A Bayesian Change Point Model for Dynamic Alternative Transcription Start Site Usage During Cellular Differentiation.

ABSTRACT An alternative transcription start site (ATSS) is a major driving force for increasing the complexity of transcripts in human tissues. As a transcriptional regulatory mechanism, ATSS has biological significance. Many studies have confirmed that ATSS plays an important role in diseases and cell development and differentiation. However, exploration of its dynamic mechanisms remains insufficient. Identifying ATSS change points during cell differentiation is critical for elucidating potential dynamic mechanisms. For relative ATSS usage as percentage data, the existing methods lack sensitivity to detect the change point for ATSS longitudinal data. In addition, some methods have strict requirements for data distribution and cannot be applied to deal with this problem. In this study, the Bayesian change point detection model was first constructed using reparameterization techniques for two parameters of a beta distribution for the percentage data type, and the posterior distributions of parameters and change points were obtained using Markov Chain Monte Carlo (MCMC) sampling. With comprehensive simulation studies, the performance of the Bayesian change point detection model is found to be consistently powerful and robust across most scenarios with different sample sizes and beta distributions. Second, differential ATSS events in the real data, whose change points were identified using our method, were clustered according to their change points. Last, for each change point, pathway and transcription factor motif analyses were performed on its differential ATSS events. The results of our analyses demonstrated the effectiveness of the Bayesian change point detection model and provided biological insights into cell differentiation.

Multiplexed bulk and single-cell RNA-seq hybrid enables cost-efficient disease modeling with chimeric organoids.

Disease modeling with isogenic Induced Pluripotent Stem Cell (iPSC)-differentiated organoids serves as a powerful technique for studying disease mechanisms. Multiplexed coculture is crucial to mitigate batch effects when studying the genetic effects of disease-causing variants in differentiated iPSCs or organoids, and demultiplexing at the single-cell level can be conveniently achieved by assessing natural genetic barcodes. Here, to enable cost-efficient time-series experimental designs via multiplexed bulk and single-cell RNA-seq of hybrids, we introduce a computational method in our Vireo Suite, Vireo-bulk, to effectively deconvolve pooled bulk RNA-seq data by genotype reference, and thereby quantify donor abundance over the course of differentiation and identify differentially expressed genes among donors. Furthermore, with multiplexed scRNA-seq and bulk RNA-seq, we demonstrate the usefulness and necessity of a pooled design to reveal donor iPSC line heterogeneity during macrophage cell differentiation and to model rare WT1 mutation-driven kidney disease with chimeric organoids. Our work provides an experimental and analytic pipeline for dissecting disease mechanisms with chimeric organoids.

Assessment of Genetic Stability in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Using Droplet Digital PCR.

Unintended genetic modifications that occur during the differentiation and proliferation of human induced pluripotent stem cells (hiPSCs) can lead to tumorigenicity. This is a crucial concern in the development of stem cell-based therapies to ensure the safety and efficacy of the final product. Moreover, conventional genetic stability testing methods are limited by low sensitivity, which is an issue that remains unsolved. In this study, we assessed the genetic stability of hiPSCs and hiPSC-derived cardiomyocytes using various testing methods, including karyotyping, CytoScanHD chip analysis, whole-exome sequencing, and targeted sequencing. Two specific genetic mutations in KMT2C and BCOR were selected from the 17 gene variants identified by whole-exome and targeted sequencing methods, which were validated using droplet digital PCR. The applicability of this approach to stem cell-based therapeutic products was further demonstrated with associated validation according to the International Council for Harmonisation (ICH) guidelines, including specificity, precision, robustness, and limit of detection. Our droplet digital PCR results showed high sensitivity and accuracy for quantitatively detecting gene mutations, whereas conventional qPCR could not avoid false positives. In conclusion, droplet digital PCR is a highly sensitive and precise method for assessing the expression of mutations with tumorigenic potential for the development of stem cell-based therapeutics.

An Efficient and Cost-Effective Approach to Generate Functional Human Inducible Pluripotent Stem Cell-Derived Astrocytes.

