Development of the Human Pancreas
Development of the Human Pancreas
Knowledge of human pancreas development underpins our interpretation and exploitation of human pluripotent stem cell (PSC) differentiation toward a β-cell fate. However, almost no information exists on the early events of human pancreatic specification in the distal foregut, bud formation, and early development. Here, we have studied the expression profiles of key lineage-specific markers to understand differentiation and morphogenetic events during human pancreas development. The notochord was adjacent to the dorsal foregut endoderm during the fourth week of development before pancreatic duodenal homeobox-1 detection. In contrast to the published data from mouse embryos, during human pancreas development, we detected only a single-phase of Neurogenin 3 (NEUROG3) expression and endocrine differentiation from approximately 8 weeks, before which Nirenberg and Kim homeobox 2.2 (NKX2.2) was not observed in the pancreatic progenitor cell population. In addition to revealing a number of disparities in timing between human and mouse development, these data, directly assembled from human tissue, allow combinations of transcription factors to define sequential stages and differentiating pancreatic cell types. The data are anticipated to provide a useful reference point for stem cell researchers looking to differentiate human PSCs in vitro toward the pancreatic β-cell so as to model human development or enable drug discovery and potential cell therapy.
The in vitro differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (IPSCs) to pancreatic β-cells is an ambitious hope for cell therapy in diabetes, a tool for mechanistic understanding of monogenic diabetes, and a potential platform for drug discovery to promote β-cell regeneration. Most stem cell differentiation protocols aim to mimic normal development by steering pluripotent cells sequentially through endoderm, foregut, pancreatic progenitor, and then endocrine cell fates. Comprehensive knowledge of this pathway has come from extensive analyses of laboratory model species, such as mouse, chick, and frog. This has demonstrated patterning of the foregut endoderm by transient proximity to the notochord that permits subsequent development of the dorsal pancreatic bud, with additional inductive influences provided by the dorsal aorta and other vasculature.
The intercellular signaling underlying these events and others has been mimicked as additives to the culture media during stem cell differentiation. In parallel, genetic inactivation in mouse has shown the sequential requirement for key transcription factors. Some of these factors, for which robust antibodies are available, have been used as phenotypic markers to indicate stages of β-cell differentiation from stem cells in vitro. Examples include SRY (sex determining region Y)-box-17 (SOX17), Gata-binding protein 4 (GATA4), forkhead box A2 (FOXA2), pancreatic and duodenal homeobox 1 (PDX1), two Nirenberg and Kim homeobox (NKX) factors, NKX6.1 and NKX2.2, and Neurogenin 3 (NEUROG3, also called NGN3). Nevertheless, if the goal is to differentiate human stem cells by mimicking human development, this approach contains an assumption; namely, that human embryogenesis is very similar, if not identical, to that which occurs in small mammalian, avian, or amphibian species.
Research on human embryos is limited by availability and appropriate ethical restrictions. Previous data mostly arise from studies initiated at 7–8 weeks of fetal development, by which time the two pancreatic buds have coalesced as a single organ, and NEUROG3 detection implies endocrine commitment has already started. Subsequent profiles of transcription factors have been reported that culminate in the development of human islets from ~12 weeks of fetal age. In stark contrast, knowledge of earlier events is exceptionally restricted; for instance, the detection of PDX1 in a single dorsal pancreatic bud at ~4 weeks of development and SOX9 transcripts by mRNA in situ hybridization soon after. Foregut development, the potential for patterning by nearby structures, and gene expression profiles during pancreatic bud formation, pancreatic growth, and early lineage differentiation has remained unexplored despite being essential for β-cell development. Here, we describe these early developmental events in a series of human embryos. The data help to minimize current interspecies assumptions made about stem cell differentiation, identify simple transcription factor combinations that signify differentiating pancreatic cell types, and provide a reference point for the validity of using stem cells at a particular stage of differentiation to model human developmental mechanisms in vitro.
