The dentate gyrus of the mammalian hippocampus continuously generates new neurons during adulthood. These adult-born neurons become functionally active and are thought to contribute to learning and memory, especially during their maturation phase, when they have extraordinary plasticity. In this Review, we discuss the molecular machinery involved in the generation of new neurons from a pool of adult neural stem cells and their integration into functional hippocampal circuits. We also summarize the potential functions of these newborn neurons in the adult brain, their contribution to behavior, and their relevance to disease.

Recent breakthroughs in pluripotent stem cell technologies have enabled a new class of in vitro systems for functional modeling of human brain development. These advances, in combination with improvements in neural differentiation methods, allow the generation of in vitro systems that reproduce many in vivo features of the brain with remarkable similarity. Here, we describe advances in the development of these methods, focusing on neural rosette and organoid approaches, and compare their relative capabilities and limitations. We also discuss current technical hurdles for recreating the cell-type complexity and spatial architecture of the brain in culture and offer potential solutions.

Neural circuit function can be drastically affected by variations in the number of cells that are produced during development or by a reduction in adult cell number owing to disease. For this reason, unique cell cycle and cell growth control mechanisms operate in the developing and adult brain. In Drosophila melanogaster and in mammalian neural stem and progenitor cells, these mechanisms are intricately coordinated with the developmental age and the nutritional, metabolic and hormonal state of the animal. Defects in neural stem cell proliferation that result in the generation of incorrect cell numbers or defects in neural stem cell differentiation can cause microcephaly or megalencephaly.

Limited access to human neurons has posed a significant barrier to progress in biological and preclinical studies of the human nervous system. The advent of cell reprogramming technologies has widely disclosed unprecedented opportunities to generate renewable sources of human neural cells for disease modeling, drug discovery, and cell therapeutics. Both somatic reprogramming into induced pluripotent stem cells (iPSCs) and directly induced Neurons (iNeurons) rely on transcription factor-based cellular conversion processes. Nevertheless, they rely on very distinct mechanisms, biological barriers, technical limitations, different levels of efficiency, and generate neural cells with distinctive properties. Human iPSCs represent a long-term renewable source of neural cells, but over time genomic aberrations might erode the quality of the cultures and the in vitro differentiation process requires extensive time. Conversely, direct neuronal reprogramming ensures a fast and straightforward generation of iNeurons endowed with functional properties. However, in this last case, conversion efficiency is reduced when starting from adult human cells, and the molecular and functional fidelity of iNeurons with respect to their corresponding native neuronal subtype is yet to be fully ascertained in many cases. For any biomedical research application, it should be carefully pondered the reprogramming method to use for generating reprogrammed human neuronal subtypes that best fit with the following analysis considering the existing limitations and gap of knowledge still present in this young field of investigation.

The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming.

Cyclins, cyclin-dependent kinases and other components of the core cell cycle machinery drive cell division. Growing evidence indicates that this machinery operates in a distinct fashion in some mammalian stem cell types, such as pluripotent embryonic stem cells. In this Review, we discuss our current knowledge of how cell cycle proteins mechanistically link cell proliferation, pluripotency and cell fate specification. We focus on embryonic stem cells, induced pluripotent stem cells and embryonic neural stem/progenitor cells.

The scarcity of live human brain cells for experimental access has for a long time limited our ability to study complex human neurological disorders and elucidate basic neuroscientific mechanisms. A decade ago, the development of methods to reprogramme somatic human cells into induced pluripotent stem cells enabled the in vitro generation of a wide range of neural cells from virtually any human individual. The growth of methods to generate more robust and defined neural cell types through reprogramming and direct conversion into induced neurons has led to the establishment of various human reprogramming-based neural disease models.

Adult neural stem cells originate from the embryonic brain, but the underlying mechanisms remain poorly known. In this issue of Neuron, Falk et al. (2017) reveal how the timely control of cleavage plane orientation during division of embryonic neural progenitors has a specific and long-lasting impact on adult neurogenesis.

