Isolation of Swine Bone Marrow Lin-/CD45-/CD133 + Cells and Cardio-protective Effects of its Exosomes

Abstract

Background: The identification in murine bone marrow (BM) of CD133 + /Lin-/CD45- cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the swine BM. Heart failure is the terminal stage of many cardiovascular diseases, and its key pathological basis is cardiac fibrosis (CF). Research showed that stem cell derived exosomes may play a critical role in cardiac fibrosis. The effect of exosomes (Exos) on CF has remained unclear. Objective: To establish an isolation and amplification method of CD133 + /Lin-/CD45- cells from newbron swine BM in vitro, explore an highly efficient method to enrich swine bone marrow derived CD133 + /Lin-/CD45- cells and probe into their biological characteristics further. Furher more, to extract exosomes from it and explore its effect on CF. Methods: The mononuclear cells isolated from swine bone marrow by red blood cell (RBC) lysing buffer were coated by adding FcR blocking solution and coupled with CD133 antibody immunomagnetic beads, obtaining CD133 + cell group via Magnetic Activated Cell Sorting (MACS). In steps, the CD133 + /Lin-/CD45- cells were collected by fluorescence-activated cell sorting (FACS) labeled with CD133, Lin and CD45 antibodies, which were cultured and amplified in vitro. The biological features of CD133 + /Lin-/CD45- cells were studied in different aspects, including morphological trait observed with inverted microscope, ultrastructural characteristics observed under transmission electron microscope, expression of pluripotent markersidentified by immunofluorescent staining and Alkaline phosphatase staining. The Exos were extracted using a sequential centrifugation approach and its effects on CF were analyzed in Angiotensin II (Ang-II) induced-cardiac fibrosis in vivo. Rats in each group were treated for 4 weeks, and 2D echocardiography was adopted to evaluate the heart function. The degree of cardiac fibrosis was assessed by Hematoxylin–Eosin (HE) and Masson's trichrome staining. Results: The CD133 + /Lin-/CD45- cells accounted for about 0.2%-0.5% of the total mononuclear cells isolated from swine bone marrow. The combination of MACS and FACS to extract CD133 + /Lin-/CD45- cells could improved efficiency and reduced cell apoptosis. The CD133 + /Lin-/CD45- cells featured typical traits of pluripotent stem cells, the nucleus is large, mainly composed of euchromatin, with less cytoplasm and larger nucleoplasmic ratio, which expressed pluripotent markers (SSEA-1, Oct-4, Nanog and Sox-2) and alkaline phosphatase staining was positive.Animal experiment indicated that the cardiac injury related indexes (BNP、cTnI、CK-MB and TNF-α), the expression of key gene Smad3 and the degree of cardiac fibrosis in Exo treatment group were significantly reduced compared with the control group. 4 weeks after the treatment, cardiac ejection fraction (EF) value in the model group showed a remarkable decrease, indicating the induction of HF model. While Exo elevated the EF values, demonstrating cardio-protective effects. Conclusion: The CD133 + /Lin-/CD45- cells derived from swine bone marrow were successfully isolated and amplified, laying a good foundation for further research on this promising therapeutic cell. The Exos may be a promising potential treatment strategy for CF. Graphical Abstract: [Figure not available: see fulltext.]