Human inducible pluripotent stem cell (hiPSC)-derived astrocytes (iAs) are critical to study astrocytes in health and disease. They provide several advantages over human fetal astrocytes in research, which include consistency, availability, disease modeling, customization, and ethical considerations. The generation of iAs is hampered by the requirement of Matrigel matrix coating for survival and proliferation. We provide a protocol demonstrating that human iAs cultured in the absence of Matrigel are viable and proliferative. Further, through a side-by-side comparison of cultures with and without Matrigel, we show significant similarities in astrocyte-specific profiling, including morphology (shape and structure), phenotype (cell-specific markers), genotype (transcriptional expression), metabolic (respiration), and functional aspects (glutamate uptake and cytokine response). In addition, we report that, unlike other CNS cell types, such as neuronal progenitor cells and neurons, iAs can withstand the absence of Matrigel coating. Our study demonstrates that Matrigel is dispensable for the culture of human iPSC-derived astrocytes, facilitating an easy, streamlined, and cost-effective method of generating these cells.

Caveolae-associated protein 3 promotes adipogenic differentiation of porcine preadipocytes by promoting extracellular signal-regulated kinase phosphorylation.

Fat deposition is one of the key factors affecting the economic development of pig husbandry. The aim of this study was to investigate the expression characteristics of caveolae-associated protein 3 (CAVIN3) and to elucidate its effect and mechanism on adipogenic differentiation of porcine preadipocytes. Cell transfection, quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot, and oil red O staining were used to detect the effect of CAVIN3 on the differentiation of porcine preadipocytes. The results showed that CAVIN3 was expressed in various tissues, with higher expression in adipose tissue, differentially expressed during cell adipogenic differentiation, and mainly distributed in the cytoplasm. Functional studies showed that, after CAVIN3 interference in preadipocytes, the expression of adipogenic factors and the content of lipid droplets were significantly decreased (p < 0.05). The results were reversed after CAVIN3 was overexpressed. The mechanism research showed that LY3214996 inhibited the extracellular signal-regulated kinase (ERK) phosphorylation and further inhibited lipogenic factors expression. Overexpression of CAVIN3 attenuates the inhibitory effect of LY3214996 on ERK phosphorylation and attenuates its inhibitory effect on adipogenic differentiation. Therefore, this study demonstrated that CAVIN3 promotes the differentiation of porcine preadipocytes by promoting ERK phosphorylation. The present study can lay a theoretical foundation for further studying the molecular mechanism of porcine fat deposition.

A review on qualifications and cost effectiveness of induced pluripotent stem cells (IPSCs)-induced cardiomyocytes in drug screening tests.

Human induced pluripotent stem cells (hIPSCs) have initiated a higher degree of successes in disease modelling, preclinical evaluation of drug therapy and pharmaco-toxicological testing. Since the discovery of iPSCs in 2006, many advanced techniques have been introduced to differentiate iPSCs to cardiomyocytes, which have been progressively improved. The disease models from iPSC-induced cardiomyocytes (iPSC-CM) have been successfully helping to study a variety of cardiac diseases such as long QT syndrome, drug-induced long QT, different cardiomyopathies related to mutations in mitochondria or desmosomal proteins and other rare genetic diseases. IPSC-CMs have also been used to screen the role of chemicals in cardiovascular drug discovery and individualisation of drug dosages. In this review, the quality of current procedures for characterisation and maturation of iPSC-CM lines will be discussed. Also, we will focus on time efficiency and cost of standard differentiation methods after reprogramming.

Depth Vision-Based Assessment of Bone Marrow Mesenchymal Stem Cell Differentiation Capacity in Patients with Congenital Scoliosis.

