Collagen Lattice Model, Populated with Heterogeneous Cancer-Associated Fibroblasts, Facilitates Advanced Reconstruction of Pancreatic Cancer Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a solid-tumor malignancy. To enhance the treatment landscape of PDAC, a 3D model optimized for rigorous drug screening is essential. Within the PDAC tumor microenvironment, a dense stroma comprising a large extracellular matrix and cancer-associated fibroblasts (CAFs) is well-known for its vital role in modulating tumor growth, cellular heterogeneity, bidirectional paracrine signaling, and chemoresistance. In this study, we employed a fibroblast-populated collagen lattice (FPCL) modeling approach that has the ability to replicate fibroblast contractility in the collagenous matrix to build dense stroma. This FPCL model allows CAF differentiation by facilitating multifaceted cell-cell interactions between cancer cells and CAFs, with the differentiation further influenced by mechanical forces and hypoxia carried within the 3D structure. Our FPCL models displayed hallmark features, including ductal gland structures and differentiated CAFs with spindle shapes. Through morphological explorations alongside in-depth transcriptomic and metabolomic profiling, we identified substantial molecular shifts from the nascent to mature model stages and potential metabolic biomarkers, such as proline. The initial pharmacological assays highlighted the effectiveness of our FPCL model in screening for improved therapeutic strategies. In conclusion, our PDAC modeling platform mirrors complex tumor microenvironmental dynamics and offers an unparalleled perspective for therapeutic exploration.

Functional engineering of human iPSC-derived parasympathetic neurons enhances responsiveness to gastrointestinal hormones.

Food-derived biological signals are transmitted to the brain via peripheral nerves through the paracrine activity of gastrointestinal (GI) hormones. The signal transduction circuit of the brain-gut axis has been analyzed in animals; however, species-related differences and animal welfare concerns necessitate investigation using in vitro human experimental models. Here, we focused on the receptors of five GI hormones (CCK, GLP1, GLP2, PYY, and serotonin (5-HT)), and established human induced pluripotent stem cell (iPSC) lines that functionally expressed each receptor. Compared to the original iPSCs, iPSCs expressing one of the receptors did not show any differences in global mRNA expression, genomic stability, or differentiation capacities of the three germ layers. We induced parasympathetic neurons from these established iPSC lines to assess vagus nerve activity. We generated GI hormone receptor-expressing neurons (CCKAR, GLP1R, and NPY2R-neuron) and tested their responsiveness to each ligand using Ca2+ imaging and microelectrode array recording. GI hormone receptor-expressing neurons (GLP2R and HTR3A) were generated directly by gene induction into iPSC-derived peripheral nerve progenitors. These receptor-expressing neurons promise to contribute to a better understanding of how the body responds to GI hormones via the brain-gut axis, aid in drug development, and offer an alternative to animal studies.

Exploring the Role of Desmoplastic Physical Stroma in Pancreatic Cancer Progression Using a Three-Dimensional Collagen Matrix Model.

Pancreatic ductal adenocarcinoma (PDAC) is a refractory tumor with a poor prognosis, and its complex microenvironment is characterized by a fibrous interstitial matrix surrounding PDAC cells. Type I collagen is a major component of this interstitial matrix. Abundant type I collagen promotes its deposition and cross-linking to form a rigid and dense physical barrier, which limits drug penetration and immune cell infiltration and provides drug resistance and metabolic adaptations. In this study, to identify the physical effect of the stroma, type I collagen was used as a 3D matrix to culture Capan-1 cells and generate a 3D PDAC model. Using transcriptome analysis, a link between type I collagen-induced physical effects and the promotion of Capan-1 cell proliferation and migration was determined. Moreover, metabolomic analysis revealed that the physical effect caused a shift in metabolism toward a glycolytic phenotype. In particular, the high expression of proline in the metabolites suggests the ability to maintain Capan-1 cell proliferation under hypoxic and nutrient-depleted conditions. In conclusion, we identified type I collagen-induced physical effects in promoting Capan-1 cells, which cause PDAC progression, providing support for the role of dense stroma in the PDAC microenvironment and identifying a fundamental method for modeling the complex PDAC microenvironment.

