Optimising methods for stem cell factor production using an in vitro cell culture system

Abstract

Stem cell factor (SCF) is a cytokine which plays an important role in the survival, propagation and self-renewal of haematopoietic stem cells (HSCs). It is therefore one of the important factors used when culturing haematopoietic erythroid progenitor cells (HEPs) in vitro. Culturing of HEPs is used for a multitude of purposes, including for research involving globin gene switching in patients with haemoglobinopathies, to induce functional genomic changes that give rise to increased foetal haemoglobin production, which can be used as an alternative form of treatment. Replacing commercially bought SCF with an in-house SCF producing method can accelerate ongoing local research regarding globin gene switching in cases of haemoglobinopathies. This can be achieved through in vitro cell culture systems of genetically altered cell lines such as HEK293 and cells which naturally produce SCF. In this study, HEK293 cells were transfected with an SCF vector (pMD18-T) using different chemical methods, and the conditioned medium was extracted at different time points. qPCR was performed on HEK293 cells to quantify SCF mRNA. Enzyme linked immunosorbent assay (ELISA) was performed on conditioned media from the HEK293 cells as well as from available mesenchymal stem cells (MSCs). The conditioned medium from MSCs were readily provided from a separate project. Findings included a modest increase in SCF, with the highest yields being found in the HEK293 cells. mRNA values from qPCR did not correlate with SCF concentrations from ELISA readings, which may possibly be explained by the complex differences in which transcriptional and translational control mechanisms function within the cell, however, did serve to show and validate overexpressed SCF.