Differentiation of Human Pluripotent Stem Cells Into Definitive Endoderm Cells in Various Flexible Three-Dimensional Cell Culture Systems: Possibilities and Limitations.

The generation of human stem cell-derived spheroids and organoids represents a major step in solving numerous medical, pharmacological, and biological challenges. Due to the advantages of three-dimensional (3D) cell culture systems and the diverse applications of human pluripotent stem cell (iPSC)-derived definitive endoderm (DE), we studied the influence of spheroid size and 3D cell culture systems on spheroid morphology and the effectiveness of DE differentiation as assessed by quantitative PCR (qPCR), flow cytometry, immunofluorescence, and computational modeling. Among the tested hydrogel-based 3D systems, we found that basement membrane extract (BME) hydrogel could not retain spheroid morphology due to dominant cell-matrix interactions. On the other hand, we found that nanofibrillar cellulose (NFC) hydrogel could maintain spheroid morphology but impeded growth factor diffusion, thereby negatively affecting cell differentiation. In contrast, suspension culture provided sufficient mass transfer and was demonstrated by protein expression assays, morphological analyses, and mathematical modeling to be superior to the hydrogel-based systems. In addition, we found that spheroid size was reversely correlated with the effectiveness of DE formation. However, spheroids of insufficient sizes failed to retain 3D morphology during differentiation in all the studied culture conditions. We hereby demonstrate how the properties of a chosen biomaterial influence the differentiation process and the importance of spheroid size control for successful human iPSC differentiation. Our study provides critical parametric information for the generation of human DE-derived, tissue-specific organoids in future studies.

RNA-based CRISPR-Mediated Loss-of-Function Mutagenesis in Human Pluripotent Stem Cells.

Current approaches for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated-9 (Cas9)-mediated genome editing in human pluripotent stem (PS) cells mainly employ plasmids or ribonucleoprotein complexes. Here, we devise an improved transfection protocol of in vitro transcribed Cas9 mRNA and crRNA:tracrRNA duplex that can effectively generate indels in four genetic loci (two active and two inactive) and demonstrate utility in four human PS cell lines (one embryonic and three induced PS cell lines). Our improved protocol incorporating a Cas9-linked selection marker and a staggered transfection strategy promotes targeting efficiency up to 85% and biallelic targeting efficiency up to 76.5% of total mutant clones. The superior targeting efficiency and the non-integrative nature of our approach underscore broader applications in high-throughput arrayed CRISPR screening and in generating custom-made or off-the-shelf cell products for human therapy.

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.

An adherent-cell depletion technique to generate human neural progenitors and neurons.

Existing methodologies to produce human neural stem cells and neurons from embryonic stem cells frequently involve multistep processes and the use of complex and expensive media components, cytokines or small molecules. Here, we report a simple technique to generate human neuroepithelial progenitors and neurons by periodic mechanical dissection and adherent-cell depletion on regular cell-culture grade plastic surfaces. This neural induction technique does not employ growth factors, small molecules or peptide inhibitors, apart from those present in serum-free supplements. Suggestive of their central nervous system origin, we found that neural progenitors formed by this technique expressed radial glia markers, and, when differentiated, expressed TUBB3, RBFOX3 (NeuN) and serotonin, but not markers for peripheral neurons. With these data, we postulate that incorporation of periodic mechanical stimuli and plastic surface-mediated cell selection could improve and streamline existing human neuron production protocols.

Human neural crest induction by temporal modulation of WNT activation.

The developmental biology of neural crest cells in humans remains unexplored due to technical and ethical challenges. The use of pluripotent stem cells to model human neural crest development has gained momentum. We recently introduced a rapid chemically defined approach to induce robust neural crest by WNT/ß-CATENIN activation. Here we investigate the temporal requirements of ectopic WNT activation needed to induce neural crest cells. By altering the temporal activation of canonical WNT/ß-CATENIN with a GSK3 inhibitor we find that a 2 Day pulse of WNT/ß-CATENIN activation via GSK3 inhibition is optimal to generate bona fide neural crest cells, as shown by their capacity to differentiate to neural crest specific fates including peripheral neurons, glia, melanoblasts and ectomesenchymal osteocytes, chondrocytes and adipocytes. Although a 2 Day pulse can impart neural crest character when GSK3 is inhibited days after seeding, optimal results are obtained when WNT is activated from the beginning, and we find that the window of competence to induce NCs from non-neural ectodermal/placodal precursors closes by day 3 of culture. The reduced requirement for exogenous WNT activation offers an approach that is cost-effective, and we show that this adherent 2-dimensional approach is efficient in a broad range of culture platforms ranging from 96-well vessels to 10 cm dishes.