Decoding Metabolic Symbiosis between Pancreatic Cancer Cells and Cancer-Associated Fibroblasts Using Cultured Tumor Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor prognosis, largely due to its unique tumor microenvironment (TME) and dense fibrotic stroma. Cancer-associated fibroblasts (CAFs) play a crucial role in promoting tumor growth and metastasis, contributing to the metabolic adaptation of PDAC cells. However, the metabolic interactions between PDAC cells and CAFs are not well-understood. In this study, an in vitro co-culture model was used to investigate these metabolic interactions. Metabolomic analysis was performed under monoculture conditions of Capan-1 PDAC cells and CAF precursor cells, as well as co-culture conditions of PDAC cells and differentiated inflammatory CAF (iCAF). Co-cultured Capan-1 cells displayed significant metabolic changes, such as increased 2-oxoglutaric acid and lauric acid and decreased amino acids. The metabolic profiles of co-cultured Capan-1 and CAFs revealed differences in intracellular metabolites. Analysis of extracellular metabolites in the culture supernatant showed distinct differences between Capan-1 and CAF precursors, with the co-culture supernatant exhibiting the most significant changes. A comparison of the culture supernatants of Capan-1 and CAF precursors revealed different metabolic processes while co-culturing the two cell types demonstrated potential metabolic interactions. In conclusion, this study emphasizes the importance of metabolic interactions between cancer cells and CAFs in tumor progression and highlights the role of TME in metabolic reprogramming.

Deciphering the Molecular Mechanisms of Autonomic Nervous System Neuron Induction through Integrative Bioinformatics Analysis.

In vitro derivation of human neurons in the autonomic nervous system (ANS) is an important technology, given its regulatory roles in maintaining homeostasis in the human body. Although several induction protocols for autonomic lineages have been reported, the regulatory machinery remains largely undefined, primarily due to the absence of a comprehensive understanding of the molecular mechanism regulating human autonomic induction in vitro. In this study, our objective was to pinpoint key regulatory components using integrated bioinformatics analysis. A protein-protein interaction network construction for the proteins encoded by the differentially expressed genes from our RNA sequencing data, and conducting subsequent module analysis, we identified distinct gene clusters and hub genes involved in the induction of autonomic lineages. Moreover, we analyzed the impact of transcription factor (TF) activity on target gene expression, revealing enhanced autonomic TF activity that could lead to the induction of autonomic lineages. The accuracy of this bioinformatics analysis was corroborated by employing calcium imaging to observe specific responses to certain ANS agonists. This investigation offers novel insights into the regulatory machinery in the generation of neurons in the ANS, which would be valuable for further understanding and precise regulation of autonomic induction and differentiation.

A Multiomics Atlas of Brown Adipose Tissue Development Over Time.

Brown adipose tissue (BAT) regulates homeostatic energy balances in response to physiological changes such as nutrition intake, calorie restriction, exercise, and environmental temperature by consuming energy to generate heat, and thus serves as an important organ for obesity and metabolic diseases. We performed an integrated transcriptomic and metabolomic characterization of developing mouse BAT from embryo to adult to obtain a time-resolved picture of BAT development. We demonstrated that there are 2 distinct developmental changes that are BAT specific. We also examined transcription factor binding sites and discovered key transcription factors in the developmental time course. A comparison of our data with other organ development transcriptome and metabolome data revealed BAT-specific transcriptome and metabolome patterns. Our findings provide an overview of mouse BAT development as well as implications for developmental and functional BAT controls.

Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells.

BACKGROUND: Canonical Wnt signaling is involved in a variety of biological processes including stem cell renewal and differentiation, embryonic development, and tissue regeneration. Previous studies reported the stage-specific roles of the Wnt signaling in heart development. Canonical Wnt signal activation by recombinant Wnt3a in the early phase of differentiation enhances the efficiency of myocardial cell production from pluripotent stem cells. However, the hydrophobicity of Wnt proteins results in high cost to produce the recombinant proteins and presents an obstacle to their preparation and application for therapeutics, cell therapy, or molecular analysis of Wnt signaling. METHODS: To solve this problem, we generated an inexpensive molecule-responsive differentiation-inducing chimeric antigen receptor (designated as diCAR) that can activate Wnt3a signaling. The extracellular domains of low-density-lipoprotein receptor-related protein 6 (LRP6) and frizzeled-8 (FZD8) were replaced with single-chain Fv of anti-fluorescein (FL) antibody, which can respond to FL-conjugated bovine serum albumin (BSA-FL) as a cognate ligand. We then analyzed the effect of this diCAR on Wnt signal activation and cardiomyocyte differentiation of mouse embryonic stem cells in response to BSA-FL treatment. RESULTS: Embryonic stem cell lines stably expressing this paired diCAR, named Wnt3a-diCAR, showed TCF/ß-catenin-dependent transactivation by BSA-FL in a dose-dependent manner. Treatment with either Wnt3a recombinant protein or BSA-FL in the early phase of differentiation revealed similar changes of global gene expressions and resulted in efficient myocardial cell differentiation. Furthermore, BSA-FL-mediated signal activation was not affected by a Wnt3a antagonist, Dkk1, suggesting that the signal transduction via Wnt3a-diCAR is independent of endogenous LRP6 or FZD8. CONCLUSION: We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.

