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.

SummaryDefects 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.

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.

In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFb)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRb and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro-and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases.

Summary 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.

The neural crest (NC) is a multipotent embryonic cell population that generates various cell types in an axial position-dependent manner. Cranial NC cells, give rise to mesoectodermal derivatives, melanocytes, neurons and glia whereas the vagal NC generates the enteric nervous system and trunk NC cells produce sympathetic neurons and neuroendocrine cells. An attractive approach for studying human NC biology and modelling NC-associated developmental disorders (neurocristopathies) involves the in vitro production of NC cells from human pluripotent stem cells (hPSCs). However, most conventional differentiation protocols generate predominantly cranial NC but fail to induce trunk NC cells. Here we describe a detailed protocol for the efficient in vitro generation of trunk NC cells and their derivatives from hPSCs. This relies on the induction of an intermediate cell population that exhibits neural and mesodermal potential, resembling the embryonic neuromesodermal progenitors, which generate the postcranial body axis in vivo.

Summary Human pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, bio-manufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells. Lay abstract Novel therapies derived from different kinds of precursor cells and stem cells are increasingly moving to clinical trials to restore tissue function in patients who have suffered injury or disease. The manufacture of these new therapies is unusually complex, which means that the manufacturing processes require great attention to assure they are safe and effective. This paper describes a conversation amongst experts in the field who are exploring therapeutic applications of two different kinds of stem cells (pluripotent stem cells and tissue-derived stem/precursor cells). It considers critical issues in developing the manufacturing process for each of these quite different cells types.