EPISOMAL VECTOR TRANSFECTION OPTIMIZATION FOR UMBILICAL CORD MESENCHYMAL STROMAL CELL (UC-MSC) REPROGRAMMING: VOLTAGE, PULSE LENGTH, AND SUPPORTING REAGENT

• Main Author: Tedjobagaskara, Bintang
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/80056
• Institution: Institut Teknologi Bandung

Induced Pluripotent Stem Cell (iPSC) is a product of cellular reprogramming from which pluripotency could be achieved without the use of bioethically issued embryonic harvesting. Episomal vector is one of the reprogramming methods of somatic cell such as umbilical cord mesenchymal stromal cell (UC-MSC) that is cost-effective, easy to reproduce, and could generate clinical grade iPSC but the reprogramming efficiency is relatively low. Transfection efficiency can be improved by optimizing the electroporation parameters and by doing so the reprogramming efficiency is expected to also improve. This experiment aims to observe the optimum voltage and pulse length to transfect UCMSC, also to observe the optimum supporting reagents to accompany the transfected cells. This experiment is done by transfecting UC-MSC with pCMV-eGFP using a variety of voltages (200 V, 250 V, 300 V, 400 V, and 500 V) supported with reagents (CloneR and P188). After 5 days the cells are counted and GFP expressions are read. Then the optimization of pulse length and supporting reagent is conducted by the same procedur with the variety of pulse lengths (7.5 ms, 8 ms, 8.5 ms, 9 ms, and 9.5 ms) accompanied by CloneR, P188, and Rock(Y) inhibitor. Furthermore, the episomal reprogramming of UC-MSC is performed using optimized electroporation parameters and reprogramming markers are read. Result shows that UC-MSC transfected with 200 V for 8.5 ms supported by CloneR generates highest viability and GFP-labelled cell population. Even though a complete iPSC is not yet to be generated, the observed morphological change indicates that a reprogramming process happened. Therefor the observed optimum voltage is 200 V, and the optimum pulse length is 8.5 ms while CloneR is the optimum supporting reagent.