Human Neural Crest Stem Cells Research Articles

Machine learning on multiple epigenetic features reveals H3K27Ac as a driver of gene expression prediction across patients with glioblastoma.

Cancer cells show remarkable plasticity and can switch lineages in response to the tumor microenvironment. Cellular plasticity drives invasiveness and metastasis and helps cancer cells to evade therapy by developing resistance to radiation and cytotoxic chemotherapy. Increased understanding of cell fate determination through epigenetic reprogramming is critical to discover how cancer cells achieve transcriptomic and phenotypic plasticity. Glioblastoma is a perfect example of cancer evolution where cells retain an inherent level of plasticity through activation or maintenance of progenitor developmental programs. However, the principles governing epigenetic drivers of cellular plasticity in glioblastoma remain poorly understood. Here, using machine learning (ML) we employ cross-patient prediction of transcript expression using a combination of epigenetic features (ATAC-seq, CTCF ChIP-seq, RNAPII ChIP-seq, H3K27Ac ChIP-seq, and RNA-seq) of glioblastoma stem cells (GSCs). We investigate different ML and deep learning (DL) models for this task and build our final pipeline using XGBoost. The model trained on one patient generalizes to another one suggesting that the epigenetic signals governing gene transcription are consistent across patients even if GSCs can be very different. We demonstrate that H3K27Ac is the epigenetic feature providing the most significant contribution to cross-patient prediction of gene expression. In addition, using H3K27Ac signals from patients-derived GSCs, we can predict gene expression of human neural crest stem cells suggesting a shared developmental epigenetic trajectory between subpopulations of these malignant and benign stem cells. Our cross-patient ML/DL models determine weighted patterns of influence of epigenetic marks on gene expression across patients with glioblastoma and between GSCs and neural crest stem cells. We propose that broader application of this analysis could reshape our view of glioblastoma tumor evolution and inform the design of new epigenetic targeting therapies.

Neural crest stem cells can be induced in vitro from human-induced pluripotent stem cells using a novel protocol free of feeder cells.

Objective: Our knowledge of human neural crest stem cells (NCSCs) is expanding, owing to recent advances in technologies utilizing human-induced pluripotent stem cells (hiPSCs) that generate NCSCs. However, the clinical application of these technologies requires the reduction of xeno-materials. To overcome this significant impediment, this study aimed to devise a novel method to induce NCSCs from hiPSCs without using a feeder cell layer.Materials and Methods: hiPSCs were cultured in feeder-free maintenance media containing the Rho-associated coiled-coil forming kinase inhibitor Y-27632. When the cells reached 50–70% confluence, differentiation was initiated by replacing the medium with knockout serum replacement (KSR) medium containing Noggin and SB431542. The KSR medium was then gradually replaced with increasing concentrations of Neurobasal medium from day 5 to 11.Results: Immunocytochemistry and flow cytometry were performed 12 days after induction of differentiation and revealed that the cells generated from hiPSCs expressed the NCSC markers p75 and HNK-1, but not the hiPSC marker SOX2.Conclusion: These findings demonstrate that hiPSCs were induced to differentiate into NCSCs in the absence of feeder cells.

Comparative Craniofacial Bone Regeneration Capacities of Mesenchymal Stem Cells Derived from Human Neural Crest Stem Cells and Bone Marrow.