In the adult rodent brain, neural stem cells (NSCs) persist in the ventricular-subventricular zone (V-SVZ) and the subgranular zone (SGZ), which are specialized niches in which young neurons for the olfactory bulb (OB) and hippocampus, respectively, are generated. Recent studies have significantly modified earlier views on the mechanisms of NSC self-renewal and neurogenesis in the adult brain. Here, we discuss the molecular control, heterogeneity, regional specification and cell division modes of V-SVZ NSCs, and draw comparisons with NSCs in the SGZ. We highlight how V-SVZ NSCs are regulated by local signals from their immediate neighbors, as well as by neurotransmitters and factors that are secreted by distant neurons, the choroid plexus and vasculature. We also review recent advances in single cell RNA analyses that reveal the complexity of adult neurogenesis. These findings set the stage for a better understanding of adult neurogenesis, a process that one day may inspire new approaches to brain repair.

Recent advancements in the research field of stem cell biology have enabled the realization of regenerative medicine in various systems of the body, including the central nervous system. However, fundamental knowledge regarding how neural stem cells divide and generate young neurons in mammals, especially in vivo, is still inadequate. In this article, we shall summarize the concept of cell cycle/division of neural stem cells that generate projection neurons in the murine cerebral cortex. We shall also review the molecular mechanisms that modulate the critical parameters related to the cell cycle regulatory mechanisms, with special reference to the cell cycle regulatory protein p27(Kip1) , an inhibitor of progression of the cell cycle at the G1 phase. A better understanding of the mechanisms controlling cell cycle progression is expected to contribute to the development of novel strategies to increase the efficiency of neural cell/tissue production, both in vivo and in vitro.

Neuroepithelial precursor cells of the vertebrate central nervous system either self-renew or differentiate into neurons, oligodendrocytes or astrocytes under the influence of a gene regulatory network that consists in transcription factors, epigenetic modifiers and microRNAs. Sox transcription factors are central to this regulatory network, especially members of the SoxB, SoxC, SoxD, SoxE and SoxF groups. These Sox proteins are widely expressed in neuroepithelial precursor cells and in newly specified, differentiating and mature neurons, oligodendrocytes and astrocytes and influence their identity, survival and development. They exert their effect predominantly at the transcriptional level but also have substantial impact on expression at the epigenetic and posttranscriptional levels with some Sox proteins acting as pioneer factors, recruiting chromatin-modifying and -remodelling complexes or influencing microRNA expression. They interact with a large variety of other transcription factors and influence the expression of regulatory molecules and effector genes in a cell-type-specific and temporally controlled manner. As versatile regulators with context-dependent functions, they are not only indispensable for central nervous system development but might also be instrumental for the development of reprogramming and cell conversion strategies for replacement therapies and for assisted regeneration after injury or degeneration-induced cell loss in the central nervous system.

Human pluripotent stem cells have the unique properties of being able to proliferate indefinitely in their undifferentiated state and to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. Critical among the applications for the latter are diseases and injuries of the nervous system, medical approaches to which have been, to date, primarily palliative in nature. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among these are methods for the generation of such divergent neural cells as dopaminergic neurons, retinal neurons, ventral motoneurons, and oligodendroglial progenitors. In this review, we attempt to fully describe most of these methods, breaking them down into five basic subdivisions: (1) starting material, (2) induction of loss of pluripotency, (3) neural induction, (4) neural maintenance and expansion, and (5) neuronal/glial differentiation. We also show data supporting the concept that undifferentiated human pluripotent stem cells appear to have an innate neural differentiation potential. In addition, we evaluate data comparing and contrasting neural stem cells differentiated from human pluripotent stem cells with those derived directly from the human brain.