Therapeutic Potential of Adipose Stem Cell-Derived Conditioned Medium on Scar Contraction Model.

Scars are composed of stiff collagen fibers, which contract strongly owing to the action of myofibroblasts. To explore the substances that modulate scar contracture, the fibroblast-populated collagen lattice (FPCL) model has been used. However, the molecular signature of the patient-derived FPCL model has not been verified. Here, we examined whether the patient-derived keloid FPCL model reflects scar contraction, analyzing detailed gene expression changes using comprehensive RNA sequencing and histological morphology, and revealed that these models are consistent with the changes during human scar contracture. Moreover, we examined whether conditioned media derived from adipose stem cells (ASC-CM) suppress the scar contracture of the collagen disc. Detailed time-series measurements of changes in disc area showed that the addition of ASC-CM significantly inhibited the shrinkage of collagen discs. In addition, a deep sequencing data analysis revealed that ASC-CM suppressed inflammation-related gene expression in the early phase of contraction; in the later phase, this suppression was gradually replaced by extracellular matrix (ECM)-related gene expression. These lines of data suggested the effectiveness of ASC-CM in suppressing scar contractures. Therefore, the molecular analysis of the ASC-CM actions found in this study will contribute to solving medical problems regarding pathological scarring in wound prognosis.

Potential Metabolite Markers for Pancreatic Cancer Identified by Metabolomic Analysis of Induced Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment perform glycolysis to produce energy, i.e., ATP. Since the origin of CAFs is unidentified, it is not determined whether the intracellular metabolism transitions from oxidative phosphorylation (OXPHOS) to glycolysis when normal tissue fibroblasts differentiate into CAFs. In this study, we established an experimental system and induced the in vitro differentiation of mesenchymal stem cells (MSCs) to CAFs. Additionally, we performed metabolomic and RNA-sequencing analyses before and after differentiation to investigate changes in the intracellular metabolism. Consequently, we discovered that OXPHOS, which was the primary intracellular metabolism in MSCs, was reprogrammed to glycolysis. Furthermore, we analyzed the metabolites in pancreatic tumor tissues in a mice model. The metabolites extracted as candidates in the in vitro experiments were also detected in the in vivo experiments. Thus, we conclude that normal tissue fibroblasts that differentiate into CAFs undergo a metabolic reprogramming from OXPHOS to glycolysis. Moreover, we identified the CAF-specific metabolites expressed during metabolic reprogramming as potential future biomarkers for pancreatic cancer.

Non-invasive cell classification using the Paint Raman Express Spectroscopy System (PRESS).

Raman scattering represents the distribution and abundance of intracellular molecules, including proteins and lipids, facilitating distinction between cellular states non-invasively and without staining. However, the scattered light obtained from cells is faint and cells have complex structures, making it difficult to obtain a Raman spectrum covering the entire cell in a short time using conventional methods. This also prevents efficient label-free cell classification. In the present study, we developed the Paint Raman Express Spectroscopy System, which uses two fast-rotating galvano mirrors to obtain spectra from a wide area of a cell. By using this system and applying machine learning, we were able to acquire broad spectra of a variety of human and mouse cell types, including pluripotent stem cells and confirmed that each cell type can be classified with high accuracy. Moreover, we classified different activation states of human T cells, despite their similar morphology. This system could be used for rapid and low-cost drug evaluation and quality management for drug screening in cell-based assays.

Applicability of Artificial Vascularized Liver Tissue to Proteomic Analysis.