Most craniofacial bones are derived from the ectodermal germ layer via neural crest stem cells, which are distinct from mesoderm-derived long bones. However, current craniofacial bone tissue engineering approaches do not account for this difference and utilize mesoderm-derived bone marrow mesenchymal stem cells (BM-MSCs) as a paradigm cell source. The effect of the embryonic origin (ontogeny) of an MSC population on its osteogenic differentiation potential and regenerative ability still remains unresolved. To clarify the effects of MSC ontogeny on bone regenerative ability, we directly compared the craniofacial bone regenerative abilities of an ecto-mesenchymal stem cell (eMSC) population, which is derived from human embryonic stem cells via a neural crest intermediate, with mesodermal adult BM-MSCs. eMSCs showed comparable osteogenic and chondrogenic ability to BM-MSCs in 2-D in vitro culture, but lower adipogenic ability. They exhibited greater proliferation than BM-MSCs and comparable construct mineralization in a well-established 3-D polycaprolactone-tricalcium phosphate (PCL-TCP) scaffold system in vitro. eMSC-derived 3D osteogenic constructs were maintained for longer in a proliferative osteoblast state and exhibited differential levels of genes related to fibroblast growth factor (FGF) signaling compared to BM-MSCs. Although both eMSC and BM-MSC-seeded scaffold constructs could promote bone regeneration in a rat calvarial defect model, eMSC-derived osseous constructs had significantly higher cellularity due to increased number of proliferative (Ki67+) cells than those seeded with BM-MSCs, and exhibited enhanced new bone formation in the defect area as compared to untreated controls. Overall, our study demonstrates the potential of human eMSCs for future clinical use in craniofacial regeneration applications and indicates the importance of considering MSC origin when selecting an MSC source for regenerative applications.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

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

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.

Directed Differentiation of Human Embryonic Stem Cells to Neural Crest Stem Cells, Functional Peripheral Neurons, and Corneal Keratocytes.

Neural crest stem cells (NCSCs) are a transient and multipotent cell population giving rise to various cell types with clinical importance. Isolation of human NCSCs is extremely challenging that limits our knowledge about neural crest development and application. Here, a defined protocol to efficiently direct human embryonic stem cells (hESCs) to NCSCs and multiple neural crest lineages is presented. A unique combination of small molecule inhibitors and growth factors is employed to generate NCSCs from hESCs through a neuroectoderm stage. The self-renewal and multipotent capacities of hESC-derived NCSCs are assessed subsequently. In the feeder-free system, hESC-derived NCSCs (P75+ /HNK1+ /AP2α+ /PAX6- ) in high purity are efficiently generated following neuroectodermal restriction. They can be propagated and differentiated toward multiple neural crest lineages in vitro, such as functional peripheral neurons (β-tubulin III+ /peripherin+ ), mesenchymal stem cells (CD73+ CD90+ CD105+ ), and corneal keratocytes (keratocan+ ). The in vivo developmental potential of hESC-derived NCSCs is confirmed using zebrafish embryos. This report is the first demonstration of efficient differentiation of hESCs into corneal keratocytes as a monolayer in a feeder-free system. Considering the high efficacy of NCSC generation, this new method will be a useful tool for future clinical organ repair and regeneration, such as peripheral nerve regeneration and corneal repair.

Quantitative Multimodal Evaluation of Passaging Human Neural Crest Stem Cells for Peripheral Nerve Regeneration.

Peripheral nerve injury is a major burden to societies worldwide, however, current therapy options (e.g. autologous nerve grafts) are unable to produce satisfactory outcomes. Many studies have shown that stem cell transplantation holds great potential for peripheral nerve repair, and human neural crest stem cells (hNCSCs), which give rise to a variety of tissues in the peripheral nervous system, are particularly promising. NCSCs are one of the best candidates for clinical translation, however, to ensure the viability and quality of NCSCs for research and clinical use, the effect of in vitro cell passaging on therapeutic effects needs be evaluated given that passaging is required to expand NCSCs to meet the demands of transplantation in preclinical research and clinical trials. To date, no study has investigated the quality of NCSCs past the 5th passage in vivo. In this study, we employed a multimodal evaluation system to investigate changes in outcomes between transplantation with 5th (p5) and 6th passage (p6) NCSCs in a 15mm rat sciatic nerve injury and repair model. Using CatWalk gait analysis, gastrocnemius muscle index, electrophysiology, immunohistochemistry, and histomorphometric analysis, we showed that p6 NCSCs demonstrated decreased cell survival, Schwann-cell differentiation, axonal growth, and functional outcomes compared to p5 NCSCs (all p < 0.05). In conclusion, p6 NCSCs showed significantly reduced therapeutic efficacy compared to p5 NCSCs for peripheral nerve regeneration.

