Human Muse cells genetically-modified by baculoviral vector for therapeutic angiogenesis

GIMÉNEZ, CARLOS SEBASTIÁN; CROTTOGINI ALBERTO; CASTILLO MARTHA GIOVANNA; BELAICH MARIANO NICOLÁS; CUNIBERTI LUIS; SIMONIN JA; OLEA FERNANDA DANIELA

Mar del Plata

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2019

SAIC-SAFIS

Muse cells are endogenous repair stem cells identified by SSEA3+. If Muse cells are not enough, the administration of exogenous Muse cells offers a prominent functional recovery. However, genetically improved Muse cells have not yet been developed.The objective was to generate human Muse (hMuse) cells that overexpress the double mutated HIF-1alpha gene through a baculoviral vector, in order to improve its angiogenic capacity.Methods. hMuse were isolated from abdominal lipoaspirates using severe cellular stress procedure. Mesenchymal stem cells (ASC´s) were harvested from matched donor samples. Recombinant AcMNPV baculovirus expressing the green fluorescent protein (Bv.CMV-GFP) or the therapeutic gene (Bv.CMV-dmHIF-alpha) were developed. The efficiency transduction (TE) of the different multiplicities of infection (MOI): 0, 25, 50, 100, 200 and 400 were determined with Bv.CMV-GFP vector by cell counting using fluorescence microscopy. The selected MOI was subsequently used to transduce hMuse with the Bv.CMV-dmHIF-1alpha vector under the same conditions. Comparative gene expression analysis for angiogenic factors and S1P receptor 2 (S1PR2) was performed by RTqPCR. Results. TE obtained for each MOI was 0%, 17.3%, 24.7%, 47.8%, 58.6% and 59.8%, respectively. The MOI 200 was selected because it yielded an ET of 58.6%. Although an MOI 400 increased TE to almost 60%, we did not consider that this increase justified duplicating the viral load of the cells. Using MOI 200, HIF-1alpha gene expression increased 5.9 fold in hMuse and 2555 fold in hMuse-HIF compared to its expression in ASCs. VEGF and FGF expression showed similar behavior; VEGF 3.3 and 7.9 and FGF 3.5 and 5.9 fold increases, respectively. Expression of S1PR2 (1.5 and 1.1) showed no changes between groups. Conclusion. hMuse cells could be effectively transduced with a baculoviral vector doubling their angiogenic potential without altering their migratory capacity mediated by S1PR2.