Gene Correction Recovers Phagocytosis in Retinal Pigment Epithelium Derived from Retinitis Pigmentosa-Human-Induced Pluripotent Stem Cells.

Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders.

Deciphering retinal diseases through the generation of three dimensional stem cell-derived organoids: Concise Review.

Three-dimensional (3D) retinal organoids, in vitro tissue structures derived from self-organizing cultures of differentiating human embryonic stem cells or induced pluripotent stem cells, could recapitulate some aspects of the cytoarchitectural structure and function of the retina in vivo. 3D retinal organoids display huge potential for the investigation of the pathogenesis of monogenic hereditary eye diseases that are related to the malfunction or degeneration of photoreceptors or retinal ganglion cells by providing an effective in vitro tool with multiple applications. In combination with recent genome editing tools, 3D retinal organoids could also represent a reliable and renewable source of transplantable cells for personalized therapies. In this review, we describe the recent advances in human pluripotent stem cells-derived retinal organoids, determination of their histoarchitecture, complexity, and maturity. We also discuss their application as a means to decipher the pathogenesis of retinal diseases, as well as the main drawbacks and challenges. Stem Cells 2019;37:1496-1504.

FM19G11 and Ependymal Progenitor/Stem Cell Combinatory Treatment Enhances Neuronal Preservation and Oligodendrogenesis after Severe Spinal Cord Injury.

Spinal cord injury (SCI) suffers from a lack of effective therapeutic strategies. We have previously shown that individual therapeutic strategies, transplantation of ependymal stem/progenitor cells of the spinal cord after injury (epSPCi) or FM19G11 pharmacological treatment, induce moderate functional recovery after SCI. Here, the combination of treatments has been assayed for functional and histological analysis. Immediately after severe SCI, one million epSPCi were intramedullary injected, and the FM19G11 compound or dimethyl sulfoxide (DMSO) (as the vehicle control) was administrated via intrathecal catheterization. The combination of treatments, epSPCi and FM19G11, improves locomotor tasks compared to the control group, but did not significantly improve the Basso, Beattie, Bresnahan (BBB) scores for locomotor analysis in comparison with the individual treatments. However, the histological analysis of the spinal cord tissues, two months after SCI and treatments, demonstrated that when we treat the animals with both epSPCi and FM19G11, an improved environment for neuronal preservation was generated by reduction of the glial scar extension. The combinatorial treatment also contributes to enhancing the oligodendrocyte precursor cells by inducing the expression of Olig1 in vivo. These results suggest that a combination of therapies may be an exciting new therapeutic treatment for more efficient neuronal activity recovery after severe SCI.

Connexin 50 modulates Sox2 expression in spinal-cord-derived ependymal stem/progenitor cells.

Ion channels included in the family of Connexins (Cx) have been reported to influence the secondary expansion of traumatic spinal cord injury (SCI) and neuropathic pain following SCI. However, Cxs also contribute to spinal cord neurogenesis during the remyelinating process and functional recovery after SCI. Certain Cxs have been recently related to the control of cell proliferation and the differentiation of neuronal progenitors. Adult spinal-cord-derived ependymal stem progenitor cells (epSPC) show high expression levels of Cx50 in non-pathological conditions and lower expression when they actively proliferate after injury (epSPCi). We explore the role of Cx50 in the ependymal population in the modulation of Sox2, a crucial factor of neural progenitor self-renewal and a promising target for promoting neuronal-cell-fate induction for neuronal tissue repair. Short-interfering-RNA ablation or over-expression of Cx50 regulates the expression of Sox2 in both epSPC and epSPCi. Interestingly, Cx50 and Sox2 co-localize at the nucleus indicating a potential role for this ion channel beyond cell-to-cell communication in the spinal cord. In vivo and in vitro experiments with Clotrimazole, a specific pharmacological modulator of Cx50, show the convergent higher expression of Cx50 and Sox2 in the isolated epSPC/epSPCi and in spinal cord tissue. Therefore, the pharmacological modulation of Cx50 might constitute an interesting mechanism for Sox2 induction to modulate the endogenous regenerative potential of neuronal tissue with a potential application in regenerative therapies.