Artificial vascularized tubular liver tissue has perfusable blood vessels that allow fluid access to the tissue interior, enabling the injection of drugs and collection of metabolites, which are valuable for drug discovery. It is amenable to standard evaluation methods, such as paraffin-embedded sectioning, qPCR, and RNA sequencing, which makes it easy to implement into existing research processes. However, the application of tissues vascularized by the self-assembly of cells, (including tubular liver tissue, has not yet been tested in comprehensive proteomic analysis relevant for drug discovery. Here, we established a method to efficiently separate cells from the tubular liver tissue by adding a pipetting step during collagenase treatment. By using this method, we succeeded in obtaining a sufficient number of cells for the proteomic analysis. In addition, to validate this approach, we compared the cells separated from the tissue with those grown in 2D culture, focusing on the proteins related to drug metabolism. We found that the levels of proteins involved in metabolic phases II and III were slightly higher in the tubular liver tissue than those in the 2D cell culture. Taken together, our suggested method demonstrates the applicability of tubular liver tissue to the proteomic analysis in drug assays.

Adipose-derived mesenchymal stem cells differentiate into heterogeneous cancer-associated fibroblasts in a stroma-rich xenograft model.

Cancer-associated fibroblasts (CAFs) are the key components of the densely proliferated stroma in pancreatic ductal adenocarcinoma (PDAC) and contribute to tumor progression and drug resistance. CAFs comprise heterogeneous subpopulations playing unique and vital roles. However, the commonly used mouse models have not been able to fully reproduce the histological and functional characteristics of clinical human CAF. Here, we generated a human cell-derived stroma-rich CDX (Sr-CDX) model, to reproduce the clinical tumor microenvironment. By co-transplanting human adipose-derived mesenchymal stem cells (AD-MSCs) and a human PDAC cell line (Capan-1) into mice, the Sr-CDX model recapitulated the characteristics of clinical pancreatic cancer, such as accelerated tumor growth, abundant stromal proliferation, chemoresistance, and dense stroma formed from the heterogeneous CAFs. Global RNA sequencing, single-cell based RNA sequencing, and histological analysis of CAFs in the Sr-CDX model revealed that the CAFs of the Sr-CDX mice were derived from the transplanted AD-MSCs and composed of heterogeneous subpopulations of CAF, including known and unknown subtypes. These lines of evidences suggest that our new tumor-bearing mouse model has the potential to address an open question in CAF research, that is the mechanism of CAF differentiation.

Exposure to small molecule cocktails allows induction of neural crest lineage cells from human adipose-derived mesenchymal stem cells.

Neural crest cells (NCCs) are a promising source for cell therapy and regenerative medicine owing to their multipotency, self-renewability, and capability to secrete various trophic factors. However, isolating NCCs from adult organs is challenging, because NCCs are broadly distributed throughout the body. Hence, we attempted to directly induce NCCs from human adipose-derived mesenchymal stem cells (ADSCs), which can be isolated easily, using small molecule cocktails. We established a controlled induction protocol with two-step application of small molecule cocktails for 6 days. The induction efficiency was evaluated based on mRNA and protein expression of neural crest markers, such as nerve growth factor receptor (NGFR) and sex-determining region Y-box 10 (SOX10). We also found that various trophic factors were significantly upregulated following treatment with the small molecule cocktails. Therefore, we performed global profiling of cell surface makers and identified distinctly upregulated markers, including the neural crest-specific cell surface markers CD271 and CD57. These results indicate that our chemical treatment can direct human ADSCs to developing into the neural crest lineage. This offers a promising experimental platform to study human NCCs for applications in cell therapy and regenerative medicine.

Adipose-derived mesenchymal stem cells differentiate into pancreatic cancer-associated fibroblasts in vitro.