Abstract A08: Using directed differentiation of human pluripotent stem cells and gene expression profiling to characterize the cell of origin of neuroblastoma

Abstract Purpose: Neuroblastoma (NB) is one of the most common childhood tumors, accounting for ~15% of all pediatric cancer deaths. Improved neuroblastoma disease modeling is needed to improve our understanding and ultimately lead to the development of more effective therapies. NB is an embryonal tumor of the sympathetic nervous system believed to arise from sympathoadrenal (SA) precursor cells belonging to the primitive neural crest. Because the neural crest is a transient embryologic structure, there have been few studies comparing NB tumors to normal human neural crest stem cells (NCSC). However, recent advances have enabled the in vitro differentiation of neural crest cells from pluripotent stem cells. We compared gene expression profiles of pluripotent stem cells, NCSC, and NB tumors to characterize the gene expression profile of the putative cell of origin of NB as the first step in the development of a human NCSC based model of NB. Methods: Human embryonic and induced pluripotent stem (iPS) cells were differentiated to early NCSC and SA precursors in vitro using modifications of previously published protocols. Specifically, undifferentiated human embryonic stem cell GFP reporter and iPS cell lines were induced to differentiate into SA cells in a sequential, stepwise manner over the course of ~4 weeks. After differentiation, SA precursors were identified by expression of the lineage-specific transcription factors ASCL1 and PHOX2B, as well as other markers including tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). We subsequently compared the gene expression profiles of these cells to early, multi-potent neural crest stem cells, autonomic and enteric neurons, 12 NB cell lines and 39 non-MYCN amplified primary NB tumors (3 stage 1, 5 stage 2, 3 stage 3, 24 stage 4, and 4 stage 4S) using Illumina HT-12 bead arrays. These profiles were also compared to the gene expression profiles of laser-capture microdissected fetal adrenal neuroblasts previously reported by De Preter et. al. (Genome Biology 2006, 7:R84). Results were normalized using quantile normalization and analyzed with principle components analysis (PCA); differential expression analysis was performed using the Linear Models for Microarray Data (Limma) method. Pathway enrichment analysis was performed using DAVID annotation tools and cross-platform normalization was performed using the CONOR package in the R software program. All analyses were corrected for multiple comparisons and results considered significant when False Discovery Rate (FDR) was &amp;lt;0.05. Results: PCA showed that undifferentiated iPS cells and early NCSC cluster relatively closely together and away from primary tumors and fetal adrenal neuroblasts. The NB cell lines and primary tumors do not cluster closely together suggesting there are significant differences between them. Gene expression profiles of in vitro differentiated ASCL1 and PHOX2B(+) SA precursor cells were similar to fetal adrenal neuroblasts and more closely resembled NB tumors than did earlier multi-potent NCSC. Comparison between SA precursor cells, NCSC, and primary tumors identified numerous differentially expressed genes (FDR&amp;lt;0.05) the majority of which are regulators of embryologic and/or neural development (SOX3, DPPA4, HES5, LIN28, IRX2, SOX21, PAX6, FABP7, and OTX2 among others). Most were overexpressed in normal neural crest derivatives relative to NB cell lines and tumors. Conclusions: This study, the first of its kind for this disease, demonstrates the feasibility and potential utility of using human pluripotent stem cells and in vitro derived sympathoadrenal precursors cells to simulate early NB pathogenesis. Gene expression profiling of NB identified down regulation of multiple developmentally important genes of normal neural crest differentiation. Further refinement of this in vitro neuroblastoma tumor model as well as investigations of its disrupted gene pathways is underway. Note: This abstract was not presented at the conference. Citation Format: Stephen S. Roberts, Yudelca Ogando, Irina Ostrovnaya, Faranak Fattahi, Irene Cheung, Nai-Kong V. Cheung, Lorenz Studer, Mark Tomishima. Using directed differentiation of human pluripotent stem cells and gene expression profiling to characterize the cell of origin of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A08.

MycN Is Critical for the Maintenance of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells.