Connexin 50 Expression in Ependymal Stem Progenitor Cells after Spinal Cord Injury Activation.

Ion channels included in the family of Connexins (Cx) help to control cell proliferation and differentiation of neuronal progenitors. Here we explored the role of Connexin 50 (Cx50) in cell fate modulation of adult spinal cord derived neural precursors located in the ependymal canal (epSPC). epSPC from non-injured animals showed high expression levels of Cx50 compared to epSPC from animals with spinal cord injury (SCI) (epSPCi). When epSPC or epSPCi were induced to spontaneously differentiate in vitro we found that Cx50 favors glial cell fate, since higher expression levels, endogenous or by over-expression of Cx50, augmented the expression of the astrocyte marker GFAP and impaired the neuronal marker Tuj1. Cx50 was found in both the cytoplasm and nucleus of glial cells, astrocytes and oligodendrocyte-derived cells. Similar expression patterns were found in primary cultures of mature astrocytes. In addition, opposite expression profile for nuclear Cx50 was observed when epSPC and activated epSPCi were conducted to differentiate into mature oligodendrocytes, suggesting a different role for this ion channel in spinal cord beyond cell-to-cell communication. In vivo detection of Cx50 by immunohistochemistry showed a defined location in gray matter in non-injured tissues and at the epicenter of the injury after SCI. epSPCi transplantation, which accelerates locomotion regeneration by a neuroprotective effect after acute SCI is associated with a lower signal of Cx50 within the injured area, suggesting a minor or detrimental contribution of this ion channel in spinal cord regeneration by activated epSPCi.

Modulation of hypoxia-inducible factors (HIF) from an integrative pharmacological perspective.

Oxygen homeostasis determines the activity and expression of a multitude of cellular proteins and the interplay of pathways that affect crucial cellular processes for development, physiology, and pathophysiology. Hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in available oxygen in the cellular environment and drives cellular adaptation to such conditions. Selective gene expression under hypoxic conditions is the result of an exquisite regulation of HIF, from the pre-transcriptional stage of the HIF gene to the final transcriptional activity of HIF protein. We provide a dissected analysis of HIF modulation with special focus on hypoxic conditions and HIF pharmacological interventions that can guide the application of any future HIF-mediated therapy.

The activation of dormant ependymal cells following spinal cord injury.

Ependymal cells, a dormant population of ciliated progenitors found within the central canal of the spinal cord, undergo significant alterations after spinal cord injury (SCI). Understanding the molecular events that induce ependymal cell activation after SCI represents the first step toward controlling the response of the endogenous regenerative machinery in damaged tissues. This response involves the activation of specific signaling pathways in the spinal cord that promotes self-renewal, proliferation, and differentiation. We review our current understanding of the signaling pathways and molecular events that mediate the SCI-induced activation of ependymal cells by focusing on the roles of some cell adhesion molecules, cellular membrane receptors, ion channels (and their crosstalk), and transcription factors. An orchestrated response regulating the expression of receptors and ion channels fine-tunes and coordinates the activation of ependymal cells after SCI or cell transplantation. Understanding the major players in the activation of ependymal cells may help us to understand whether these cells represent a critical source of cells contributing to cellular replacement and tissue regeneration after SCI. A more complete understanding of the role and function of individual signaling pathways in endogenous spinal cord progenitors may foster the development of novel targeted therapies to induce the regeneration of the injured spinal cord.

Alzheimer's disease and synapse Loss: What can we learn from induced pluripotent stem Cells?

BACKGROUND: Synaptic dysfunction is a major contributor to Alzheimers disease (AD) pathogenesis in addition to the formation of neuritic ß-amyloid plaques and neurofibrillary tangles of hyperphosphorylated Tau protein. However, how these features contribute to synaptic dysfunction and axonal loss remains unclear. While years of considerable effort have been devoted to gaining an improved understanding of this devastating disease, the unavailability of patient-derived tissues, considerable genetic heterogeneity, and lack of animal models that faithfully recapitulate human AD have hampered the development of effective treatment options. Ongoing progress in human induced pluripotent stem cell (hiPSC) technology has permitted the derivation of patient- and disease-specific stem cells with unlimited self-renewal capacity. These cells can differentiate into AD-affected cell types, which support studies of disease mechanisms, drug discovery, and the development of cell replacement therapies in traditional and advanced cell culture models. AIM OF REVIEW: To summarize current hiPSC-based AD models, highlighting the associated achievements and challenges with a primary focus on neuron and synapse loss. KEY SCIENTIFIC CONCEPTS OF REVIEW: We aim to identify how hiPSC models can contribute to understanding AD-associated synaptic dysfunction and axonal loss. hiPSC-derived neural cells, astrocytes, and microglia, as well as more sophisticated cellular organoids, may represent reliable models to investigate AD and identify early markers of AD-associated neural degeneration.

