Swine Bone Marrow Derived Multipotent Adult Progenitor Cells

We have previously reported that multipotent adult progenitor cells (MAPCs) can be isolated from the bone marrow of rodents and humans. Here we describe isolation of similar cells, fetal swine MAPCs (fsMAPCs) from fetal swine bone marrow (BM). BM was obtained from 5 fetal swine donors. BM mononuclear cells (BMMNCs) were plated into fibronectin-coated T150 flasks at 2x104 cells/cm2 in 58% LG-DMEM and 40% MCDB-201, supplemented with ITS, LA-BSA, 10−8 M dexamethasone, 10−4 M ascorbic acid 2-phosphate, 2% fetal bovine serum (FBS), 10ng/ml EGF and 10ng/ml PDGF-BB. Small clusters of cells appeared by day 5, were detached with 0.25% trypsin-EDTA on day 7 and sub-cloned at 1 cell/well in 96 well plates. Once populations of small cells appeared (40% of wells), they were culture expanded by plating at 500 cells/cm2. Cells were replated at 500cells/cm2 once they divided 2–3 times. Population doubling (PD) time was 24h for the initial 30–40 PDs, and 48 h at higher PDs. 3/5 fetal swine populations have been maintained for more than 100 PDs without cytogenetic abnormalities. For two of the populations, telomere length was evaluated on day-0, 40PDs and 80PDS, and no telomere shortening was seen. The phenotype of cultured cells before 30 PDs is CD45 and MHC class I negative, CD44 and MHC class II positive, while the phenotype after 60 PDs was CD44, CD45, MHC class I and II negative. FsMAPCs, cultured on Matrigel with fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), differentiated into epitheloid cells that expressed cytokeratin 18 (CK18), hepatocyte nuclear factor-1 (HNF-1), albumin protein on day 14–28. Cells differentiated for 21 days secreted 10pg/cell/hour urea (Urea Production Test), as is seen for primary hepatocytes. More functional tests for hepatocytes are ongoing, including LDL uptake, glycogen storage, cytochrome-p450 activity. When treated with vascular endothelial growth factor (VEGF), fsMAPCs differentiated into endothelium-like cells that expressed CD31, CD34, VEGF-Receptor, vascular endothelial cadherin (VE-Cadherin) and von willebrand factor (VWF) protein and mRNA at day 14–28, formed vascular tube on extracellular matrix and took up LDL. Neuroectoderm differentiation studies are ongoing. Finally, we have preliminary evidence that fsMAPCs may differentiate into cells with cardiomyocyte markers. FsMAPCs, plated at 1,500 cells/cm2, were cultured with dickkopf-1 (Dkk-1) and 0.75% dimethyl sulfoxide (DMSO) for 1 day, followed by bone morphogenetic protein-2 (BMP-2), FGF-8, FGF-4, Dkk-1 for 21 days. Cells acquired muscle morphology after d6. By day 21, cells expressed cardiac Troponin T, cardiac myosin heavy chain (cMHC), phospholamban protein, and Nkx2.5, Cardiac Troponin T, cMHC, GATA-4, atrial natriuretic peptide (ANP) mRNA. Although suggestive that the cells may differentiate into cardiomyocytes, they did not beat regularly. Electrophysiology studies are ongoing to determine whether the cells have functional characteristics of cardiomyocytes.