The biologic studies of human neural crest stem cells (hNCSCs) are extremely challenging due to the limited source of hNCSCs as well as ethical and technical issues surrounding isolation of early human embryonic tissues. On the other hand, vast majority of studies on MycN have been conducted in human tumor cells, thus, the role of MycN in normal human neural crest development is completely unknown. In the present study, we determined the role of MycN in hNCSCs isolated from in vitro-differentiating human embryonic stem cells (hESCs). For the first time, we show that suppression of MycN in hNCSCs inhibits cell growth and cell cycle progression. Knockdown of MycN in hNCSCs increases the expression of Cdkn1a, Cdkn2a and Cdkn2b, which encodes the cyclin-dependent kinases p21CIP1, p16 INK4a and p15INK4b. In addition, MycN is involved in the regulation of human sympathetic neurogenesis, as knockdown of MycN enhances the expression of key transcription factors involved in sympathetic neuron differentiation, including Phox2a, Phox2b, Mash1, Hand2 and Gata3. We propose that unlimited source of hNCSCs provides an invaluable platform for the studies of human neural crest development and diseases.

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles.

The hair follicle undergoes lifelong cycling and growth. Previous studies have been focused on epithelial stem cells in the hair follicles. Neural crest stem cells (NCSCs) are pluripotent cells that can persist in adult tissues. We have previously demonstrated that human NCSCs can be isolated from hair follicles. Here, we present a protocol to isolate NCSCs from human hair follicles based on their specific surface-marker expression of CD271/HNK1 or CD271/CD49D (alpha4 integrin). NCSCs can be expanded in the culture as neural spheres or attached cells.

Human neural crest stem cells derived from human pluripotent stem cells.

The neural crest cells give rise to neurons and glia in the peripheral nervous system, which is an important component of the nervous system. Here we developed a scalable process of inducing neural crest stem cells (NCSCs) from hESCs/iPSCs by a combination of growth factors in medium conditioned on stromal cells, and showed that NCSCs could be purified by p75 using FACS. In vitro-expanded NCSCs were able to differentiate into neurons and glia (Schwann cells) of the peripheral nervous system (PNS) as well as mesenchymal derivatives.

Enhanced differentiation of human neural crest stem cells towards the Schwann cell lineage by aligned electrospun fiber matrix

Human pluripotent stem cell-derived neural crest stem cells (NCSCs) provide a promising cell source for generating Schwann cells in the treatment of neurodegenerative diseases and traumatic injuries in the peripheral nervous system. Influencing cell behavior through a synthetic matrix topography has been shown to be an effective approach to directing stem cell proliferation and differentiation. Here we have investigated the effect of nanofiber topography on the differentiation of human embryonic stem cell-derived NCSCs towards the Schwann cell lineage. Using electrospun fibers of different diameters and alignments we demonstrated that aligned fiber matrices effectively induced cell alignment, and that fiber matrices with average diameters of 600nm and 1.6μm most effectively promoted NCSC differentiation towards the Schwann cell lineage compared with random fibers and two-dimensional tissue culture plates. More importantly, human NCSCs that were predifferentiated in Schwann cell medium for 2weeks exhibited higher sensitivity to the aligned fiber topography than undifferentiated NCSCs. This study provides an efficient protocol for Schwann cell derivation by combining an aligned nanofiber matrix and an optimized differentiation medium, and highlights the importance of matching extrinsic matrix signaling with cell intrinsic programming in a temporally specific manner.

Human induced pluripotent stem cell-derived neural crest stem cells integrate into the injured spinal cord in the fetal lamb model of myelomeningocele

Background/PurposeNeurological function in patients with myelomeningocele (MMC) is limited even after prenatal repair. Neural crest stem cells (NCSCs) can improve neurological function in models of spinal cord injury. We aimed to evaluate the survival, integration, and differentiation of human NCSCs derived from induced pluripotent stem cells (iPSC-NCSCs) in the fetal lamb model of MMC. MethodsHuman iPSCs derived from skin fibroblasts were differentiated into NCSCs in vitro, mixed with hydrogel, and seeded on nanofibrous scaffolds for surgical transplantation. Fetal lambs (n=2) underwent surgical MMC creation and repair with iPSC-NCSC seeded scaffolds. Gross necropsy and immunohistochemistry were performed at term. ResultsIPSC-NCSCs expressed NCSC markers, maintained > 95% viability, and demonstrated neuronal differentiation in vitro. Immunohistochemical analysis of repaired spinal cords thirty days after transplantation demonstrated the co-localization of human nuclear mitotic apparatus protein (NuMA) and Neurofilament M subunit (NFM) in the area of spinal cord injury. No gross tumors were identified. ConclusionsHuman iPSC-NCSCs survived, integrated, and differentiated into neuronal lineage in the fetal lamb model of MMC. This is the first description of human stem cell engraftment in a model of fetal MMC and supports the concept of using NCSCs to address spinal cord damage in MMC.

