Therapeutic function of iPSCs-derived primitive neuroepithelial cells in a rat model of Parkinson's disease.

Induced pluripotent stem cells (iPSCs) are a promising unlimited source for cell replacement therapy of neurodegenerative disorders, including Parkinson's disease (PD). In the present study, rat iPSCs-derived primitive neuroepithelial cells (RiPSCs-iNECs) were successfully induced from rat iPSCs (RiPSCs) following two major developmental stages, and could generate neurospheres and differentiated into both neurons and astrocytes in vitro. Then, the RiPSCs-iNECs-GFP+ were unilaterally transplanted into the right substantia nigra (SN) of 6-hydroxydopamine-lesioned rat models of PD. The results demonstrated that the grafted RiPSCs-iNECs could survive in parkinsonian rat brain for at least 150 days, and many of them differentiated into tyrosine hydroxylase (TH)-positive cells. Furthermore, the PD model rats grafted with RiPSCs-iNECs exhibited a significant functional recovery from their parkinsonian behavioral defects. Histological studies showed that RiPSCs-iNECs could differentiate into multiple types of neurons including dopaminergic neurons, GFAP, Pax6, FoxA2 and DAT-positive cells, and induced dopaminergic neurons extended dense neurites into the host striatum. Thus, iPSCs derived primitive neuroepithelial cells could be an attractive candidate as a source of donor material for the treatment of PD, but the molecular mechanism needs further clarification.

Initial cell maturity changes following transplantation in a hyaluronan-based hydrogel and impacts therapeutic success in the stroke-injured rodent brain.

Ischemic stroke results in a loss of neurons for which there are no available clinical strategies to stimulate regeneration. While preclinical studies have demonstrated that functional recovery can be obtained by transplanting an exogenous source of neural progenitors into the brain, it remains unknown at which stage of neuronal maturity cells will provide the most benefit. We investigated the role of neuronal maturity on cell survival, differentiation, and long-term sensorimotor recovery in stroke-injured rats using a population of human cortically-specified neuroepithelial progenitor cells (cNEPs) delivered in a biocompatible, bioresorbable hyaluronan/methylcellulose hydrogel. We demonstrate that transplantation of immature cNEPs result in the greatest tissue and functional repair, relative to transplantation of more mature neurons. The transplantation process itself resulted in the least cell death and phenotypic changes in the immature cNEPs, and the greatest acute cell death in the mature cells. The latter negatively impacted host tissue and negated any potential positive effects associated with cell maturity and the hydrogel vehicle, which itself showed some functional and tissue benefit. Moreover, we show that more mature cell populations are drastically altered during the transplantation process itself. The phenotype of the cells before and after transplantation had an enormous impact on their survival and the consequent tissue and behavioral response, emphasizing the importance of characterizing injected cells in transplantation studies more broadly.

Grafted Miniature-Swine Neural Stem Cells of Early Embryonic Mesencephalic Neuroepithelial Origin can Repair the Damaged Neural Circuitry of Parkinson's Disease Model Rats.

Although recent progress in the use of human iPS cell-derived midbrain dopaminergic progenitors is remarkable, alternatives are essential in the strategies of treatment of basal-ganglia-related diseases. Attention has been focused on neural stem cells (NSCs) as one of the possible candidates of donor material for neural transplantation, because of their multipotency and self-renewal characteristics. In the present study, miniature-swine (mini-swine) mesencephalic neuroepithelial stem cells (M-NESCs) of embryonic 17 and 18 days grafted in the parkinsonian rat striatum were assessed immunohistochemically, behaviorally and electrophysiologically to confirm their feasibility for the neural xenografting as a donor material. Grafted mini-swine M-NESCs survived in parkinsonian rat striatum at 8 weeks after transplantation and many of them differentiated into tyrosine hydroxylase (TH)-positive cells. The parkinsonian model rats grafted with mini-swine M-NESCs exhibited a functional recovery from their parkinsonian behavioral defects. The majority of donor-derived TH-positive cells exhibited a matured morphology at 8 weeks. Whole-cell recordings from donor-derived neurons in the host rat brain slices incorporating the graft revealed the presence of multiple types of neurons including dopaminergic. Glutamatergic and GABAergic post-synaptic currents were evoked in the donor-derived cells by stimulation of the host site, suggesting they receive both excitatory and inhibitory synaptic inputs from host area. The present study shows that non-rodent mammalian M-NESCs can differentiate into functionally active neurons in the diseased xenogeneic environment and could improve the parkinsonian behavioral defects over the species. Neuroepithelial stem cells could be an attractive candidate as a source of donor material for neural transplantation.