Differences in definitive endoderm induction approaches using growth factors and small molecules.

Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (Activin A and Wnt-3a) and small molecules (sodium butyrate and IDE 1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapid DE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications. We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications.

Next generation organoids for biomedical research and applications.

Organoids are in vitro cultures of miniature fetal or adult organ-like structures. Their potentials for use in tissue and organ replacement, disease modeling, toxicology studies, and drug discovery are tremendous. Currently, major challenges facing human organoid technology include (i) improving the range of cellular heterogeneity for a particular organoid system, (ii) mimicking the native micro- and matrix-environment encountered by cells within organoids, and (iii) developing robust protocols for the in vitro maturation of organoids that remain mostly fetal-like in cultures. To tackle these challenges, we advocate the principle of reverse engineering that replicates the inner workings of in vivo systems with the goal of achieving functionality and maturation of the resulting organoid structures with the input of minimal intrinsic (cellular) and environmental (matrix and niche) constituents. Here, we present an overview of organoid technology development in several systems that employ cell materials derived from fetal and adult tissues and pluripotent stem cell cultures. We focus on key studies that exploit the self-organizing property of embryonic progenitors and the role of designer matrices and cell-free scaffolds in assisting organoid formation. We further explore the relationship between adult stem cells, niche factors, and other current developments that aim to enhance robust organoid maturation. From these works, we propose a standardized pipeline for the development of future protocols that would help generate more physiologically relevant human organoids for various biomedical applications.

The Molecular Pathway Regulating Bergmann Glia and Folia Generation in the Cerebellum.

Evolution of complex behaviors in higher vertebrates and primates require the development of sophisticated neuronal circuitry and the expansion of brain surface area to accommodate the vast number of neuronal and glial populations. To achieve these goals, the neocortex in primates and the cerebellum in amniotes have developed specialized types of basal progenitors to aid the folding of their cortices. In the cerebellum, Bergmann glia constitute such a basal progenitor population, having a distinctive morphology and playing a critical role in cerebellar corticogenesis. Here, we review recent studies on the induction of Bergmann glia and their crucial role in mediating folding of the cerebellar cortex. These studies uncover a key function of FGF-ERK-ETV signaling cascade in the transformation of Bergmann glia from radial glia in the ventricular zone. Remarkably, in the neocortex, the same signaling axis operates to facilitate the transformation of ventricular radial glia into basal radial glia, a Bergmann glia-like basal progenitor population, which have been implicated in the establishment of neocortical gyri. These new findings draw a striking similarity in the function and ontogeny of the two basal progenitor populations born in distinct brain compartments.

Analogous mechanism regulating formation of neocortical basal radial glia and cerebellar Bergmann glia.

Neocortical basal radial glia (bRG) and cerebellar Bergmann glia (BG) are basal progenitors derived from ventricular apical radial glia (aRG) that selectively lose their apical processes. bRG and BG have been implicated in the expansion and folding of the cerebrum and cerebellum, respectively. Here, we analyzed the molecular characteristics and development of bRG and BG. Transcriptomic comparison revealed striking similarity of the molecular features of bRG and BG. We found that heightened ERK signaling activity in aRG is tightly linked to the temporal formation and the relative abundance of bRG in human and mouse cortices. Forced activation of an FGF-ERK-ETV axis that is crucial to BG induction specifically induced bRG with canonical human bRG features in mice. Therefore, our data point to a common mechanism of bRG and BG generation, bearing implications to the role for these basal progenitors in the evolution of cortical folding of the cerebrum and cerebellum.

WNT/ß-catenin signaling mediates human neural crest induction via a pre-neural border intermediate.

Neural crest (NC) cells arise early in vertebrate development, migrate extensively and contribute to a diverse array of ectodermal and mesenchymal derivatives. Previous models of NC formation suggested derivation from neuralized ectoderm, via meso-ectodermal, or neural-non-neural ectoderm interactions. Recent studies using bird and amphibian embryos suggest an earlier origin of NC, independent of neural and mesodermal tissues. Here, we set out to generate a model in which to decipher signaling and tissue interactions involved in human NC induction. Our novel human embryonic stem cell (ESC)-based model yields high proportions of multipotent NC cells (expressing SOX10, PAX7 and TFAP2A) in 5 days. We demonstrate a crucial role for WNT/ß-catenin signaling in launching NC development, while blocking placodal and surface ectoderm fates. We provide evidence of the delicate temporal effects of BMP and FGF signaling, and find that NC development is separable from neural and/or mesodermal contributions. We further substantiate the notion of a neural-independent origin of NC through PAX6 expression and knockdown studies. Finally, we identify a novel pre-neural border state characterized by early WNT/ß-catenin signaling targets that displays distinct responses to BMP and FGF signaling from the traditional neural border genes. In summary, our work provides a fast and efficient protocol for human NC differentiation under signaling constraints similar to those identified in vivo in model organisms, and strengthens a framework for neural crest ontogeny that is separable from neural and mesodermal fates.