Human Neural Crest Stem Cells Derived from Human ESCs and Induced Pluripotent Stem Cells: Induction, Maintenance, and Differentiation into Functional Schwann Cells

The neural crest (NC) is a transient, multipotent, migratory cell population unique to vertebrates that gives rise to diverse cell lineages. Much of our knowledge of NC development comes from studies of organisms such as chicken and zebrafish because human NC is difficult to obtain because of its transient nature and the limited availability of human fetal cells. Here we examined the process of NC induction from human pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). We showed that NC cells could be efficiently induced from hESCs by a combination of growth factors in medium conditioned on stromal cells and that NC stem cells (NCSCs) could be purified by p75 using fluorescence-activated cell sorting (FACS). FACS-isolated NCSCs could be propagated in vitro in five passages and cryopreserved while maintaining NCSC identity characterized by the expression of a panel of NC markers such as p75, Sox9, Sox10, CD44, and HNK1. In vitro-expanded NCSCs were able to differentiate into neurons and glia (Schwann cells) of the peripheral nervous system, as well as mesenchymal derivatives. hESC-derived NCSCs appeared to behave similarly to endogenous embryonic NC cells when injected in chicken embryos. Using a defined medium, we were able to generate and propagate a nearly pure population of Schwann cells that uniformly expressed glial fibrillary acidic protein, S100, and p75. Schwann cells generated by our protocol myelinated rat dorsal root ganglia neurons in vitro. To our knowledge, this is the first report on myelination by hESC- or iPSC-derived Schwann cells.

Abstract SY15-01: Modeling the initiation of Ewing sarcoma in human neural crest stem cells

Abstract SY15-01: Modeling the initiation of Ewing sarcoma in human neural crest stem cells

Abstract 4239: Ectopic expression of EWS-FLI1 in human neural crest stem cells induces polycomb genes and maintenance of stemness

Abstract Ewing sarcoma family of tumors (ESFT) are aggressive bone and soft tissue tumors of unknown cellular origin. Most ESFT express EWS-FLI1, a chimeric protein which functions as a growth-promoting oncogene in ESFT but is toxic to most normal cells. A major difficulty in understanding EWS-FLI1 function has been the lack of an adequate model in which to study EWS-FLI1-induced transformation. Although the cell of origin of ESFT remains elusive, both mesenchymal (MSC) and neural crest (NCSC) have been implicated. We recently developed the tools to generate NCSC from human embryonic stem cells (hNCSC). In the current study we have used this model to test the hypothesis that NCSC are the cells of origin of ESFT and to evaluate the consequences of EWS-FLI1 expression on human NCSC biology. ESFT demonstrate variable degrees of neuro-mesenchymal differentiation potential. Similarly, we have found that hNCSC can be induced to differentiate into neural, glial and mesenchymal progeny. Significantly, Affymetrix whole genome expression profiling of 32 primary ESFT, 11 normal adult tissues, bone marrow-derived MSC and hNCSC revealed that ESFT are more similar to hNCSC than any other normal tissue, including MSC, thus further implicating NCSC in the origin of ESFT. To evaluate the consequences of EWS-FLI1 on these multipotent stem cells, hNCSC were stably transduced with an EWS-FLI1 lentivirus. Unlike most normal cells, hNCSC tolerated expression of the oncoprotein. Moreover, EWS-FLI1-transduced hNCSC continued to proliferate and maintain EWS-FLI1 expression in culture for several weeks after transduction. Affymetrix HuEx 1.0 expression profiling of hNCSC cells five days post-transduction with EWS-FLI1 demonstrated the expected induction and repression of well-established EWS-FLI1 targets and identified numerous other novel EWS-FLI1-regulated genes that are likely to be cell-type and situation specific. In contrast to control vector-transduced cells, EWS-FLI1 transduced hNCSC reproducibly maintained expression of the NCSC markers p75 and HNK-1, even after transfer to differentiation-inducing conditions. In addition, expression of the polycomb genes BMI1 and EZH2 was consistently and significantly upregulated in EWS-FLI1-transduced cells. In keeping with persistent expression of these stem cell-associated genes, EWS-FLI1-expressing cells retained the ability to form neurospheres upon transfer to non-adherent conditions and also the ability to differentiate into neural and mesenchymal lineages several weeks following transduction. In contrast, after six weeks in differentiation media, control cells had upregulated p16 and had undergone senescence. Together these data implicate NCSC in the origin of ESFT and suggest that EWS-FLI1 enables malignant transformation by inducing maintenance of their multipotent, stem cell state through deregulation of polycomb genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4239.

