Abstract
A challenge facing the human pluripotent stem cell (hPSC) field is the variability observed in differentiation potential of hPSCs. Variability can lead to time consuming and costly optimisation to yield the cell type of interest. This is especially relevant for the differentiation of hPSCs towards the endodermal lineages. Endodermal cells have the potential to yield promising new knowledge and therapies for diseases affecting multiple organ systems, including lung, thymus, intestine, pancreas and liver, as well as applications in regenerative medicine and toxicology. Providing a means to rapidly, cheaply and efficiently assess the differentiation potential of multiple hPSCs is of great interest. To this end, we have developed a rapid small molecule based screen to assess the endodermal potential (EP) of hPSCs, based solely on definitive endoderm (DE) morphology. This drastically reduces the cost and time to identify lines suitable for use in deriving endodermal lineages. We demonstrate the efficacy of this screen using 10 different hPSCs, including 4 human embryonic stem cell lines (hESCs) and 6 human induced pluripotent stem cell lines (hiPSCs). The screen clearly revealed lines amenable to endodermal differentiation, and only lines that passed our morphological assessment were capable of further differentiation to hepatocyte like cells (HLCs).
Highlights
With human embryonic stem cell derived retinal pigmented epithelium[14,15]
It has been reported that insulin/PI3K signaling can be inhibitory with respect to the efficient production of DE8
A critical challenge that faces the human pluripotent stem cells (hPSCs) field is the issue of cell line variability with respect to differentiation potential
Summary
With human embryonic stem cell (hESC) derived retinal pigmented epithelium[14,15]. To date a plethora of hESC lines and more recently hiPSC lines have been derived in order to dissect disease[13]. With respect to the endodermal lineage, the Yamanaka group observed that hiPSC lines derived from a number of different donors demonstrated a high degree of variability in terms of their capacity to differentiate to hepatocytes[25] This highlights the necessity for efficient, unambiguous, cost effective and rapid methods to assess if a line is up to the job, with respect to differentiation potential. By the end of second day, there has been extensive cellular migration and proliferation, with the cells taking on a typical petal/cobblestone like morphology These observed morphological changes are concomitant with dramatic transcriptional change, including the rapid induction of NODAL within 4 hours of administration of CHIR99021, demonstrating a transition towards Primitive Streak (PS). We report a simple, robust, cost effective and rapid screen capable of assessing multiple hPSC lines for their EP purely by morphology
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