Congenital scoliosis (CS) is a lateral curvature of one or more segments of the spine due to spinal dysplasia during fetal life. CS is clinically defined as a curvature of the spine >10° due to structural abnormalities of the vertebrae during the embryonic period. Its etiology is unknown, but recent studies suggest that it may be closely related to genetic factors, environmental factors, and developmental abnormalities. The induction methods and modern applications of bone marrow MSCs provide a reference for in-depth human research on the induction of differentiation of bone marrow MSCs into osteoblasts. In this paper, by reviewing and organizing the literature on bone marrow MSCs, we summarized and analyzed the biological properties and preparation of bone marrow MSCs, the methods of inducing osteoblasts, the applications in tissue engineering bone, the problems faced, and the future research directions and proposed a method to assess the differentiation ability of bone marrow MSCs in patients with congenital scoliosis based on depth visual characteristics and the change of the method. The method reveals and evaluates the multidirectional differentiation potential of bone marrow MSCs, which can be induced to differentiate into osteoblasts in vitro and can be used to construct bone tissue engineering scaffolds in vitro using tissue engineering techniques. Based on the properties of bone marrow MSCs, their application in congenital scoliosis patients for trauma repair, cell replacement therapy, hematopoietic support, and gene therapy is quite promising. It is necessary to carry out research on the mechanism of osteogenic differentiation of bone marrow MSCs to provide guidance and reference value for their induced differentiation into osteoblasts.

Simulation-based inference of differentiation trajectories from RNA velocity fields.

We report Cytopath, a method for trajectory inference that takes advantage of transcriptional activity information from the RNA velocity of single cells to perform trajectory inference. Cytopath performs this task by defining a Markov chain model, simulating an ensemble of possible differentiation trajectories, and constructing a consensus trajectory. We show that Cytopath can recapitulate the topological and molecular characteristics of the differentiation process under study. In our analysis, we include differentiation trajectories with varying bifurcated, circular, convergent, and mixed topologies studied in single-snapshot as well as time-series single-cell RNA sequencing experiments. We demonstrate the capability to reconstruct differentiation trajectories, assess the association of RNA velocity-based pseudotime with actually elapsed process time, and identify drawbacks in current state-of-the art trajectory inference approaches.

A Simple, Rapid, and Cost-Effective Method for Loss-of-Function Assays in Pluripotent Cells.

Embryonic stem cells (ESCs) are derived from the inner cell mass of the preimplantation blastocyst and can be maintained indefinitely in vitro without losing their properties. Given their self-renewal and pluripotency, ESCs not only represent a key tool to study early embryonic development in a dish, but also an unlimited source of material for tissue replacement in regenerative medicine. Loss-of-function assays using RNA interference are a powerful tool to understand the roles of specific genes and are facilitated by lentiviral-mediated delivery of vector-encoded shRNAs which allows long-term silencing of single or multiple genes. Here, we describe the steps for rapid and cost-effective production and testing of lentiviral particles with vector-encoded shRNAs for gene silencing in ESCs. This protocol can be easily adapted for loss-of-function assays in other pluripotent cells or culture conditions of interest.

Osteogenesis Imperfecta: The Impact of Genotype and Clinical Phenotype on Adiposity and Resting Energy Expenditure.

CONTEXT: Mutations in type I collagen or collagen-related proteins cause osteogenesis imperfecta (OI). Energy expenditure and body composition in OI could reflect reduced mobility or intrinsic defects in osteoblast differentiation increasing adipocyte development. OBJECTIVE: This study compares adiposity and resting energy expenditure (REE) in OI and healthy controls (HC), for OI genotype- and Type-associated differences. METHODS: We studied 90 participants, 30 with OI (11 COL1A1 Gly, 8 COL1A2 Gly, 4 COL1A1 non-Gly, 1 COL1A2 non-Gly, 6 non-COL; 8 Type III, 16 Type IV, 4 Type VI, 1 Type VII, 1 Type XIV) and 60 HC with sociodemographic characteristics/BMI/BMIz similar to the OI group. Participants underwent dual-energy x-ray absorptiometry to determine lean mass and fat mass percentage (FM%) and REE. FM% and REE were compared, adjusting for covariates, to examine the relationship of OI genotypes and phenotypic Types. RESULTS: FM% did not differ significantly in all patients with OI vs HC (OI: 36.6% ± 1.9%; HC: 32.7% ± 1.2%; P = 0.088). FM% was, however, greater than HC for those with non-COL variants (P = 0.016). FM% did not differ from HC among OI Types (P values > 0.05).Overall, covariate-adjusted REE did not differ significantly between OI and HC (OI: 1376.5 ± 44.7 kcal/d; HC: 1377.0 ± 96 kcal/d; P = 0.345). However, those with non-COL variants (P = 0.016) and Type VI OI (P = 0.04) had significantly lower REE than HC. CONCLUSION: Overall, patients with OI did not significantly differ in either extra-marrow adiposity or REE from BMI-similar HC. However, reduced REE among those with non-COL variants may contribute to greater adiposity.