Isolation and Characterization of Neural Crest Stem Cells Derived From In Vitro–Differentiated Human Embryonic Stem Cells

The neural crest is a transient structure of vertebrate embryos that initially generates neural crest stem cells (NCSCs) which then migrate throughout the body to produce a diverse array of mature tissue types. Due to the rarity of adult NCSCs as well as ethical and technical issues surrounding isolation of early embryonic tissues, biologic studies of human NCSCs are extremely challenging. Thus, much of what is known about human neural crest development has been inferred from model organisms. In this study, we report that functional NCSCs can be rapidly generated and isolated from in vitro-differentiated human embryonic stem cells (hESCs). Using the stromal-derived inducing activity (SDIA) of PA6 fibroblast co-culture we have induced hESCs to differentiate into neural crest. Within 1 week, migrating cells that express the early neural crest markers p75 and HNK1 as well as numerous other genes associated with neural crest induction such as SNAIL, SLUG, and SOX10 are detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the p75-positive population enriches for cells with genetic, phenotypic, and functional characteristics of NCSCs. These p75-enriched cells readily form neurospheres in suspension culture, self-renew to form secondary spheres, and give rise under differentiation conditions to multiple neural crest lineages including peripheral nerves, glial, and myofibroblastic cells. Importantly, these cells differentiate into neural crest derivatives when transplanted into developing chick embryos in vivo. Thus, this SDIA protocol can be used to successfully and efficiently isolate early human NCSCs from hESCs in vitro. This renewable source of NCSCs provides an invaluable source of cells for studies of both normal and disordered human neural crest development.

Human neural crest stem cells transplanted in rat penile corpus cavernosum to repair erectile dysfunction

To investigate the feasibility of applying neural crest stem cells (NCSCs), with multipotent capacity, to repair injury in the penile cavernosum, the HNC10.K10 (K10) immortalized NCSC line was transplanted into the penile cavernosum of adult rats, as one of the causes of erectile dysfunction is damaged penile cavernous smooth muscle cells and sinus endothelial cells. The K10 human NCSC line was generated via transfection of primary cultured NCSC with a retroviral vector encoding v-myc. K10 NCSCs were transplanted into the cavernosum of adult rats. The expression of cell type-specific markers for endothelial cells (CD31 and von Willebrand factor), and specific markers for smooth muscle cells (smooth muscle cell actin, calponin, and desmin) was determined immunohistochemically in the penile cavernosum of rats 2 weeks after transplantation. In the rat cavernosum, transplanted K10 NCSCs identified by human nuclear antigen labelling expressed cell type-specific markers for endothelial cells (CD31 and von Willebrand factor), and specific markers for smooth muscle cells (smooth muscle cell actin, calponin, and desmin) 2 weeks after transplantation. Human NCSCs transplanted into the rat penile corpus cavernosum differentiated into endothelial cells or smooth muscle cells, as shown by their expression of cell type-specific markers for the cell types. It appears that NCSCs are an ideal cell source for reconstructing endothelial and smooth muscle cells in the corpus cavernosum in cell therapy for patients with erectile dysfunction.

Production of immortalized human neural crest stem cells.

Production of immortalized human neural crest stem cells.