Analysis of Differentiation Protocols Defines a Common Pancreatic Progenitor Molecular Signature and Guides Refinement of Endocrine Differentiation

Abstract

Several distinct differentiation protocols for deriving pancreatic progenitors (PPs) from human pluripotent stem cells have been described, but it remains to be shown how similar the PPs are across protocols and how well they resemble their in vivo counterparts. Here, we evaluated three differentiation protocols, performed RNA and assay for transposase-accessible chromatin using sequencing on isolated PPs derived with these, and compared them with fetal human pancreas populations. This enabled us to define a shared transcriptional and epigenomic signature of the PPs, including several genes not previously implicated in pancreas development. Furthermore, we identified a significant and previously unappreciated cross-protocol variation of the PPs through multi-omics analysis and demonstrate how such information can be applied to refine differentiation protocols for derivation of insulin-producing beta-like cells. Together, our study highlights the importance of a detailed characterization of defined cell populations derived from distinct differentiation protocols and provides a valuable resource for exploring human pancreatic development.

Islet organoid as a promising model for diabetes

“…They omitted BMP inhibitor treatment during the induction of pancreatic progenitors to prevent premature endocrine commitment, instead treating progenitors with the BMP inhibitor noggin to induce endocrine precursors, allowing spontaneous differentiation into ~20% monohormonal glucose-responding β-like cells in basal medium (Russ et al, 2015 ). However, omission of noggin during the early pancreatic progenitor step was recently found to induce the expression of CDX2, a marker of intestinal development, at the expense of losing NKX6.1 expression and the ability for further differentiation into endocrine cells (Wesolowska-Andersen et al, 2020 ). However, different cell lines were employed in these studies; therefore, the action of noggin could be a cell-specific effect, as also observed with other factors, including TPB or PdBU for PKC activation, and dorsomorphin or noggin for BMP inhibition (Kunisada et al, 2012 ; Rezania et al, 2012 ; Pagliuca et al, 2014 ; Nostro et al, 2015 ).…”

Human pluripotent stem cell-derived insulin-producing cells: A regenerative medicine perspective

“…These findings highlighted once more the importance of tight control of the differentiation cultures to avoid the generation of off-target cells. Indeed, Wesolowska-Andersen et al performed transcriptomic and epigenomic analyses of pancreatic progenitors derived in vitro using modified versions of three different differentiation protocols (Nostro et al, 2015;Rezania et al, 2014;Russ et al, 2015) to assess their similarity to one another and to embryonic development (Wesolowska-Andersen et al, 2020). Gene expression profiles of pancreatic progenitor cells generated with the Rezania et al (2014) and Nostro et al (2015) protocols showed significant similarity to pancreatic progenitor cells isolated from human fetal pancreas, suggesting that the timing and modulation of signaling pathways reported by these protocols indeed recapitulated several aspects of human pancreas development and limited the formation of off-target cells, like CDX2-expressing endoderm.…”

“…Gene expression profiles of pancreatic progenitor cells generated with the Rezania et al (2014) and Nostro et al (2015) protocols showed significant similarity to pancreatic progenitor cells isolated from human fetal pancreas, suggesting that the timing and modulation of signaling pathways reported by these protocols indeed recapitulated several aspects of human pancreas development and limited the formation of off-target cells, like CDX2-expressing endoderm. These studies constitute a great resource for the beta cell field, as they provide the transcriptional profiles of progenitor cells and their trajectory to beta-like cells using current differentiation protocols and can support hypothesis-driven experiments to improve differentiation protocols to generate specific cell types (Wesolowska-Andersen et al, 2020).…”

Harnessing Proliferation for the Expansion of Stem Cell-Derived Pancreatic Cells: Advantages and Limitations

“…These cells contain differentiation capacity towards all major pancreatic cell lineages (exocrine, ductal, and endocrine) and bypass the polyhormonal cell fate observed in C-peptideexpressing cells that have no detectable NKX6-1 (41)(42)(43)(44). A significant portion of end-stage pancreatic progenitor cells have been found to still contain the replication marker KI67 (~30%-50%) (5,45,46). Although KI67 expression is present, pancreatic progenitors demonstrate up-regulation of the cyclin dependent kinase (CDK) inhibitors p21 and p16, suggesting that these cells may be exiting the cell cycle (47).…”