Mesenchymal Stem Cells (MSCs) are a bone marrow-derived population present in adult tissues that possess the important property of dividing when called upon and of differentiating into specialized cells. The evidence that MSCs were able to transdifferentiate into specialized cells of tissues different from bone marrow, in particular into nervous cells, opened up the possibility of using MSCs to substitute damaged neurons, that are normally not replaced but lost, in order to repair the Nervous System. The first neuronal differentiation protocols were based on the use of a mixture of toxic drugs which induced MSCs to rapidly acquire a neuronal-like morphology with the expression of specific neuronal markers. However, many subsequent studies demonstrated that the morphological and molecular modifications of MSCs were probably due to a stress response, rather than to a real differentiation into neuronal cells, thus throwing into question the possible use of MSCs to repair the nervous system. Currently, some papers are suggesting again that it may be possible to induce neuronal differentiation of MSCs by using several differentiation protocols, and by accompanying the morphological evidence of differentiation with functional evidence, thus demonstrating that MSC-derived cells not only seem to be neurons, but that they also function like neurons. In this review, we have attempted to shed light on the capacity of MSCs to genuinely differentiate into nervous cells, and to identify the most reliable protocols for obtaining neurons from MSCs for nervous system repair.

While the pace of traumatic brain injury (TBI) research has accelerated, the treatment options remain limited. Clinical trials are yet to yield successful treatment options, leading to innovative strategies to overcome the severe debilitating consequences of TBI. Stem cells may act as a potential treatment option. They have two key characteristics, the ability of self-renewal and the ability to give rise to daughter cells, which in the case of neural stem cells (NSCs) includes neurons, astrocytes and oligodendrocytes. They respond to the injury environment providing trophic support and have been shown to differentiate and integrate into the host brain. In this review, we introduce the notion of an NSC and describe the two neurogenic niches in the mammalian brain. The literature supporting the activation of an NSC in rodent models of TBI, both in vivo and in vitro, is detailed. This endogenous activation of NSCs may be augmented by exogenous transplantation of NSCs. Delivery of NSCs to assist the host nervous system has become an attractive option, with either fetal or adult NSC. This has resulted in cognitive and functional improvement in rodents, and current animal studies are using human NSCs. While no NSC clinical trials are currently ongoing for TBI, this review touches upon other neurological diseases and discuss how this may move forward into TBI.

With the recent start of the first clinical trial evaluating a human embryonic stem cell-derived therapy for the treatment of acute spinal cord injury, it is important to review the current literature examining the use of embryonic stem cells for neural tissue engineering applications with a focus on diseases and disorders that affect the central nervous system. Embryonic stem cells exhibit pluripotency and thus can differentiate into any cell type found in the body, including those found in the nervous system. A range of studies have investigated how to direct the differentiation of embryonic cells into specific neural phenotypes using a variety of cues to achieve the goal of replacing diseased or damaged neural tissue. Additionally, the recent development of induced pluripotent stem cells provides an intriguing alternative to the use of human embryonic stem cell lines for these applications. This review will discuss relevant studies that have used embryonic stem cells to replicate the tissue found in the central nervous system as well as evaluate the potential of induced pluripotent stem cells for the aforementioned applications.

Motor neuron diseases (MNDs) are a group of neurological disorders that selectively affect motor neurons. There are currently no cures or efficacious treatments for these diseases. In recent years, significant developments in stem cell research have been applied to MNDs, particularly regarding neuroprotection and cell replacement. However, a consistent source of motor neurons for cell replacement is required. Human embryonic stem cells (hESCs) could provide an inexhaustible supply of differentiated cell types, including motor neurons that could be used for MND therapies. Recently, it has been demonstrated that induced pluripotent stem (iPS) cells may serve as an alternative source of motor neurons, since they share ES characteristics, self-renewal, and the potential to differentiate into any somatic cell type. In this review, we discuss several reproducible methods by which hESCs or iPS cells are efficiently isolated and differentiated into functional motor neurons, and possible clinical applications.