Cancer-associated fibroblasts (CAFs) are key components of the dense, proliferating stroma observed in pancreatic ductal adenocarcinoma (PDAC), and CAF subpopulations drive tumor heterogeneity and play a major role in PDAC progression and drug resistance. CAFs consist of heterogenous subpopulations such as myoblastic CAF (myCAF) and inflammatory CAF (iCAF), and each has distinct essential roles. However, it is not clear how CAF subpopulations are formed in PDAC. Adipose-derived MSCs (AD-MSCs), which possess a high multilineage potential and self-renewal capacity, are reported to be one of the in vivo CAF sources. Here, we aimed to investigate whether AD-MSCs can act as precursors for CAFs in vitro. We recorded morphological features and collected omics data from two in vitro co-culture models for recapitulating clinical PDAC. Additionally, we tested the advantages of the co-culture model in terms of accurately modeling morphology and CAF heterogeneity. We showed that AD-MSCs differentiate into two distinct CAF subpopulations: Direct contact co-culture with PDAC cell line Capan-1 induced differentiation into myCAFs and iCAFs, while indirect co-culture induced differentiation into only iCAFs. Using these co-culture systems, we also identified novel CAF markers that may be helpful for elucidating the mechanisms of CAFs in the tumor microenvironment (TME). In conclusion, AD-MSCs can differentiate into distinct CAF subtypes depending on the different co-culture conditions in vitro, and the identification of potential CAF markers may aid in future investigations of the mechanisms underlying the role of CAFs in the TME.

Expression of genes involved in drug metabolism differs between perfusable 3D liver tissue and conventional 2D-cultured hepatocellular carcinoma cells.

Tubular 3D liver tissue with enhanced capillary-like structures branching from a large main channel is potentially useful for drug discovery because the perfusable main channel and capillary-like structures enable mass transfer into and out from the tissue. Tubular liver tissue is comprised of the hepatocellular carcinoma cell line HepG2, human umbilical vein endothelial cells (HUVECs), and mesenchymal stem cells (MSCs), using a perfusion device functioning as the interface for an external pump. This study aimed to compare the expression of genes involved in drug metabolism between 2D-cultured hepatocellular carcinoma cells and 3D-cultured tubular liver tissue. Gene expression profiles of 2D-cultured cells and tubular liver tissue were compared using RNA sequencing. Multidimensional scaling analysis revealed that culture dimensionality had a more prominent effect on gene expression profiles than perfusion conditions. More specifically, genes involved in drug metabolism such as CYP2D6, CYP2E1, NNMT, and SLC28A1 were slightly upregulated in the 3D cultures, while certain genes such as ALDH1B1, ALDH1A2, and SULT1E1 were downregulated. These results indicate that gene expression profiles are largely influenced by culture dimensionality and are potentially useful to researchers intending to switch from 2D culture to 3D culture of hepatocellular carcinoma or other tissue types.

Selective Induction of Human Autonomic Neurons Enables Precise Control of Cardiomyocyte Beating.

The autonomic nervous system (ANS) regulates tissue homeostasis and remodelling through antagonistic effects of noradrenergic sympathetic and cholinergic parasympathetic signalling. Despite numerous reports on the induction of sympathetic neurons from human pluripotent stem cells (hPSCs), no induction methods have effectively derived cholinergic parasympathetic neurons from hPSCs. Considering the antagonistic effects of noradrenergic and cholinergic inputs on target organs, both sympathetic and parasympathetic neurons are expected to be induced. This study aimed to develop a stepwise chemical induction method to induce sympathetic-like and parasympathetic-like ANS neurons. Autonomic specification was achieved through restricting signals inducing sensory or enteric neurogenesis and activating bone morphogenetic protein (BMP) signals. Global mRNA expression analyses after stepwise induction, including single-cell RNA-seq analysis of induced neurons and functional assays revealed that each induced sympathetic-like or parasympathetic-like neuron acquired pharmacological and electrophysiological functional properties with distinct marker expression. Further, we identified selective induction methods using appropriate seeding cell densities and neurotrophic factor concentrations. Neurons were individually induced, facilitating the regulation of the beating rates of hiPSC-derived cardiomyocytes in an antagonistic manner. The induction methods yield specific neuron types, and their influence on various tissues can be studied by co-cultured assays.

Fabrication of Perfusable Vascular Channels and Capillaries in 3D Liver-like Tissue.

Although various production methods for 3D vascularised tissues have been developed, constructing capillary-like structures branching from perfusable large channels remains difficult. This study describes a method to fabricate tube-shaped 3D liver-like tissue (tubular liver tissue) with large channels and capillary-like structures using a perfusion device. The perfusion device functions as an interface between the tissue and an external pump, as it has connectors equipped with anchors that hold the tissue in response to its shrinkage, which is accompanied by the self-organisation of capillary-like structures. Histological analysis revealed that perfusion via the large channel induced capillary formation around the channel and maintained proper tissue functions. Accompanied by structural examinations, global gene expression analysis supported this finding; specifically, genes involved in angiogenesis were enriched in the perfused condition. Furthermore, we confirmed the penetrability of the capillary-like structures by infusing India ink, as well as substance exchange by measuring the amounts of secreted albumin. These lines of evidence indicate that our method can be used to construct 3D tissues, which is useful for fields of in vitro tissue regeneration for drug development and regenerative medicine.

