rather simplified isolation method of CB-VSELs, capable of in vitro hematopoiesis.
Authors: Mariusz Z Ratajczak; Alison Domingues; Suman Suman; Alex R Straughn; Sham S Kakar; Malwina Suszynska Journal: Proc Stem Cell Res Oncog Date: 2019-06-17
Authors: Andrzej K Ciechanowicz; Katarzyna Sielatycka; Monika Cymer; Marta Skoda; Malwina Suszyńska; Kamila Bujko; Mariusz Z Ratajczak; Diane S Krause; Magdalena Kucia Journal: Cells Date: 2021-06-22 Impact factor: 6.600