Nestin-expressing stem cells of the hair follicle, discovered by our laboratory, have been shown to be able to form neurons and other nonfollicle cell types. We have shown that the nestin-expressing stem cells from the hair follicle can effect the repair of peripheral nerve and spinal cord injury. The hair follicle stem cells differentiate into neuronal and glial cells after transplantation to the injured peripheral nerve and spinal cord, and enhance injury repair and locomotor recovery. We have termed these cells hair follicle-accessible pluripotent (HAP) stem cells. When the excised hair follicle with its nerve stump was placed in Gelfoam 3D histoculture, HAP stem cells grew and extended the hair follicle nerve which consisted of βIII-tubulin-positive fibers with F-actin expression at the tip. These findings indicate that βIII-tubulin-positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in HAP stem cells, which appeared to play a major role in its elongation and interaction with other nerves in 3D Gelfoam histoculture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. Our results suggest that a major function of the HAP stem cells in the hair follicle is for growth of the follicle sensory nerve. HAP stem cells have critical advantages over embryonic stem cells and induced pluripotent stem cells in that they are highly accessible, require no genetic manipulation, are nontumorigenic, and do not present ethical issues for regenerative medicine.

Pluripotent stem cells (embryonic stem cells, ESCs, and induced pluripotent stem cells, iPSCs) have the capacity to generate neural progenitors that are intrinsically patterned to undergo differentiation into specific neuronal subtypes and express in vivo properties that match the ones formed during normal embryonic development. Remarkable progress has been made in this field during recent years thanks to the development of more refined protocols for the generation of transplantable neuronal progenitors from pluripotent stem cells, and the access to new tools for tracing of neuronal connectivity and assessment of integration and function of grafted neurons. Recent studies in brains of neonatal mice or rats, as well as in rodent models of brain or spinal cord damage, have shown that ESC- or iPSC-derived neural progenitors can be made to survive and differentiate after transplantation, and that they possess a remarkable capacity to extend axons over long distances and become functionally integrated into host neural circuitry. Here, we summarize these recent developments in the perspective of earlier studies using intracerebral and intraspinal transplants of primary neurons derived from fetal brain, with special focus on the ability of human ESC- and iPSC-derived progenitors to reconstruct damaged neural circuitry in cortex, hippocampus, the nigrostriatal system and the spinal cord, and we discuss the intrinsic and extrinsic factors that determine the growth properties of the grafted neurons and their capacity to establish target-specific long-distance axonal connections in the damaged host brain.

In 2003, human amniotic fluid has been shown to contain stem cells expressing Oct-4, a marker for pluripotency. This finding initiated a rapidly growing and very promising new stem cell research field. Since then, amniotic fluid stem (AFS) cells have been demonstrated to harbour the potential to differentiate into any of the three germ layers and to form three-dimensional aggregates, so-called embryoid bodies, known as the principal step in the differentiation of pluripotent stem cells. Marker selection and minimal dilution approaches allow the establishment of monoclonal AFS cell lineages with high proliferation potential. AFS cells have a lower risk for tumour development and do not raise the ethical issues of embryonic stem cells. Compared to induced pluripotent stem cells, AFS cells do not need exogenic treatment to induce pluripotency, are chromosomal stable and do not harbour the epigenetic memory and accumulated somatic mutations of specific differentiated source cells. Compared to adult stem cells, AFS can be grown in larger quantities and show higher differentiation potential. Accordingly, in the recent past, AFS became increasingly accepted as an optimal tool for basic research and probably also for specific cell-based therapies. Here, we review the current knowledge on the neurogenic differentiation potential of AFS cells.

Spinal cord injury (SCI) often results in significant dysfunction and disability. A series of treatments have been proposed to prevent and overcome the formation of the glial scar and inhibitory factors to axon regrowth. In the last decade, cell therapy has emerged as a new tool for several diseases of the nervous system. Stem cells act as minipumps providing trophic and immunomodulatory factors to enhance axonal growth, to modulate the environment, and to reduce neuroinflammation. This capability can be boosted by genetical manipulation to deliver trophic molecules. Different types of stem cells have been tested, according to their properties and the therapeutic aims. They differ from each other for origin, developmental stage, stage of differentiation, and fate lineage. Related to this, stem cells differentiating into neurons could be used for cell replacement, even though the feasibility that stem cells after transplantation in the adult lesioned spinal cord can differentiate into neurons, integrate within neural circuits, and emit axons reaching the muscle is quite remote. The timing of cell therapy has been variable, and may be summarized in the acute and chronic phases of disease, when stem cells interact with a completely different environment. Even though further experimental studies are needed to elucidate the mechanisms of action, the therapeutic, and the side effects of cell therapy, several clinical protocols have been tested or are under trial. Here, we report the state-of-the-art of cell therapy in SCI, in terms of feasibility, outcome, and side effects.