Transplanted oligodendrocytes and motoneuron progenitors generated from human embryonic stem cells promote locomotor recovery after spinal cord transection.

Human embryonic stem cells (hESC) hold great promise for the treatment of patients with many neurodegenerative diseases particularly those arising from cell loss or neural dysfunction including spinal cord injury. This study evaluates the therapeutic effects of transplanted hESC-derived oligodendrocyte progenitors (OPC) and/or motoneuron progenitors (MP) on axonal remyelination and functional recovery of adult rats after complete spinal cord transection. OPC and/or MP were grafted into the site of injury in the acute phase. Based on Basso-Beattie-Bresnahan scores recovery of locomotor function was significantly enhanced in rats treated with OPC and/or MP when compared with control animals. When transplanted into the spinal cord immediately after complete transection, OPC and MP survived, migrated, and differentiated into mature oligodendrocytes and neurons showing in vivo electrophysiological activity. Taken together, these results indicate that OPC and MP derived from hESC could be a useful therapeutic strategy to repair injured spinal cord.

Activation of Neurogenesis in Multipotent Stem Cells Cultured In Vitro and in the Spinal Cord Tissue After Severe Injury by Inhibition of Glycogen Synthase Kinase-3.

The inhibition of glycogen synthase kinase-3 (GSK-3) can induce neurogenesis, and the associated activation of Wnt/ß-catenin signaling via GSK-3 inhibition may represent a means to promote motor function recovery following spinal cord injury (SCI) via increased astrocyte migration, reduced astrocyte apoptosis, and enhanced axonal growth. Herein, we assessed the effects of GSK-3 inhibition in vitro on the neurogenesis of ependymal stem/progenitor cells (epSPCs) resident in the mouse spinal cord and of human embryonic stem cell-derived neural progenitors (hESC-NPs) and human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPs) and in vivo on spinal cord tissue regeneration and motor activity after SCI. We report that the treatment of epSPCs and human pluripotent stem cell-derived neural progenitors (hPSC-NPs) with the GSK-3 inhibitor Ro3303544 activates ß-catenin signaling and increases the expression of the bIII-tubulin neuronal marker; furthermore, the differentiation of Ro3303544-treated cells prompted an increase in the number of terminally differentiated neurons. Administration of a water-soluble, bioavailable form of this GSK-3 inhibitor (Ro3303544-Cl) in a severe SCI mouse model revealed the increased expression of bIII-tubulin in the injury epicenter. Treatment with Ro3303544-Cl increased survival of mature neuron types from the propriospinal tract (vGlut1, Parv) and raphe tract (5-HT), protein kinase C gamma-positive neurons, and GABAergic interneurons (GAD65/67) above the injury epicenter. Moreover, we observed higher numbers of newly born BrdU/DCX-positive neurons in Ro3303544-Cl-treated animal tissues, a reduced area delimited by astrocyte scar borders, and improved motor function. Based on this study, we believe that treating animals with epSPCs or hPSC-NPs in combination with Ro3303544-Cl deserves further investigation towards the development of a possible therapeutic strategy for SCI.

Activated spinal cord ependymal stem cells rescue neurological function.

