Seven days in the life cycle of Homo sapiens.

The second week of embryonic development is a critical phase of the human life cycle and one that has been largely inaccessible to scientific investigation. Recent studies of human embryo models built from stem cells promise to yield dramatic insights into the key events of cell specification and morphogenesis that occur during this brief window of embryogenesis.

An ethical framework for human embryology with embryo models.

A human embryo's legal definition and its entitlement to protection vary greatly worldwide. Recently, human pluripotent stem cells have been used to form in vitro models of early embryos that have challenged legal definitions and raised questions regarding their usage. In this light, we propose a refined legal definition of an embryo, suggest "tipping points" for when human embryo models could eventually be afforded similar protection to that of embryos, and then revisit basic ethical principles that might help to draft a roadmap for the gradual, justified usage of embryo models in a manner that aims to maximize benefits to society.

Germline competent embryonic stem cells derived from rat blastocysts.

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.

A case for revisiting Nodal signaling in human pluripotent stem cells.

Nodal is a transforming growth factor-ß (TGF-ß) superfamily member that plays a number of critical roles in mammalian embryonic development. Nodal is essential for the support of the peri-implantation epiblast in the mouse embryo and subsequently acts to specify mesendodermal fate at the time of gastrulation and, later, left-right asymmetry. Maintenance of human pluripotent stem cells (hPSCs) in vitro is dependent on Nodal signaling. Because it has proven difficult to prepare a biologically active form of recombinant Nodal protein, Activin or TGFB1 are widely used as surrogates for NODAL in hPSC culture. Nonetheless, the expression of the components of an endogenous Nodal signaling pathway in hPSC provides a potential autocrine pathway for the regulation of self-renewal in this system. Here we review recent studies that have clarified the role of Nodal signaling in pluripotent stem cell populations, highlighted spatial restrictions on Nodal signaling, and shown that Nodal functions in vivo as a heterodimer with GDF3, another TGF-ß superfamily member expressed by hPSC. We discuss the role of this pathway in the maintenance of the epiblast and hPSC in light of these new advances.

A Splicing Mutation in Slc4a5 Results in Retinal Detachment and Retinal Pigment Epithelium Dysfunction.

Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood-retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in tvrm77 mice, which exhibit hypopigmented patches in the central retina. Mapping and nucleotide sequencing of tvrm77 mice revealed a disrupted 5' splice donor sequence in Slc4a5, a sodium bicarbonate cotransporter gene. Slc4a5 expression was reduced 19.7-fold in tvrm77 RPE relative to controls, and alternative splice variants were detected. SLC4A5 was localized to the Golgi apparatus of cultured human RPE cells and in apical and basal membranes. Fundus imaging, optical coherence tomography, microscopy, and electroretinography (ERG) of tvrm77 mice revealed retinal detachment, hypopigmented patches corresponding to neovascular lesions, and retinal folds. Detachment worsened and outer nuclear layer thickness decreased with age. ERG a- and b-wave response amplitudes were initially normal but declined in older mice. The direct current ERG fast oscillation and light peak were reduced in amplitude at all ages, whereas other RPE-associated responses were unaffected. These results link a new Slc4a5 mutation to subretinal fluid accumulation and altered light-evoked RPE electrophysiological responses, suggesting that SLC4A5 functions at the outer blood-retinal barrier.

Human embryo research and the 14-day rule.

In many jurisdictions, restrictions prohibit the culture of human embryos beyond 14 days of development. However, recent reports describing the successful maintenance of embryos in vitro to this stage have prompted many in the field to question whether the rule is still appropriate. This Spotlight article looks at the original rationale behind the 14-day rule and its relevance today in light of advances in human embryo culture and in the derivation of embryonic-like structures from human pluripotent stem cells.

The pluripotent state in mouse and human.

In the mouse, naïve pluripotent stem cells (PSCs) are thought to represent the cell culture equivalent of the late epiblast in the pre-implantation embryo, with which they share a unique defining set of features. Recent studies have focused on the identification and propagation of a similar cell state in human. Although the capture of an exact human equivalent of the mouse naïve PSC remains an elusive goal, comparative studies spurred on by this quest are lighting the path to a deeper understanding of pluripotent state regulation in early mammalian development.

What if stem cells turn into embryos in a dish?

Recent studies show that pluripotent stem cells can undergo self-organized development in vitro into structures that mimic the body plan of the post-implantation embryo. Modeling human embryogenesis in a dish opens up new possibilities for the study of early development and developmental disorders, but it may also raise substantial ethical concerns.

Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system.

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3ß and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.

Extrinsic regulation of pluripotent stem cells.