PURPOSE OF REVIEW:To review the current medical and stem-cell therapy for spinal muscular atrophy (SMA) and prenatal transplantation of amniotic fluid stem cells in the future. RECENT FINDINGS:SMA is an autosomal recessive inheritance of neurodegenerative disease, which is caused of the mutation in survival motor neuron. The severe-type SMA patients usually die from the respiratory failure within 2 years after birth. Recently, researchers had found that 3-methyladenine could inhibit the autophagy and had the capacity to reduce death of the neurons. The first food and drug administration-approved drug to treat SMA, Nusinersen, is a modified antisense oligonucleotide to target intronic splicing silencer N1 just recently launched. Not only medical therapy, but also stem cells including neural stem cells, embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells could show the potential to repair the injured tissue and differentiate into neuron cells to rescue the SMA animal models. Human amniotic fluid stem cells (HAFSCs) share the potential of mesenchymal stem cells and could differentiate into tri-lineage-relative cells, which are also having the ability to restore the injured neuro-muscular function. In this review, we further demonstrate the therapeutic effect of using HAFSCs to treat type III SMA prenatally. HAFSCs, similar to other stem cells, could also help the improvement of SMA with even longer survival. SUMMARY:The concept of prenatal stem-cell therapy preserves the time window to treat disease in utero with much less cell number. Stem cell alone might not be enough to correct or cure the SMA but could be applied as the additional therapy combined with antisense oligonucleotide in the future.

PURPOSE:Currently, the only available and approved treatments for glaucoma are various pharmacologic, laser-based, and surgical procedures that lower IOP. Although these treatments can be effective, they are not always sufficient, and they cannot restore vision that has already been lost. The goal of this review is to briefly assess current developments in the application of stem cell biology to the study and treatment of glaucoma and other forms of optic neuropathy. METHODS:A combined literature review and summary of the glaucoma-related discussion at the 2015 "Sight Restoration Through Stem Cell Therapy" meeting that was sponsored by the Ocular Research Symposia Foundation (ORSF). RESULTS:Ongoing advancements in basic and eye-related developmental biology have enabled researchers to direct murine and human stem cells along specific developmental paths and to differentiate them into a variety of ocular cell types of interest. The most advanced of these efforts involve the differentiation of stem cells into retinal pigment epithelial cells, work that has led to the initiation of several human trials. More related to the glaucoma field, there have been recent advances in developing protocols for differentiation of stem cells into trabecular meshwork and retinal ganglion cells. Additionally, efforts are being made to generate stem cell-derived cells that can be used to secrete neuroprotective factors. CONCLUSIONS:Advancing stem cell technology provides opportunities to improve our understanding of glaucoma-related biology and develop models for drug development, and offers the possibility of cell-based therapies to restore sight to patients who have already lost vision.

Dental pulp stem cells (DPSC) are a relatively new alternative stem cell source for the study of neurogenetic disorders. DPSC can be obtained non-invasively and collected from long-distances remaining viable during transportation. These highly proliferative cells express stem cell markers and retain the ability to differentiate down multiple cell lineages including chondrocytes, adipocytes, osteoblasts, and multiple neuronal cell types. The neural crest origin of DPSC makes them a useful source of primary cells for modeling neurological disorders at the molecular level. In this brief review, we will discuss recent developments in DPSC research that highlight the molecular etiology of DPSC derived neurons and how they may contribute to our understanding of neurogenetic disorders.