Spinal cord injury (SCI) is a major cause of paralysis. Currently, there are no effective therapies to reverse this disabling condition. The presence of ependymal stem/progenitor cells (epSPCs) in the adult spinal cord suggests that endogenous stem cell-associated mechanisms might be exploited to repair spinal cord lesions. epSPC cells that proliferate after SCI are recruited by the injured zone, and can be modulated by innate and adaptive immune responses. Here we demonstrate that when epSPCs are cultured from rats with a SCI (ependymal stem/progenitor cells injury [epSPCi]), these cells proliferate 10 times faster in vitro than epSPC derived from control animals and display enhanced self renewal. Genetic profile analysis revealed an important influence of inflammation on signaling pathways in epSPCi after injury, including the upregulation of Jak/Stat and mitogen activated protein kinase pathways. Although neurospheres derived from either epSPCs or epSPCi differentiated efficiently to oligodendrocites and functional spinal motoneurons, a better yield of differentiated cells was consistently obtained from epSPCi cultures. Acute transplantation of undifferentiated epSPCi or the resulting oligodendrocyte precursor cells into a rat model of severe spinal cord contusion produced a significant recovery of motor activity 1 week after injury. These transplanted cells migrated long distances from the rostral and caudal regions of the transplant to the neurofilament-labeled axons in and around the lesion zone. Our findings demonstrate that modulation of endogenous epSPCs represents a viable cell-based strategy for restoring neuronal dysfunction in patients with spinal cord damage.

Glaucoma as a Neurodegenerative Disease Caused by Intrinsic Vulnerability Factors.

Glaucoma, one of the most common causes of blindness in developing countries today, involves a progressive loss of neural cells in the optic nerve that leads to progressive, irreversible vision loss. Increased intraocular pressure (IOP) presents as a major risk factor for glaucoma, although there exist cases of glaucoma patients with normal IOP that exhibit damage to retinal ganglion cells (RGCs) and the optic nerve. However, treatment approaches have maintained their focus on modifying IOP due to a lack of other modifiable risks factors. Traditional concepts in glaucoma involve the neuronal environment and external effects as a source of causative factors; however, studies have yet to investigate whether the molecular profile of RGCs in glaucoma patients makes them more vulnerable and/or susceptible to external damage. Our hypothesis states that molecular changes at the whole cell, gene expression, and electrophysiological level of the neurons can contribute to their degeneration. Herein, we briefly describe different types of glaucoma and any similarities to different molecular and cellular features of neurodegeneration. To test our hypothesis, we describe human induced pluripotent stem cells (hiPSCs) as a reliable cellular tool to model neurodegenerative aspects of glaucoma to reveal the multiple pathological molecular mechanisms underlying disease development.

Hypoxia causes downregulation of mismatch repair system and genomic instability in stem cells.

The DNA mismatch repair (MMR) system maintains genomic integrity by correcting replication errors: its malfunction causes genomic instability in several tumor types. Hypoxia-inducible factor-1alpha (HIF1alpha), the major regulator of the processes that occur in hypoxia and certain epigenetic events downregulate the expression of MMR genes in cancer cells. However, there is a lack of information regarding MMR regulation and the genetic stability of stem cells under hypoxic conditions. The expression of the MMR system is downregulated in murine and human stem cells cultured in hypoxia, which correlates with lower DNA repair activity in neural stem cells. We observed, through the use of short hairpin loop RNAi expression constructs, that HIF1alpha positively regulated MLH1 and MSH6 when the C17.2 neural stem cells were exposed to short-term hypoxia. However, in prolonged exposure to oxygen depletion, the reduced transcriptional activation of MMR genes was directed by specific epigenetic events. Chromatin immunoprecipitation experiments showed a hypoacetylated/hypermethylated histone H3 and lower SP1 binding within MLH1 and MSH6 adjacent promoter regions. Treatment with the histone deacetylase inhibitor trichostatin A increased histone H3 acetylation and SP1 occupancy and enhanced MMR expression. Sequencing of microsatellite markers revealed genomic instability in the murine and human stem cells grown under hypoxia. Thus, the present article reports, for the first time in the stem cell field, experimental data that indicate that hypoxic niches are an environment in which stem cells might undergo genomic instability, which could lie at the origin of subpopulations with cancer stem cell properties. Disclosure of potential conflicts of interest is found at the end of this article.

Organized Neurogenic-Niche-Like Pinwheel Structures Discovered in Spinal Cord Tissue-Derived Neurospheres.