Abstract and Introduction
Abstract
Knowledge of human pancreas development underpins our interpretation and exploitation of human pluripotent stem cell (PSC) differentiation toward a β-cell fate. However, almost no information exists on the early events of human pancreatic specification in the distal foregut, bud formation, and early development. Here, we have studied the expression profiles of key lineage-specific markers to understand differentiation and morphogenetic events during human pancreas development. The notochord was adjacent to the dorsal foregut endoderm during the fourth week of development before pancreatic duodenal homeobox-1 detection. In contrast to the published data from mouse embryos, during human pancreas development, we detected only a single-phase of Neurogenin 3 (NEUROG3) expression and endocrine differentiation from approximately 8 weeks, before which Nirenberg and Kim homeobox 2.2 (NKX2.2) was not observed in the pancreatic progenitor cell population. In addition to revealing a number of disparities in timing between human and mouse development, these data, directly assembled from human tissue, allow combinations of transcription factors to define sequential stages and differentiating pancreatic cell types. The data are anticipated to provide a useful reference point for stem cell researchers looking to differentiate human PSCs in vitro toward the pancreatic β-cell so as to model human development or enable drug discovery and potential cell therapy.
Introduction
The in vitro differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (IPSCs) to pancreatic β-cells is an ambitious hope for cell therapy in diabetes, a tool for mechanistic understanding of monogenic diabetes, and a potential platform for drug discovery to promote β-cell regeneration. Most stem cell differentiation protocols aim to mimic normal development by steering pluripotent cells sequentially through endoderm, foregut, pancreatic progenitor, and then endocrine cell fates. Comprehensive knowledge of this pathway has come from extensive analyses of laboratory model species, such as mouse, chick, and frog. This has demonstrated patterning of the foregut endoderm by transient proximity to the notochord that permits subsequent development of the dorsal pancreatic bud, with additional inductive influences provided by the dorsal aorta and other vasculature.
The intercellular signaling underlying these events and others has been mimicked as additives to the culture media during stem cell differentiation. In parallel, genetic inactivation in mouse has shown the sequential requirement for key transcription factors. Some of these factors, for which robust antibodies are available, have been used as phenotypic markers to indicate stages of β-cell differentiation from stem cells in vitro. Examples include SRY (sex determining region Y)-box-17 (SOX17), Gata-binding protein 4 (GATA4), forkhead box A2 (FOXA2), pancreatic and duodenal homeobox 1 (PDX1), two Nirenberg and Kim homeobox (NKX) factors, NKX6.1 and NKX2.2, and Neurogenin 3 (NEUROG3, also called NGN3). Nevertheless, if the goal is to differentiate human stem cells by mimicking human development, this approach contains an assumption; namely, that human embryogenesis is very similar, if not identical, to that which occurs in small mammalian, avian, or amphibian species.
Research on human embryos is limited by availability and appropriate ethical restrictions. Previous data mostly arise from studies initiated at 7–8 weeks of fetal development, by which time the two pancreatic buds have coalesced as a single organ, and NEUROG3 detection implies endocrine commitment has already started. Subsequent profiles of transcription factors have been reported that culminate in the development of human islets from ~12 weeks of fetal age. In stark contrast, knowledge of earlier events is exceptionally restricted; for instance, the detection of PDX1 in a single dorsal pancreatic bud at ~4 weeks of development and SOX9 transcripts by mRNA in situ hybridization soon after. Foregut development, the potential for patterning by nearby structures, and gene expression profiles during pancreatic bud formation, pancreatic growth, and early lineage differentiation has remained unexplored despite being essential for β-cell development. Here, we describe these early developmental events in a series of human embryos. The data help to minimize current interspecies assumptions made about stem cell differentiation, identify simple transcription factor combinations that signify differentiating pancreatic cell types, and provide a reference point for the validity of using stem cells at a particular stage of differentiation to model human developmental mechanisms in vitro.