Adult human stem cells have gained progressive interest as a promising source of autologous cells to be used as therapeutic vehicles. Particularly, mesenchymal stem cells (MSCs) represent a great tool in regenerative medicine because of their ability to differentiate into a variety of specialized cells. Among adult tissues in which MSCs are resident, adipose tissue has shown clear advantages over other sources of MSCs (ease of surgical access, availability, and isolation), making adipose tissue the ideal large-scale source for research on clinical applications. Stem cells derived from the adipose tissue (adipose-derived stem cells = ADSCs) possess a great and unique regenerative potential: they are self-renewing and can differentiate along several mesenchymal tissue lineages (adipocytes, osteoblasts, myocytes, chondrocytes, endothelial cells, and cardiomyocytes), among which neuronal-like cells gained particular interest. In view of the promising clinical applications in tissue regeneration, research has been conducted towards the creation of a successful protocol for achieving cells with a well-defined neural phenotype from adipose tissue. The promising results obtained open new scenarios for innovative approaches for a cell-based treatment of neurological degenerative disorders.

Human pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine as they are an important source of functional cells for potential cell replacement. These human PSCs, similar to their counterparts of mouse, have the full potential to give rise to any type of cells in the body. However, for the promise to be fulfilled, it is necessary to convert these PSCs into functional specialized cells. Using the developmental principles of neural lineage specification, human ESCs and iPSCs have been effectively differentiated to regional and functional specific neurons and glia, such as striatal gama-aminobutyric acid (GABA)-ergic neurons, spinal motor neurons and myelin sheath forming oligodendrocytes. The human PSCs, in general differentiate after the similar developmental program as that of the mouse: they use the same set of cell signaling to tune the cell fate and they share a conserved transcriptional program that directs the cell fate transition. However, the human PSCs, unlike their counterparts of mouse, tend to respond divergently to the same set of extracellular signals at certain stages of differentiation, which will be a critical consideration to translate the animal model based studies to clinical application.

The abilities of stem cells to self-renew and form different mature cells expand the possibilities of applications in cell-based therapies such as tissue recomposition in regenerative medicine, drug screening, and treatment of neurodegenerative diseases. In addition to stem cells found in the embryo, various adult organs and tissues have niches of stem cells in an undifferentiated state. In the central nervous system of adult mammals, neurogenesis occurs in two regions: the subventricular zone and the dentate gyrus in the hippocampus. The generation of the different neural lines originates in adult neural stem cells that can self-renew or differentiate into astrocytes, oligodendrocytes, or neurons in response to specific stimuli. The regulation of the fate of neural stem cells is a finely controlled process relying on a complex regulatory network that extends from the epigenetic to the translational level and involves extracellular matrix components. Thus, a better understanding of the mechanisms underlying how the process of neurogenesis is induced, regulated, and maintained will provide elues for development of novel for strategies for neurodegenerative therapies. In this review, we focus on describing the mechanisms underlying the regulation of the neuronal differentiation process by transcription factors, microRNAs, and extracellular matrix components.

Various types of stem cells reside in the skin, including keratinocyte progenitor cells, melanocyte progenitor cells, skin-derived precursors (SKPs), and nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells. HAP stem cells, located in the bulge area of the hair follicle, have been shown to differentiate to nerve cells, glial cells, keratinocytes, smooth muscle cells, cardiac muscle cells, and melanocytes. HAP stem cells are positive for the stem-cell marker CD34, as well as K15-negative, suggesting their relatively undifferentiated state. Therefore, HAP stem cells may be the most primitive stem cells in the skin. Moreover, HAP stem cells can regenerate the epidermis and at least parts of the hair follicle. These results suggest that HAP stem cells may be the origin of other stem cells in the skin. Transplanted HAP stem cells promote the recovery of peripheral-nerve and spinal-cord injuries and have the potential for heart regeneration as well. HAP stem cells are readily accessible from everyone, do not form tumors, and can be cryopreserved without loss of differentiation potential. These results suggest that HAP stem cells may have greater potential than iPS or ES cells for regenerative medicine.

