A high-content small molecule screen identifies novel inducers of definitive endoderm

Abstract

ObjectivesHuman embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can generate any given cell type in the human body. One challenge for cell-replacement therapy is the efficient differentiation and expansion of large quantities of progenitor cells from pluripotent stem cells produced under good manufacturing practice (GMP). FOXA2 and SOX17 double positive definitive endoderm (DE) progenitor cells can give rise to all endoderm-derived cell types in the thymus, thyroid, lung, pancreas, liver, and gastrointestinal tract. FOXA2 is a pioneer transcription factor in DE differentiation that is also expressed and functionally required during pancreas development and islet cell homeostasis. Current differentiation protocols can successfully generate endoderm; however, generation of mature glucose-sensitive and insulin-secreting β-cells is still a challenge. As a result, it is of utmost importance to screen for small molecules that can improve DE and islet cell differentiation for cell-replacement therapy for diabetic patients.MethodsThe aim of this study was to identify and validate small molecules that can induce DE differentiation and further enhance pancreatic progenitor differentiation. Therefore, we developed a large scale, high-content screen for testing a chemical library of 23,406 small molecules to identify compounds that induce FoxA2 in mouse embryonic stem cells (mESCs).ResultsBased on our high-content screen algorithm, we selected 84 compounds that directed differentiation of mESCs towards the FoxA2 lineage. Strikingly, we identified ROCK inhibition (ROCKi) as a novel mechanism of endoderm induction in mESCs and hESCs. DE induced by the ROCK inhibitor Fasudil efficiently gives rise to PDX1+ pancreatic progenitors from hESCs.ConclusionTaken together, DE induction by ROCKi can simplify and improve current endoderm and pancreatic differentiation protocols towards a GMP-grade cell product for β-cell replacement.

NOTO Transcription Factor Directs Human Induced Pluripotent Stem Cell-Derived Mesendoderm Progenitors to a Notochordal Fate

“…The results confirmed the presence of T+/SOX9+ NLC up to day 7 when NOTO was transfected (7.6%, Figure 5C,D and Figure S1). In the course of hiPSCs differentiation, FOXA2+ is also indicative of the presence of nascent definitive endoderm cells, which co-express SOX17 at early stages [51,52]. Consistent with our RT-qPCR results, FOXA2-transfected cells had a propensity to differentiate into definitive endoderm cells as compared to those transfected with NOTO (45.6% and 26.2% of FOXA2+/SOX17+ cells at day 7 respectively; Figure 5C,D and Figure S1).…”

Emergence and patterning dynamics of mouse definitive endoderm

“…Limiting cell movement, by inhibition of Rho-associated protein kinase (ROCK), an important controller of actin microfilaments, did not affect the differentiation to Sox17+ cells nor their proliferation, regardless of the timing of inhibition. It was previously shown that ROCK inhibition and cytoskeletal reorganization can mediate activation of Wnt signaling and the induction of EMT, and can affect cell differentiation in vitro [33][34][35]. While ROCK inhibition early in differentiation may induce EMT and mesendoderm differentiation and by that enhance Sox17 expression later on, the emergence of a similar fraction of Sox17+ cells when ROCKi is added later in differentiation indicates that Sox17 expression does not require cell movement.…”

Small Molecule-Induced Pancreatic β-Like Cell Development: Mechanistic Approaches and Available Strategies

“…They can efficiently promote cell differentiation and govern other cellular activities like common promoters and growth factors which are expensive, and can be harmful. Small molecules are cell permeable chemical compounds which are generally safer, inexpensive, pure, amenable to scaling up, and are devoid of animal derived products which makes them more suitable candidates to be used in in vitro practices [29][30][31][32]. The main motivation behind using small molecules is that they can be synthesized in large amounts with higher purity and stored in a way that ensures their reproducible activity [30].…”

“…Small molecules are cell permeable chemical compounds which are generally safer, inexpensive, pure, amenable to scaling up, and are devoid of animal derived products which makes them more suitable candidates to be used in in vitro practices [29][30][31][32]. The main motivation behind using small molecules is that they can be synthesized in large amounts with higher purity and stored in a way that ensures their reproducible activity [30]. Small molecules have been efficiently used in differentiation processes, as they regulate cellular processes by inflecting signal transduction pathways, metabolism, or gene expression.…”

“…CHIR99021 is preferred over WNT3A, due to its high efficiency, stability, and cost, for the differentiation of stem cells into DE [19]. MCX-928 has also been used as GSK3βi to induce DE differentiation [23,30].…”