Development of a rapid screen for the endodermal differentiation potential of human pluripotent stem cell lines

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).

Rapid Screening of the Endodermal Differentiation Potential of Human Pluripotent Stem Cells

“…Importantly, in the first 24 hr there is little to no migration of cells out of the colonies. In our previous studies, we demonstrated that cells at day 1 represent a mesendodermal/primitive streak (PS) identity, corroborated by expression of PS-specific markers such as FOXA2, CER1, NODAL, GSC, and T Siller et al, 2015;Siller et al, 2016). At the endpoint of the EP screen (day 2), successful lines and conditions are evidenced by a monolayer of cells exhibiting a cobblestone-or petal-like morphology (See Figs.…”

A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates

“…H1 and H9 were obtained from WiCell Research Institute (Madison, Wisconsin, WiCell) and well characterized as previously described 1 . Detroit 551-A fibroblasts were obtained from ATCC (American Type Culture Collection), AG05836B-15 fibroblasts were obtained from the Coriell Cell Repositories and reprogramed into hiPSCs and characterized as previously described in Mathipathi et al 19 , 20 .…”

Direct On-Chip Differentiation of Intestinal Tubules from Induced Pluripotent Stem Cells

“…For the differentiation of the iPSC cells towards definitive endoderm in the OrganoPlate, we used a short pulse of small molecule Wnt activator CHIR99021 instead of the recombinant Activin A molecule [ 17 ] based protocol. GSK-3 inhibition by CHIR99021 alone is sufficient for the generation of SOX17 and FOXA2 positive cells [ 19 , 47 ]. This step shortens the definitive endoderm phase from 5 days to 2 days.…”

“…They usually follow a step wise addition of growth factors or small molecules to mimic intestinal morphogenesis and cytodifferentiation. In the first step definitive endoderm (DE) is usually acquired by applying an activin or Wnt-based protocol [ 18 , 19 ]. In the second step the culture is directed towards hindgut specification through FGF/Wnt induced posterior endoderm patterning [ 20 , 21 , 22 ].…”