Sympathetic neurons are crucial for maintenance of body homeostasis and regulation of all organs. Diseases can arise from malfunction of sympathetic neurons, including malignancies, hypertension, and genetic disorders. Human pluripotent stem cells (hPSCs) allow modeling of human diseases and the in-depth study of pathologies of specific cell types associated with such disorders. Advances in the ability to differentiate hPSCs in vitro has allowed the generation of specific cell types such as sympathetic neurons, which provides the novel opportunity to study diseases affecting the sympathetic nervous system in the human context. Here, we compare selected recent publications that have achieved the goal of generating sympathetic neurons from hPSCs. We discuss strengths and weaknesses of each approach and debate future improvements and the next steps for using these neurons to better our understanding of sympathetic neuron disorders and their treatments. © 2019 by John Wiley & Sons, Inc.

BACKGROUND:The neural stem cells discovered in the adult ciliary epithelium (CE) in higher vertebrates have emerged as an accessible source of retinal progenitors; these cells can self-renew and possess retinal potential. However, recent studies have cast doubt as to whether these cells could generate functional neurons and differentiate along the retinal lineage. Here, we have systematically examined the pan neural and retinal potential of CE stem cells. RESULTS:Molecular and cellular analysis was carried out to examine the plasticity of CE stem cells, obtained from mice expressing green fluorescent protein (GFP) under the influence of the promoter of the rod photoreceptor-specific gene, Nrl, using the neurospheres assay. Differentiation was induced by specific culture conditions and evaluated by both transcripts and protein levels of lineage-specific regulators and markers. Temporal pattern of their levels were examined to determine the expression of genes and proteins underlying the regulatory hierarchy of cells specific differentiation in vitro. Functional attributes of differentiation were examined by the presence of current profiles and pharmacological mobilization of intracellular calcium using whole cell recordings and Fura-based calcium imaging, respectively. We demonstrate that stem cells in adult CE not only have the capacity to generate functional neurons, acquiring the expression of sodium and potassium channels, but also respond to specific cues in culture and preferentially differentiate along the lineages of retinal ganglion cells (RGCs) and rod photoreceptors, the early and late born retinal neurons, respectively. The retinal differentiation of CE stem cells was characterized by the temporal acquisition of the expression of the regulators of RGCs and rod photoreceptors, followed by the display of cell type-specific mature markers and mobilization of intracellular calcium. CONCLUSIONS:Our study demonstrates the bonafide retinal potential of adult CE stem cells and suggests that their plasticity could be harnessed for clinical purposes once barriers associated with any lineage conversion, i.e., low efficiency and fidelity is overcome through the identification of conducive culture conditions.

OBJECTIVE:To explore whether hypoxic condition could promote the olfactory mucosa mesenchymal stem cells (OM-MSCs) to differentiate into neurons with the olfactory ensheathing cells (OECs) supernatant and the potential mechanisms.
 Methods: The OM-MSCs and OECs were isolated and cultured, and they were identified by flow cytometry and immunofluorescence. The OM-MSCs were divided into three groups: a 3%O2+ HIF-1α inhibitors (lificiguat: YC-1) + OECs supernatant group (Group A) , a 3%O2 + OECs supernatant group (Group B) and a 21%O2 + OECs supernatant group (Control group). The neurons, which were differentiated from OM-MSCs, were assessed by immunofluorescence test. The mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), βIII-tubulin and glial fibrillary acidic portein (GFAP) were detected by quantitative polymerase chain reaction (Q-PCR) and Western blot. The potassium channels were analyzed by patch clamp.
 Results: The neurons differentiated from OM-MSCs expressed the most amount of βIII-tubulin, and the result of Q-PCR showed that HIF-1α expression in the Group B was significantly higher than that in the other groups (all P<0.05). Western blot result showed that the βIII-tubulin protein expression was significantly higher and GFAP protein expression was obviously decreased in the Group B (both P<0.05). The patch clamp test confirmed that the potassium channels in the neurons were activated.
 Conclusion: Hypoxic condition can significantly increase the neuronal differentiation of OM-MSCs by the OECs supernatant and decrease the production of neuroglia cells, which is associated with the activation of HIF-1 signal pathway.