The neurogenic niche of the subventricular zone (SVZ) in adult brain tissue takes the form of a pinwheel-like cytoarchitectural structure, with mono-ciliated astrocytes displaying neural stem cell (NSC) characteristics present in the core surrounded by ciliated ependymal cells. For the first time, we have demonstrated the formation of similar pinwheel structures in spinal cord and SVZ tissue-derived neurospheres cultured in vitro. To investigate whether the organization and integrity of these pinwheel structures depends on the appropriate organization of ciliated astrocytes and ependymal cells, we modified neurosphere cell arrangements via the application of the methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dc) or the antiviral drug ganciclovir (GCV) in transgenic mice expressing herpes simplex virus thymidine kinase from the GFAP promoter (GFAP-TK). Treatment of neurospheres with 5-aza-dc increased FoxJ1 expression, a crucial factor for ciliogenesis, by reducing methylation of the FoxJ1 CpG island. 5-aza-dc also increased the expression of the astrocyte marker GFAP and caused aberrant accumulation of ciliated astrocytes. However, the ablation of dividing astrocytes within neurospheres by GCV treatment led to an increase in the accumulation of ciliated ependymal cells, as evidenced by the increased expression of the ependymal cell markers Vimentin or CD24. While 5-aza-dc and GCV treatment differentially affected cell arrangement, both compounds significantly diminished the number of pinwheel structures present in neurospheres. Thus, we suggest that the ratio of ciliated astrocytes to ependymal cells plays a crucial role in the correct formation of the pinwheel structures in spinal cord tissue-derived neurospheres in vitro.

The identification of small molecules that stimulate retinal pigment epithelial cells: potential novel therapeutic options for treating retinopathies.

INTRODUCTION: Combinatory strategies using pharmacology and stem cell therapy have emerged due to their potential in the treatment of retinal pigment epithelium (RPE) cell related diseases, and a variety of different stem cell sources have been evaluated both in animal models and in humans. RPE cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (hiPSCs) are already in clinical trials, holding great promise for the treatment of age-related macular disease (AMD) and hereditary RPE-related retinal dystrophies. Highly efficient protocol for RPE generations have been developed, but they are still time-consuming and laborious. Areas covered: The authors review RPE related diseases, as well as the known functions of RPE cells in retinal homeostasis. The authors also discuss small molecules that target RPE in vivo as well as in vitro to aid RPE differentiation from pluripotent stem cells clinically. The authors base this review on literature searches performed through PubMed. Expert opinion: Using high-throughput systems, technology will provide the possibility of identifying and optimizing molecules/drugs that could lead to faster and simpler protocols for RPE differentiation. This could be crucial in moving forward to create safer and more efficient RPE-based personalized therapies.

Assessment of Toxic Effects of Ochratoxin A in Human Embryonic Stem Cells.

Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species, and it is considered a common contaminant in food and animal feed worldwide. On the other hand, human embryonic stem cells (hESCs) have been suggested as a valuable model for evaluating drug embryotoxicity. In this study, we have evaluated potentially toxic effects of OTA in hESCs. By using in vitro culture techniques, specific cellular markers, and molecular biology procedures, we found that OTA produces mild cytotoxic effects in hESCs by inhibiting cell attachment, survival, and proliferation in a dose-dependent manner. Thus, we suggest that hESCs provide a valuable human and cellular model for toxicological studies regarding preimplantation stage of human fetal development.

Overexpression of SPARC protein contrasts with its transcriptional silencing by aberrant hypermethylation of SPARC CpG-rich region in endometrial carcinoma.

Secreted protein acidic and rich in cysteine (SPARC) is a secreted matricellular glycoprotein involved in crucial processes that occur during cancer. This study explored the occurrence of deregulated expression of SPARC in endometrial carcinomas, since it has been associated with the progression of other tumor types. We analyzed the expression of SPARC in endometrial carcinomas by TaqMan, Western blotting and immunohistochemistry. The CpG island methylation status of SPARC was evaluated by bisulfite sequencing method. A significant down-regulation of SPARC mRNA expression (p<0.001) was observed in endometrial tumor tissues, regardless of their microsatellite instability status (MSI). The down-regulation can be accounted for by aberrant hypermethylation of its CpG-rich region, since we demonstrate that SPARC is a frequent target of this epigenetic event in this pathology. Although, differential expression of SPARC is already known in other cancer types, we report that down-regulation of the SPARC gene in endometrial tumors, formed by at least 80% of epithelial tumor cells, contrasts with a frequent overexpression of SPARC protein, with strong immunoreactivity in stromal cells. These results indicate a cell type specific expression of SPARC in endometrial carcinomas. Accumulation of SPARC protein in most tumors compared to normal tissues (p<0.025), suggests an important role in the carcinogenesis of endometrial tumors. SPARC overexpression can be a useful molecular tool that may contribute to the diagnosis of this disease.