The osteogenic differentiation of human bone marrow stromal cells induced by nanofiber scaffolds using bioinformatics.

This article aims to investigate the mechanism of behaviors of human bone marrow stromal cells (hBMSCs) affected by scaffold structure combining Monte Carlo feature selection (MFCS), incremental feature selection (IFS) and support vector machine (SVM). The specific differentially expressed genes (DEGs) of hBMSCs cultured on nanofiber (NF) scaffolds and freeform fabrication (FFF) scaffolds were obtained. Key genes were screened from common genes between osteogenic DEGs and NF specific DEGs with MFCS, IFS and SVM. The results demonstrated that NF scaffolds induced hBMSCs to express more genes related to osteogenic differentiation. Finally, 16 key genes were identified among the common genes. The common genes were significantly enriched in Rap1 signaling pathway, extracellular matrix and ossification. The results in this study suggested that the gene expression of hBMSCs was sensitive to NF scaffolds and FFF scaffolds, and the osteogenic differentiation of hBMSCs could be enhanced by NF scaffolds.

Functional Assessment of Direct Reprogrammed Neurons In Vitro and In Vivo.

Direct reprogramming is an emerging research field where you can generate neurons from a somatic cell, such as a skin or glial cell by overexpressing neurogenic transcription factors. This technique allows fast generation of subtype-specific and functional neurons from both human and mouse cells. Despite the fact that neurons have been successfully generated both in vitro and in vivo, a more extensive analysis of the induced neurons including phenotypic functional identity or gradual maturity is still lacking. This is an important step for a further development of induced neurons towards cell therapy or disease modeling of neurological diseases. In this protocol, we describe a method for functional assessment of direct reprogrammed neuronal cells both in vitro and in vivo. Using a synapsin-driven reporter, our protocol allows for a direct identification of the reprogrammed neurons that permits functional assessment using patch-clamp electrophysiology. For in vitro reprogramming we further provide an optimized coating condition that allows a long-term maturation of human induced neurons in vitro.

Molecular phenotyping and functional assessment of smooth muscle-like cells with pathogenic variants in aneurysm genes ACTA2, MYH11, SMAD3 and FBN1.

Aortic aneurysms (AAs) are pathological dilatations of the aorta. Pathogenic variants in genes encoding for proteins of the contractile machinery of vascular smooth muscle cells (VSMCs), genes encoding proteins of the transforming growth factor beta signaling pathway and extracellular matrix (ECM) homeostasis play a role in the weakening of the aortic wall. These variants affect the functioning of VSMC, the predominant cell type in the aorta. Many variants have unknown clinical significance, with unknown consequences on VSMC function and AA development. Our goal was to develop functional assays that show the effects of pathogenic variants in aneurysm-related genes. We used a previously developed fibroblast transdifferentiation protocol to induce VSMC-like cells, which are used for all assays. We compared transdifferentiated VSMC-like cells of patients with a pathogenic variant in genes encoding for components of VSMC contraction (ACTA2, MYH11), transforming growth factor beta (TGFß) signaling (SMAD3) and a dominant negative (DN) and two haploinsufficient variants in the ECM elastic laminae (FBN1) to those of healthy controls. The transdifferentiation efficiency, structural integrity of the cytoskeleton, TGFß signaling profile, migration velocity and maximum contraction were measured. Transdifferentiation efficiency was strongly reduced in SMAD3 and FBN1 DN patients. ACTA2 and FBN1 DN cells showed a decrease in SMAD2 phosphorylation. Migration velocity was impaired for ACTA2 and MYH11 cells. ACTA2 cells showed reduced contractility. In conclusion, these assays for showing effects of pathogenic variants may be promising tools to help reclassification of variants of unknown clinical significance in AA-related genes.

Natural Extracts to Augment Energy Expenditure as a Complementary Approach to Tackle Obesity and Associated Metabolic Alterations.

