USE OF SMALL MOLECULE SB4 AS A POTENTIAL ANALOG OF BMP4 IN DIFFERENTIATION PROTOCOLS INVOLVING HUMAN INDUCED PLURIPOTENT STEM CELLS

ROMANO FLORIT, CAROLINA; CHIRINO FELKER GONZALO; AMIN, GUADALUPE; SMUCLER, JOAQUIN; SEVLEVER, FEDERICO; WAISMAN ARIEL; CASTAÑEDA SHEILA; MIRIUKA SANTIAGO; MORO LUCIA; CAMILLETTI, MARIA ANDREA

Mar del Planta

Congreso; REUNIÓN CONJUNTA SAIC SAB AAFE AACYTAL 2023; 2023

SOCIEDAD ARGENTINA DE INVESTIGACION CLINICA

Bone morphogenetic protein-4 (BMP4) mediates essential signalling pathways in the early steps ofhuman development. Recombinant BMPs have been synthesised for in vitro studies and therapeuticpurposes, but they are very costly and display low stability. In this context, a small molecule knownas Sb4 was identified as a potential analog of BMP4 in human renal cells. Since this has not beenstudied in human induced Pluripotent Stem Cells (hiPSCs), we set out to evaluate the ability of Sb4to activate the BMP signalling pathway in hiPSCs and induce differentiation toward the mesodermallineage. First, the cytotoxicity of Sb4 in hiPSCs was determined by calculating the percentage ofliving cells after 24h of Sb4 treatment using Trypan Blue and Neubauer’s chamber. Forconcentrations 0.01-50uM, cell viability was above 80%, whereas 100uM caused it to drop below30%. Next, for mesoderm-differentiation assays, hiPSCs were incubated in RPMI supplemented withActivin(A)+rBMP4 or A+Sb4(10 uM) for 72hrs, and RNA samples were collected for RT-qPCR.Results revealed an upregulation of mesoderm-associated genes (TBXT,EOMES,MIXL1) and adownregulation of pluripotency genes (OCT4,NANOG) in both conditions. To explore if Sb4 effectsare partly mediated by a SMADs-dependent BMP pathway, SMAD1 expression was evaluated bywestern blot in hiPSCs treated with Sb4 alone or in combination with rBMP4. Interestingly, SMAD1increased after Sb4 treatment, and this effect was more significant for the combined group (vs.controlhiPSCs). Additionally, preliminary results showed positive pSMAD1/5 immunostaining in hiPSCs after30’ treatment with Sb4, mirroring the response with rBMP4, suggesting an activation of the pathway.Finally, biological BMP4 targets ID1 and ID3 were upregulated after 24h of Sb4 treatment. To sumup, our findings indicate that Sb4 activates the BMP pathway in hiPSCs, and might be used as acheaper alternative to rBMP4 for hiPSCs differentiation towards mesodermal lineages.