Biological and clinical significance of MMP-2, MMP-9, TIMP-1 and TIMP-2 in non-small cell lung cancer.

Our main aim consists of investigating the clinical usefulness of gelatinases and their tissue inhibitors in non-small cell lung cancer (NSCLC). Thus, we have analysed in 111 NSCLCs, levels and activity of MMP-2, MMP-9, TIMP-1 and TIMP-2, by Enzymoimmunoassay and Gelatine zymography, respectively. Our data revealed higher MMP-2 net activity in the NSCLC population analyzed in this study, this parameter showing a significant association with the TNM stage of tumours (P=0.002). Moreover, MMP-9 levels were significantly associated with poor clinical evolution of patients (P=0.02). Also, disease-free survival time was higher for patients whose tumours showed TIMP-1 increased levels (P=0.04). Of interest, Cox multivariate analysis revealed that TIMP-1 levels can be considered as an independent prognostic factor in NSCLC. Relative Risk (RR) to tumour relapse was more than two times lower for patients showing high TIMP-1 levels (RR=0.420, P=0.041). Therefore, according to our results, we conclude that MMP-9 and TIMP-1 levels of synthesis could be useful for the selection of patients with potentially unfavourable clinical evolution in order to establish adjuvant therapy protocols. Among these parameters, TIMP-1 level evaluation emerges as the main factor to predict the clinical outcome of patients.

hiPSC Disease Modeling of Rare Hereditary Cerebellar Ataxias: Opportunities and Future Challenges.

Cerebellar ataxias are clinically and genetically heterogeneous diseases affecting primary cerebellar cells. The lack of availability of affected tissue from cerebellar ataxias patients is the main obstacle in investigating the pathogenicity of these diseases. The landmark discovery of human-induced pluripotent stem cells (hiPSC) has permitted the derivation of patient-specific cells with an unlimited self-renewing capacity. Additionally, their potential to differentiate into virtually any cell type of the human organism allows for large amounts of affected cells to be generated in culture, converting this hiPSC technology into a revolutionary tool in the study of the mechanisms of disease, drug discovery, and gene correction. In this review, we will summarize the current studies in which hiPSC were utilized to study cerebellar ataxias. Describing the currently available 2D and 3D hiPSC-based cellular models, and due to the fact that extracerebellar cells were used to model these diseases, we will discuss whether or not they represent a faithful cellular model and whether they have contributed to a better understanding of disease mechanisms.

Highly Efficient Neural Conversion of Human Pluripotent Stem Cells in Adherent and Animal-Free Conditions.

Neural differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can produce a valuable and robust source of human neural cell subtypes, holding great promise for the study of neurogenesis and development, and for treating neurological diseases. However, current hESCs and hiPSCs neural differentiation protocols require either animal factors or embryoid body formation, which decreases efficiency and yield, and strongly limits medical applications. Here we develop a simple, animal-free protocol for neural conversion of both hESCs and hiPSCs in adherent culture conditions. A simple medium formula including insulin induces the direct conversion of >98% of hESCs and hiPSCs into expandable, transplantable, and functional neural progenitors with neural rosette characteristics. Further differentiation of neural progenitors into dopaminergic and spinal motoneurons as well as astrocytes and oligodendrocytes indicates that these neural progenitors retain responsiveness to instructive cues revealing the robust applicability of the protocol in the treatment of different neurodegenerative diseases. The fact that this protocol includes animal-free medium and human extracellular matrix components avoiding embryoid bodies makes this protocol suitable for the use in clinic. Stem Cells Translational Medicine 2017;6:1217-1226.