Obesity is the epidemic of the 21st century. In developing countries, the prevalence of obesity continues to rise, and obesity is occurring at younger ages. Obesity and associated metabolic stress disrupt the whole-body physiology. Adipocytes are critical components of the systemic metabolic control, functioning as an endocrine organ. The enlarged adipocytes during obesity recruit macrophages promoting chronic inflammation and insulin resistance. Together with the genetic susceptibility (single nucleotide polymorphisms, SNP) and metabolic alterations at the molecular level, it has been highlighted that key modifiable risk factors, such as those related to lifestyle, contribute to the development of obesity. In this scenario, urgent therapeutic options are needed, including not only pharmacotherapy but also nutrients, bioactive compounds, and natural extracts to reverse the metabolic alterations associated with obesity. Herein, we first summarize the main targetable processes to tackle obesity, including activation of thermogenesis in brown adipose tissue (BAT) and in white adipose tissue (WAT-browning), and the promotion of energy expenditure and/or fatty acid oxidation (FAO) in muscles. Then, we perform a screening of 20 natural extracts (EFSA approved) to determine their potential in the activation of FAO and/or thermogenesis, as well as the increase in respiratory capacity. By means of innovative technologies, such as the study of their effects on cell bioenergetics (Seahorse bioanalyzer), we end up with the selection of four extracts with potential application to ameliorate the deleterious effects of obesity and the chronic associated inflammation.

Uncovering the cell fate decision in lysis-lysogeny transition and stem cell development via Markov state modeling.

Understanding the behavior of a complex gene regulatory network is a fundamental but challenging task in systems biology. How to reduce the large number of degrees of freedom of a specific network and identify its main biological pathway is the key issue. In this paper, we utilized the transition path theory (TPT) and Markov state modeling (MSM) framework to numerically study two typical cell fate decision processes: the lysis-lysogeny transition and stem cell development. The application of TPT to the lysis-lysogeny decision-making system reveals that the competitions of CI and Cro dimer binding play the major role in determining the cell fates. We also quantified the transition rates from the lysogeny to lysis state under different conditions. The overall computational results are consistent with biological intuitions but with more detailed information. For the stem cell developmental system, we applied the MSM to reduce the original dynamics to a moderate-size Markov chain. Further spectral analysis showed that the reduced system exhibits nine metastable states, which correspond to the refinement of the five known typical cell types in development. We further investigated the dominant transition pathways corresponding to the cell differentiation, reprogramming, and trans-differentiation. A similar approach can be applied to study other biological systems.

Melatonin has a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/secretion / Melatonina tem efeito estimulador em osteoblastos, pela regulação positiva da expressão/secreção de COL-I e OPN

ABSTRACT Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.

RESUMO A melatonina (MLT) é uma molécula potencial de sinalização na homeostase do metabolismo ósseo e pode ser um importante mediador da formação e estimulação óssea. O objetivo deste estudo in vitro foi avaliar o efeito da MLT na viabilidade, na expressão do mRNA da proteína e mineralização de células préosteoblásticas. As concentrações de MLT 5, 2,5, 1, 0,1 e 0,01 mM foram testadas em células pré-osteoblásticas da linhagem MC3T3 em comparação ao controle (sem MLT), avaliando a proliferação e a viabilidade celular (C50), expressão gênica (rtPCR) e secreção (Elisa) de Colágeno tipo 1 (COL-I) e osteopontina (OPN) às 24, 48 e 72 horas, além da formação de nódulos minerais por meio do teste vermelho de Alizarina fast red após 10 dias de tratamento. MLT a 5 e 2,5 mM provou ser tóxico (C50). Portanto, as concentrações de 0,01, 0,1 e 1 mM foram utilizadas para as análises subsequentes. A expressão do mRNA da OPN aumentou com MLT a 0,1 mM-1mM, seguida pela secreção aumentada de OPN às 24 e 72 horas em comparação aos demais grupos (p<0,05). O mRNA de COL-I e a secreção de COL-I seguiram o mesmo padrão do OPN a 0,1 mM de MLT em 72 horas de tratamento (p<0,05). Em relação à mineralização, todas as doses de MLT (exceto 1mM) causaram aumento (p<0,05) na formação de nódulos minerais em comparação ao controle. A MLT na concentração entre 0,01mM a 1 mM teve um efeito estimulador sobre os osteoblastos, ao regular positivamente a expressão e secreção de COL-I e OPN, além da mineralização, favorecendo a osteogênese.

