ROCKII inhibition promotes the maturation of human pancreatic beta-like cells

Abstract

Diabetes is linked to loss of pancreatic beta-cells. Pluripotent stem cells offer a valuable source of human beta-cells for basic studies of their biology and translational applications. However, the signalling pathways that regulate beta-cell development and functional maturation are not fully understood. Here we report a high content chemical screen, revealing that H1152, a ROCK inhibitor, promotes the robust generation of insulin-expressing cells from multiple hPSC lines. The insulin expressing cells obtained after H1152 treatment show increased expression of mature beta cell markers and improved glucose stimulated insulin secretion. Moreover, the H1152-treated beta-like cells show enhanced glucose stimulated insulin secretion and increased capacity to maintain glucose homeostasis after transplantation. Conditional gene knockdown reveals that inhibition of ROCKII promotes the generation and maturation of glucose-responding cells. This study provides a strategy to promote human beta-cell maturation and identifies an unexpected role for the ROCKII pathway in the development and maturation of beta-like cells.

Single-Cell RNA-Seq Reveals that CD9 Is a Negative Marker of Glucose-Responsive Pancreatic β-like Cells Derived from Human Pluripotent Stem Cells

“…Although current protocols produce human b-like cells with some functionality (Pagliuca et al, 2014;Rezania et al, 2014;Russ et al, 2015), the differentiation efficiency varies greatly among different hPSC lines. For instance, most studies utilize HUES8 (Pagliuca et al, 2014;Veres et al, 2019) or H1 (Ghazizadeh et al, 2017;Rezania et al, 2014) hESCs, which have been shown to be efficient in b cell lineage commitment. The same protocol, when applied to other cell lines, including patient-specific iPSCs, could result in poor b cell specification (Ghazizadeh et al, 2017).…”

“…Modifications of these stepwise pathways facilitate the generation of neurogenin 3 (NGN3 + ) and/or neurogenic differentiation factor 1 (NEUROD1 + ) endocrine progenitor cells (Rezania et al, 2014), as well as monohormonal endocrine cells, including insulin-expressing b-like cells that are glucose responsive in vitro (Pagliuca et al, 2014;Rezania et al, 2014). More recently, Rho-associated coiled-coil containing protein kinase 2 (ROCKII) inhibition has also been found to promote the maturation of hPSC-derived b-like cells (Ghazizadeh et al, 2017).…”

“…This might attenuate the therapeutic potential of patient-specific induced PSCs (iPSCs), as we and others have demonstrated that their efficiency in lineage commitment varies greatly among different hiPSC lines (Chetty et al, 2015;Sahara et al, 2014;Xu et al, 2012). Moreover, the generated b-like cells express fewer maturation markers, such as musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA) and insulin, compared with human islets (Ghazizadeh et al, 2017;Johnson, 2016;Millman and Pagliuca, 2017). Nevertheless, identification of specific cell-surface markers that enables the purification of mature and functional human b-like cells for transplantation awaits urgent investigations.…”

A high-content screen identifies the vulnerability of MYC-overexpressing cells to dimethylfasudil

“…diMF has been extensively studied as a ROCK inhibitor and characterized in pre-clinical models. It promotes generation of pancreatic beta-like cells from human pluripotent stem cells [30], augments neurite extension [31], enhances proliferation and migration of endothelial progenitor cells [32], and triggers cell-cycle arrest and cellular senescence of mouse embryo fibroblasts [33]. However, ROCKindependent activity has also been ascribed to diMF.…”

Hydrogel platform for in vitro three-dimensional assembly of human stem cell-derived β cells and endothelial cells

“…Several reports by us and others have detailed approaches for making insulin-producing β-like cells from human embryonic stem cells (hESCs) with the goal that these cells could be used for both cell replacement therapy and drug screening for diabetes [3][4][5][6][7][8]. These hESC-derived β (SC-β) cells are capable of undergoing glucose-stimulated insulin secretion and express markers found in β cells.…”