Chimeric G-CSF Receptor-Mediated STAT3 Activation Contributes to Efficient Induction of Cardiomyocytes from Mouse Induced Pluripotent Stem Cells.

Producing a sufficient number of cardiomyocytes from pluripotent stem cells has been of great demand for cardiac regeneration therapy. However, it remains challenging to efficiently differentiate cardiomyocytes with low costs. Reportedly, granulocyte colony-stimulating factor (G-CSF) receptor (GCSFR) signaling activates signal transducers and activators of transcription (STAT) signaling and enhances cardiac differentiation from embryonic stem cells or induced pluripotent stem cells (iPSCs). To economically and efficiently produce cardiomyocytes from iPSCs through GCSFR/STAT axis activation, we constructed antibody/receptor chimeras that can respond to an inexpensive small molecule. Single-chain Fv of anti-fluorescein (FL) antibody was ligated to transmembrane/cytoplasmic domains of GCSFRs, enabling transduction of GCSFR signaling in response to FL-conjugated bovine serum albumin (BSA-FL) as an alternative ligand. Mouse iPSC lines constitutively expressing these chimeric receptors exhibited increased BSA-FL-induced STAT3 phosphorylation in a dose-dependent manner, which was abolished by an inhibitor of Janus tyrosine kinase (JAK). In addition, BSA-FL stimulation also increased the incidence of beating embryoid bodies and upregulated cardiac-specific gene expressions after differentiation in these iPSC lines. Therefore, the chimeric GCSFRs activated endogenous GCSFR signaling at least via the JAK/STAT3 pathway, thereby enhancing cardiac differentiation from iPSCs. This approach, as an economical strategy, could contribute to stem cell-based cardiac regeneration therapy.

Notch and Hippo signaling converge on Strawberry Notch 1 (Sbno1) to synergistically activate Cdx2 during specification of the trophectoderm.

The first binary cell fate decision occurs at the morula stage and gives rise to two distinct types of cells that constitute the trophectoderm (TE) and inner cell mass (ICM). The cell fate determinant, Cdx2, is induced in TE cells and plays an essential role in their differentiation and maintenance. Notch and Hippo signaling cascades are assumed to converge onto regulatory elements of Cdx2, however, the underlying molecular mechanisms are largely unknown. Here, we show involvement of Strawberry Notch1 (Sbno1), a novel chromatin factor of the helicase superfamily 2, during preimplantation development. Sbno1 knockout embryos die at the preimplantation stage without forming a blastocoel, and Cdx2 is not turned on even though both Yap and Tead4 reside normally in nuclei. Accordingly, Sbno1 acts on the trophectoderm-enhancer (TEE) of Cdx2, ensuring its robust and synergistic activation by the Yap/Tead4 and NICD/Rbpj complexes. Interestingly, this synergism is enhanced when cells are mechanically stretched, which might reflect that TE cells are continuously stretched by the expanding ICM and blastocoel cavity. In addition, the histone chaperone, FACT (FAcilitates Chromatin Transcription) physically interacts with Sbno1. Our data provide new evidence on TE specification, highlighting unexpected but essential functions of the highly conserved chromatin factor, Sbno1.

Brief exposure to small molecules allows induction of mouse embryonic fibroblasts into neural crest-like precursors.

In this study, we propose a novel method for inducing neuronal cells by briefly exposing them to small-molecule cocktails in a step-by-step manner. Global gene expression analysis with immunohistochemical staining and calcium flux assays reveal the generation of neurons from mouse embryonic fibroblasts. In addition, time-lapse imaging of neural precursor-specific enhancer expression and global gene expression analyses show that the neurons are generated by passing through a neural crest-like precursor stage. Consistent with these results, the neural crest-like cells are able to differentiate into neural crest lineage cells, such as sympathetic neurons, adipocytes, osteocytes, and smooth muscle cells. Therefore, these results indicate that brief exposure to chemical compounds could expand and induce a substantial multipotent cell population without viral transduction.