Human hair follicle-associated pluripotent (hHAP) stem cells were isolated from the upper parts of human hair follicles. hHAP stem cells were suspended in DMEM containing 10% fetal bovine serum (FBS) where they differentiated to cardiac muscle cells as well as neurons, glial cells, keratinocytes, and smooth muscle cells. The methods of this chapter are appropriate for use of human hair follicles to produce hHAP stem cells in sufficient quantities for future heart, nerve, and spinal cord regeneration in the clinic.

Human adipose-derived stem cells (hADSCs) have great capacity to differentiate into mesodermal origins as well as nonmesodermal lineages, including neural cells. This valuable feature paves the way for the therapeutic application of hADSCs for neurodegenerative maladies such as Parkinson's disease (PD). We tested the capacity of choroid plexus epithelial cell-conditioned medium (CPEC-CM) alone or cocktailed with knockout serum (KS) to induce dopaminergic (DAergic) differentiation of hADSCs. To this end, hADSCs from lipoaspirate were phenotypically characterized and shown to maintain mesodermal multipotency so that selected media easily differentiated them into osteoblasts, chondrocytes, and adipocytes. To begin inducing hADSC neuronal differentiation, we isolated CPECs from rat brain and expanded them in culture to obtain CPEC-CM. We then treated hADSCs with optimized quantities of collected CPEC-CM, KS, or both. The ADSCs treated with either CPEC-CM or CPEC-CM and KS displayed morphological changes typical of neuron-like phenotypes. As revealed by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), and immunostaining analyses, hADSCs cotreated with CPEC-CM and KS expressed significantly higher levels of neuronal and DAergic markers in comparison with single-treated groups. Moreover, the hADSCs began expressing dopamine-biosynthesizing enzymes mainly after cotreatment with CPEC-CM and KS. Consequently, only cotreated hADSCs were capable of synthesizing and releasing dopamine detectable by high-performance liquid chromatography (HPLC). Finally, hADSCs growing in an ordinary medium were found positive for astrocytic marker glial fibrillary acidic protein (GFAP), but stopped GFAP expression on either single or cotreatments. These combined results suggest that CPEC-CM and KS can synergize to remarkably augment DAergic induction of hADSCs, an effect that has implications for cell replacement therapy for PD and related disorders.

We have previously shown that nestin-expressing hair follicle stem cells from the mouse and human are multipotent and can differentiate into many cell types, including neurons and glial cells. The nestin-expressing hair follicle stem cells can effect nerve and spinal cord repair upon transplantation in mouse models. In the present study, nestin-expressing hair follicle stem cells expressing red fluorescent protein (RFP) were induced by retinoic acid and fetal bovine serum to differentiate and then transplanted together with Matrigel into the transected distal sciatic or tibial nerve stump of transgenic nude mice ubiquitously expressing green fluorescent protein (GFP). Control mice were transplanted with Matrigel only. The transplanted cells appeared neuron like, with large round nuclei and long extensions. Immunofluorescence staining showed that some of the transplanted cells in the distal nerve stump expressed the neuron marker Tuj1 as well as motor neuron markers Isl 1/2 and EN1. These transplanted cells contacted each other as well as host nerve fibers. Two weeks post-transplantation, nerve fibers in the distal sciatic nerve stump of the transplanted mice had greater expression of motor neuron markers and neurotrophic factor-3 than those in the Matrigel-only transplanted mice. Muscle fiber areas in the nestin-expressing stem cell plus Matrigel-transplanted animals were much bigger than that in the Matrigel-only transplanted animals after 4 weeks. The present results suggest that transplanted nestin-expressing hair follicle stem cells can differentiate into motor neurons and reduce muscle atrophy after sciatic nerve transection. This study demonstrates a new and accessible neuron source to reduce muscle atrophy after nerve injury.