Shp2-dependent ERK signaling is essential for induction of Bergmann glia and foliation of the cerebellum.

Folding of the cortex and the persistence of radial glia (RG)-like cells called Bergmann glia (BG) are hallmarks of the mammalian cerebellum. Similar to basal RG in the embryonic neocortex, BG maintain only basal processes and continuously express neural stem cell markers. Past studies had focused on the function of BG in granule cell migration and how granule cell progenitors (GCP) regulate cerebellar foliation. The molecular control of BG generation and its role in cerebellar foliation are less understood. Here, we have analyzed the function of the protein tyrosine phosphatase Shp2 in mice by deleting its gene Ptpn11 in the entire cerebellum or selectively in the GCP lineage. Deleting Ptpn11 in the entire cerebellum by En1-cre blocks transformation of RG into BG but preserves other major cerebellar cell types. In the absence of BG, inward invagination of GCP persists but is uncoupled from the folding of the Purkinje cell layer and the basement membrane, leading to disorganized lamination and an absence of cerebellar folia. In contrast, removing Ptpn11 in the GCP lineage by Atoh1-cre has no effect on cerebellar development, indicating that Shp2 is not cell autonomously required in GCP. Furthermore, we demonstrate that Ptpn11 interacts with Fgf8 and is essential for ERK activation in RG and nascent BG. Finally, expressing constitutively active MEK1 rescues BG formation and cerebellar foliation in Shp2-deficient cerebella. Our results demonstrate an essential role of Shp2 in BG specification via fibroblast growth factor/extracellular signal-regulated protein kinase signaling, and reveal a crucial function of BG in organizing cerebellar foliation.

Differential BMP signaling controls formation and differentiation of multipotent preplacodal ectoderm progenitors from human embryonic stem cells.

Sensory and endoneurocrine tissues as diverse as the lens, the olfactory epithelium, the inner ear, the cranial sensory ganglia, and the anterior pituitary arise from a common pool of progenitors in the preplacodal ectoderm (PPE). Around late gastrulation, the PPE forms at the border surrounding the anterior neural plate, and expresses a unique set of evolutionarily conserved transcription regulators including Six1, Eya 1 and Eya2. Here, we describe the first report to generate and characterize the SIX1(+) PPE cells from human embryonic stem (ES) cells by adherent differentiation. Before forming PPE cells, differentiating cultures first expressed the non-neural ectoderm specific transcriptional factors TFAP2A, GATA2, GATA3, DLX3, and DLX5, which are crucial in establishing the PPE competence. We demonstrated that bone morphogenetic protein (BMP) activity plays a transient but essential role in inducing expression of these PPE competence factors and eventually the PPE cells. Interestingly, we found that attenuating BMP signaling after establishing the competence state induces anterior placode precursors. By manipulating BMP and hedgehog signaling pathways, we further differentiate these precursors into restricted lineages including the lens placode and the oral ectoderm (pituitary precursor) cells. Finally, we also show that sensory neurons can be generated from human PPE cells, demonstrating the multipotency of the human ES-derived PPE cells.

Loss of procollagen IIA from the anterior mesendoderm disrupts the development of mouse embryonic forebrain.

Morphogenesis of the mammalian forebrain is influenced by the patterning activity of signals emanating from the anterior mesendoderm. In this study, we show that procollagen IIA (IIA), an isoform of the cartilage extracellular matrix protein encoded by an alternatively spliced transcript of Col2a1, is expressed in the prechordal plate and the anterior definitive endoderm. In the absence of IIA activity, the null mutants displayed a partially penetrant phenotype of loss of head tissues, holoprosencephaly, and loss of mid-facial structures, which is associated with reduced sonic hedgehog (Shh) expression in the prechordal mesoderm. Genetic interaction studies reveal that IIA function in forebrain and face development does not involve bone morphogenetic protein receptor 1A (BMPR1A)- or NODAL-mediated signaling activity.