During early mammalian development, as the pluripotent cells that give rise to all of the tissues of the body proliferate and expand in number, they pass through transition states marked by a stepwise restriction in developmental potential and by changes in the expression of key regulatory genes. Recent findings show that cultured stem-cell lines derived from different stages of mouse development can mimic these transition states. They further reveal that there is a high degree of heterogeneity and plasticity in pluripotent populations in vitro and that these properties are modulated by extrinsic signalling. Understanding the extrinsic control of plasticity will guide efforts to use human pluripotent stem cells in research and therapy.

Human pluripotent stem cell strategies for age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of severe vision loss in the Western world and is increasing exponentially as the population ages. Despite enormous worldwide efforts, the earliest pathogenic pathways involved in AMD are still not fully understood. It is essential to develop research tools for effective modeling of AMD pathogenesis and for subsequent drug discovery and cell or molecular therapies. This review will focus on the current progress in human pluripotent stem cells for understanding and treating AMD.

An in vitro neurogenetics platform for precision disease modeling in the mouse.

The power and scope of disease modeling can be markedly enhanced through the incorporation of broad genetic diversity. The introduction of pathogenic mutations into a single inbred mouse strain sometimes fails to mimic human disease. We describe a cross-species precision disease modeling platform that exploits mouse genetic diversity to bridge cell-based modeling with whole organism analysis. We developed a universal protocol that permitted robust and reproducible neural differentiation of genetically diverse human and mouse pluripotent stem cell lines and then carried out a proof-of-concept study of the neurodevelopmental gene DYRK1A. Results in vitro reliably predicted the effects of genetic background on Dyrk1a loss-of-function phenotypes in vivo. Transcriptomic comparison of responsive and unresponsive strains identified molecular pathways conferring sensitivity or resilience to Dyrk1a1A loss and highlighted differential messenger RNA isoform usage as an important determinant of response. This cross-species strategy provides a powerful tool in the functional analysis of candidate disease variants identified through human genetic studies.

Complex regulatory networks influence pluripotent cell state transitions in human iPSCs.

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network are significantly enriched for regulatory variants with large effects, suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks, shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity, and suggests that genetic factors may contribute to cell state transitions in human iPSC lines.

Defining pluripotency.

Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties.

The GCTM-5 epitope associated with the mucin-like glycoprotein FCGBP marks progenitor cells in tissues of endodermal origin.

Monoclonal antibodies against cell surface markers are powerful tools in the study of tissue regeneration, repair, and neoplasia, but there is a paucity of specific reagents to identify stem and progenitor cells in tissues of endodermal origin. The epitope defined by the GCTM-5 monoclonal antibody is a putative marker of hepatic progenitors. We sought to analyze further the distribution of the GCTM-5 antigen in normal tissues and disease states and to characterize the antigen biochemically. The GCTM-5 epitope was specifically expressed on tissues derived from the definitive endoderm, in particular the fetal gut, liver, and pancreas. Antibody reactivity was detected in subpopulations of normal adult biliary and pancreatic duct cells, and GCTM-5-positive cells isolated from the nonparenchymal fraction of adult liver expressed markers of progenitor cells. The GCTM-5-positive cell populations in liver and pancreas expanded greatly in numbers in disease states such as biliary atresia, cirrhosis, and pancreatitis. Neoplasms arising in these tissues also expressed the GCTM-5 antigen, with pancreatic adenocarcinoma in particular showing strong and consistent reactivity. The GCTM-5 epitope was also strongly displayed on cells undergoing intestinal metaplasia in Barrett's esophagus, a precursor to esophageal carcinoma. Biochemical, mass spectrometry, and immunochemical studies revealed that the GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP. The GCTM-5 epitope on the mucin-like glycoprotein FCGBP is a cell surface marker for the study of normal differentiation lineages, regeneration, and disease progression in tissues of endodermal origin.

Analysis of regulatory network modules in hundreds of human stem cell lines reveals complex epigenetic and genetic factors contribute to pluripotency state differences between subpopulations.

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discovered 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which were highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlied the coordinated expression of genes in the GNMs. Epigenetic analyses revealed that regulatory networks underlying self-renewal and pluripotency have a surprising level of complexity. Genetic analyses identified thousands of regulatory variants that overlapped predicted transcription factor binding sites and were associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network were significantly enriched for regulatory variants with large effects, suggesting that they may play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work captures the coordinated activity of tens of thousands of regulatory elements in hiPSCs and bins these elements into discrete functionally characterized regulatory networks, shows that regulatory elements in pluripotency networks harbor variants with large effects, and provides a rich resource for future pluripotent stem cell research.

ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

Pluripotent cell states and unexpected fates.

Pluripotent stem cells provide a powerful model for the study of human development and its disorders. Recent studies, including two in this issue of Stem Cell Reports, raise important questions concerning the developmental potential of human pluripotent stem cells, and how the behavior of these cells in vitro mirrors normal embryogenesis.