Assessment of Expression of Homeobox A5 in Endometrial Cancer on the mRNA and Protein Level.

BACKGROUND: Endometrial cancer is one of the most common gynecological cancer in the developed countries and occurs mainly in postmenopausal women. Angiogenesis is important for cancer formation as it provides nutrients for growing tumor mass. Most tumors do not show detectable Homeobox A5 (HOXA5 level), suggesting its potential role as a cancer suppressor. It was demonstrated that HOXA5 is involved in the progression of various types of cancer and the loss of its expression correlates with higher pathological grade and poorer outcome. OBJECTIVE: The aim of the study was to evaluate HOXA5 expression at transcriptome and protein levels. MATERIALS AND METHODS: The study enrolled 45 women diagnosed with endometrial cancer and 15 without neoplastic changes. The histopathological examination allowed us to divide cancer tissue samples according to the degree of histological differentiation: G1, 17; G2, 15; G3, 13. The expression of the HOXA5 protein was determined by immunohistochemistry. Microarray and RT-qPCR techniques were used to assess HOXA5 expression at the mRNA level. RESULTS: The reaction to the HOXA5 protein was only visible in glandular cells in G1 endometrial cancer and was lower compared to the control. In grades 2 and 3, reactions were noted at the limit of the method's sensitivity. In addition, reduced HOXA5 expression was observed at the transcriptome level. CONCLUSION: HOXA5 may become a potential complementary molecular marker, allowing early detection of neoplastic changes in the endometrium. It also seems that detection of HOXA5 at the mRNA and protein levels may be helpful in improving the accuracy of diagnosis and planning effective oncological therapy.

A novel dual reporter embryonic stem cell line for toxicological assessment of teratogen-induced perturbation of anterior-posterior patterning of the heart.

Reliable in vitro models to assess developmental toxicity of drugs and chemicals would lead to improvement in fetal safety and a reduced cost of drug development. The validated embryonic stem cell test (EST) uses cardiac differentiation of mouse embryonic stem cells (mESCs) to predict in vivo developmental toxicity, but does not take into account the stage-specific patterning of progenitor populations into anterior (ventricular) and posterior (atrial) compartments. In this study, we generated a novel dual reporter mESC line with fluorescent reporters under the control of anterior and posterior cardiac promoters. Reporter expression was observed in nascent compartments in transgenic mouse embryos, and mESCs were used to develop differentiation assays in which chemical modulators of Wnt (XAV939: 3, 10 µM), retinoic acid (all-trans retinoic acid: 0.1, 1, 10 µM; 9-cis retinoic acid: 0.1, 1, 10 µM; bexarotene 0.1, 1, 10 µM), and Tgf-ß (SB431542: 3, 10 µM) pathways were tested for stage- and dose-dependent effects on in vitro anterior-posterior patterning. Our results suggest that with further development, the inclusion of anterior-posterior reporter expression could be part of a battery of high-throughput tests used to identify and characterize teratogens.

A genome-wide assessment of the ancestral neural crest gene regulatory network.

The neural crest (NC) is an embryonic cell population that contributes to key vertebrate-specific features including the craniofacial skeleton and peripheral nervous system. Here we examine the transcriptional and epigenomic profiles of NC cells in the sea lamprey, in order to gain insight into the ancestral state of the NC gene regulatory network (GRN). Transcriptome analyses identify clusters of co-regulated genes during NC specification and migration that show high conservation across vertebrates but also identify transcription factors (TFs) and cell-adhesion molecules not previously implicated in NC migration. ATAC-seq analysis uncovers an ensemble of cis-regulatory elements, including enhancers of Tfap2B, SoxE1 and Hox-α2 validated in the embryo. Cross-species deployment of lamprey elements identifies the deep conservation of lamprey SoxE1 enhancer activity, mediating homologous expression in jawed vertebrates. Our data provide insight into the core GRN elements conserved to the base of the vertebrates and expose others that are unique to lampreys.