Abstract 52: Isolation, Characterization and Partial Direct Differentiation of Patient Derived Human Primary Cardiac and Placental Fibroblasts From Surgical Discard Tissue into Induced Mesoderm Progenitor Cell

Abstract

Background: Human primary cells more closely mimic the physiological state of cells in vivo and generate more relevant data representing living systems. Access to the surgical discard tissue enables the possibility of patient derived, disease specific, cellular bio-bank. In achieving this goal, target cellular lines must be isolated, characterized and fully expanded under specific Standard Operating Procedures(SOP). In this project, we aim in achieving and implementing such goal for Fibroblasts derived from two distinct patient tissues. In parallel, in light of cardiac regenerative therapy, with reference to the published work on Cardiomyocyte Direct Differentiation on mouse fibroblasts into induced cardiomyocyte-like cells (iCMs) by transduction with MicroRNAs (1,133a, 208a and 499) we sought to determine whether primary human cardiac and placental fibroblasts could have the potential in direct differentiation to induced cardiomyocyte-like cells (iCM) through sequential administration of small molecule (ITD-1) in combination with MicroRNAs. Methods and Results: Primary right atrial appendage cardiac and smooth chorion laeve placental fibroblast isolates were generated from surgical discard patient derived tissues subjected for identification of the most efficient isolation method through available enzymatic and mechanical techniques. We have shown through FACS analysis that both groups extracellular marker expression can be limited to CD90+CD31-CD44+FBSP+ which constitutes the majority of cultured primary fibroblasts. Respectively, intercellular expression of DDR2+ VIM+ COL1+CD90+ markers was validated through immunofluorescence microscopy. Transcriptional profiling of Noted lines showed expression of Fibroblast specific genes at higher rate.Furthermore, sorted placental fibroblast isolates were subjected to (ITD-1)treatment and mature MicroRNA mimic cocktail. Transcriptional profiling analysis through RT-PCR concluded down regulation of most distinct fibroblast markers and partial expression of MESP1.