hPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung's disease.

The enteric nervous system (ENS) is derived from both the vagal and sacral component of the neural crest (NC). Here, we present the derivation of sacral ENS precursors from human PSCs via timed exposure to FGF, WNT, and GDF11, which enables posterior patterning and transition from posterior trunk to sacral NC identity, respectively. Using a SOX2::H2B-tdTomato/T::H2B-GFP dual reporter hPSC line, we demonstrate that both trunk and sacral NC emerge from a double-positive neuro-mesodermal progenitor (NMP). Vagal and sacral NC precursors yield distinct neuronal subtypes and migratory behaviors in vitro and in vivo. Remarkably, xenografting of both vagal and sacral NC lineages is required to rescue a mouse model of total aganglionosis, suggesting opportunities in the treatment of severe forms of Hirschsprung's disease.

Retinoic Acid Accelerates the Specification of Enteric Neural Progenitors from In-Vitro-Derived Neural Crest.

The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1 and 7. Defects in the development and function of the ENS cause a range of enteric neuropathies, including Hirschsprung disease. Little is known about the signals that specify early ENS progenitors, limiting progress in the generation of enteric neurons from human pluripotent stem cells (hPSCs) to provide tools for disease modeling and regenerative medicine for enteric neuropathies. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and, following in vivo transplantation, achieved long-term colonization of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.

Top-Down Inhibition (TDi) and Baseline Activation (BLa): Controlling Signal Transduction When Endogenous Cytokines are Ruining Your Differentiation.

In the 20 years since the first human pluripotent stem cell (hPSC) lines were established, there have been a plethora of protocols developed that allow us to generate a wide range of human cell types in vitro. Efforts to achieve a greater degree of specificity and efficiency in generating desired cell types have resulted in increasingly complex approaches. The magnitude and timing of signals has become key, and the concept of a "fully defined" system is a forever sought-after goal with shifting goalposts. This overview discusses two related approaches that can be used to deliver a tightly regulated, intermediate-strength signal, and which can also manage the impact of endogenous signaling variation and enable a switch away from bovine serum albumin-containing medium to a better-defined system without suffering a subsequent loss of robustness or efficiency. The approaches, referred to as top-down inhibition and baseline activation, were developed to deliver intermediate levels of BMP and WNT signaling during neural crest induction from hPSC, but could be applied to a variety of other signals and differentiation systems. © 2019 by John Wiley & Sons, Inc.

WNT/ß-catenin modulates the axial identity of embryonic stem cell-derived human neural crest.

WNT/ß-catenin signaling is crucial for neural crest (NC) formation, yet the effects of the magnitude of the WNT signal remain ill-defined. Using a robust model of human NC formation based on human pluripotent stem cells (hPSCs), we expose that the WNT signal modulates the axial identity of NCs in a dose-dependent manner, with low WNT leading to anterior OTX+ HOX- NC and high WNT leading to posterior OTX- HOX+ NC. Differentiation tests of posterior NC confirm expected derivatives, including posterior-specific adrenal derivatives, and display partial capacity to generate anterior ectomesenchymal derivatives. Furthermore, unlike anterior NC, posterior NC exhibits a transient TBXT+/SOX2+ neuromesodermal precursor-like intermediate. Finally, we analyze the contributions of other signaling pathways in posterior NC formation, which suggest a crucial role for FGF in survival/proliferation, and a requirement of BMP for NC maturation. As expected retinoic acid (RA) and FGF are able to modulate HOX expression in the posterior NC. Surprisingly, early RA supplementation prohibits NC formation. This work reveals for the first time that the amplitude of WNT signaling can modulate the axial identity of NC cells in humans.

FGF Modulates the Axial Identity of Trunk hPSC-Derived Neural Crest but Not the Cranial-Trunk Decision.

The neural crest is a transient embryonic tissue that gives rise to a multitude of derivatives in an axially restricted manner. An in vitro counterpart to neural crest can be derived from human pluripotent stem cells (hPSCs) and can be used to study neural crest ontogeny and neurocristopathies, and to generate cells for therapeutic purposes. In order to successfully do this, it is critical to define the specific conditions required to generate neural crest of different axial identities, as regional restriction in differentiation potential is partly cell intrinsic. WNT and FGF signaling have been implicated as inducers of posterior fate, but the exact role that these signals play in trunk neural crest formation remains unclear. Here, we present a fully defined, xeno-free system for generating trunk neural crest from hPSCs and show that FGF signaling directs cells toward different axial identities within the trunk compartment while WNT signaling is the primary determinant of trunk versus cranial identity.

Fully Defined and Xeno-Free Induction of hPSCs into Neural Crest Using Top-Down Inhibition of BMP Signaling.

The neural crest is a transient embryonic tissue that originates from the border of the neural plate prior to delamination and migration throughout the developing embryo, where it contributes to a wide range of different tissues. Defects in neural crest development have been implicated in a variety of different disorders (neurocristopathies) including cancers, neuropathies, craniofacial malformations, and pigment disorders. The differentiation of human pluripotent stem cells (hPSCs) into an in vitro counterpart to neural crest cells holds huge potential for the study of neural crest development, as well as modeling neurocristopathy, carrying out drug discovery experiments and eventually cell replacement therapy. Here we describe a method for generating human neural crest cells from hPSCs that is fully defined and free from animal-derived components. We found that in the absence of serum or bovine serum albumin (BSA), variability in endogenous BMP expression leads to unpredictable differentiation efficiency. In order to control against this issue, we have developed a system termed "top-down inhibition" (TDi) that allows robust neural crest induction as described below.

Human axial progenitors generate trunk neural crest cells in vitro.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

Identification and Single-Cell Functional Characterization of an Endodermally Biased Pluripotent Substate in Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESCs we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single-cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long-term self-renewal. When clonal stem cell colonies were formed from GATA6-positive and GATA6-negative cells, more of those derived from GATA6-positive cells contained spontaneously differentiated endoderm cells than similar colonies derived from the GATA6-negative cells. We characterized these discrete cellular states using single-cell transcriptomic analysis, identifying a potential role for SOX17 in the establishment of the endoderm-biased stem cell state.

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

Defects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.