New markers for tracking endoderm induction and hepatocyte differentiation from human pluripotent stem cells

Abstract

The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population, broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study, we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach, we identified two endoderm-specific antibodies, HDE1 and HDE2, which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential, whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination, the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line.

Single-cell time-series mapping of cell fate trajectories reveals an expanded developmental potential for human PSC-derived distal lung progenitors

“…Given these results, detailed mapping of the developmental path or paths that progenitor cells take during differentiation to their end state or fate both in vivo as well as in PSC-derived systems has now become a primary objective of the field. Similar challenges in obtaining pure cell populations from PSC differentiation protocols were also recently observed for other tissue types such as the renal epithelium (Holtzinger et al, 2015;Schwartzentruber et al, 2018;Wu et al, 2018).…”

Activation of Wnt/β-catenin signalling via GSK3 inhibitors direct differentiation of human adipose stem cells into functional hepatocytes

“…In the liver development, definitive endoderm specification is the essential early and the most important step to generate of hepatocytes. Thus, a better understanding and control of the definitive endoderm differentiation process in vitro should result in enhanced efficiency and higher fidelity in the resulting cells67. The efficient and reproducible production of definitive endoderm is dependent on our ability to recapitulate key stages of embryonic lineage development in differentiation cultures.…”

Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

“…While the studies mentioned above have all utilized monolayer culture, embryoid body (EB)-based protocols are also commonly used (Craft et al, 2015;Holtzinger et al, 2015;Kattman et al, 2011;Lee et al, 2017;Protze et al, 2016;Witty et al, 2014). EB-based methods for mesoderm induction (cardiac myocytes or chondrocytes) utilize one day of BMP4 treatment followed by an additional two days of BMP4, Activin A and FGF2 treatment (Craft et al, 2015;Kattman et al, 2011;Lee et al, 2017;Protze et al, 2016;Witty et al, 2014).…”

“…The specific concentration of growth factors is dependent on the targeted cell lineage and the cell line used for induction. Definitive endodermderived lineages, including pancreatic cells and hepatocytes, can also be produced from EBs using a similar growth factor cocktail, albeit with higher levels of Activin A (Holtzinger et al, 2015;Nostro et al, 2011).…”

“…ng/mL (Bernardo et al, 2011;Brown et al, 2011;Holtzinger et al, 2015;Nostro et al, 2015). For induction of mesoderm, much lower levels of both Activin A and BMP4 have been utilized (1-10 ng/mL, depending on the cell line and target lineage; (Craft et al, 2015;Kattman et al, 2011;Lee et al, 2017;Protze et al, 2016;Witty et al, 2014)).…”