Olfactory Derived Stem Cells Delivered in a Biphasic Conduit Promote Peripheral Nerve Repair In Vivo.

Peripheral nerve injury presents significant therapeutic challenges for recovery of motor and sensory function in patients. Different clinical approaches exist but to date there has been no consensus on the most effective method of treatment. Here, we investigate a novel approach to peripheral nerve repair using olfactory derived stem (ONS) cells delivered in a biphasic collagen and laminin functionalized hyaluronic acid based nerve guidance conduit (NGC). Nerve regeneration was studied across a 10-mm sciatic nerve gap in Sprague Dawley rats. The effect of ONS cell loading of NGCs with or without nerve growth factor (NGF) supplementation on nerve repair was compared to a cell-free NGC across a variety of clinical, functional, electrophysiological, and morphologic parameters. Animals implanted with ONS cell loaded NGCs demonstrated improved clinical and electrophysiological outcomes compared to cell free NGC controls. The nerves regenerated across ONS cell loaded NGCs contained significantly more axons than cell-free NGCs. A return of the nocioceptive withdrawal reflex in ONS cell treated animals indicated an advanced repair stage at a relatively early time point of 8 weeks post implantation. The addition of NGF further improved the outcomes of the repair indicating the potential beneficial effect of a combined stem cell/growth factor treatment strategy delivered on NGCs. Stem Cells Translational Medicine 2017;6:1894-1904.

Conversion of monkey fibroblasts to transplantable telencephalic neuroepithelial stem cells.

Non-human primates provide optimal models for the development of stem cell therapies. Although somatic cells have been converted into neural stem/progenitor cells, it is unclear whether telencephalic neuroepithelial stem cells (NESCs) with stable properties can be generated from fibroblasts in primate. Here we report that a combination of transcription factors (Oct4, Sox2, Klf4) with a new culture medium induces rhesus monkey fibroblasts into NESCs, which can develop into miniature neural tube (NT)-like structures at a cell level. Furthermore, single induced NESCs (iNESCs) can generate later-stage 3D-NTs after grown on matrigel in suspension culture. iNESCs express NT cell markers, have a unique gene expression pattern biasing towards telencephalic patterning, and give rise to cortical neurons. Via transplantation, single iNESCs can extensively survive, regenerate myelinated neuron axons and synapse structures in adult monkey striatum and cortex, and differentiate into cortical neurons. Successful transplantation is closely associated with graft regions and grafted cell identities. The ability to generate defined and transplantable iNESCs from primate fibroblasts under a defined condition with predictable fate choices will facilitate disease modeling and cell therapy.

Specification of transplantable astroglial subtypes from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have been differentiated efficiently to neuronal cell types. However, directed differentiation of hPSCs to astrocytes and astroglial subtypes remains elusive. In this study, hPSCs were directed to nearly uniform populations of immature astrocytes (>90% S100ß(+) and GFAP(+)) in large quantities. The immature human astrocytes exhibit similar gene expression patterns as primary astrocytes, display functional properties such as glutamate uptake and promotion of synaptogenesis, and become mature astrocytes by forming connections with blood vessels after transplantation into the mouse brain. Furthermore, hPSC-derived neuroepithelia, patterned to rostral-caudal and dorsal-ventral identities with the same morphogens used for neuronal subtype specification, generate immature astrocytes that express distinct homeodomain transcription factors and display phenotypic differences of different astroglial subtypes. These human astroglial progenitors and immature astrocytes will be useful for studying astrocytes in brain development and function, understanding the roles of astrocytes in disease processes and developing novel treatments for neurological disorders.

Oligodendrocyte precursors originate in the parabasal band of the basal plate in prosomere 1 and migrate into the alar prosencephalon during chick development.

Oligodendrocytes are the myelin-forming cells in the central nervous system of vertebrates. Oligodendrocyte precursors arise from multiple restricted foci distributed along the antero-posterior axis of the developing brain. In chick and mouse embryos, oligodendrocyte precursors of the anterior forebrain emerge from neuroepithelial cells of the subpallium and migrate tangentially to invade the entire telencephalon (Olivier et al. (2001) Development 128:1757-1769). In the diencephalon, oligodendrocyte neuroepithelial precursors seem to be mainly located in the basal plate of caudal prosomeres, but very little is known about their distribution and maturation at later stages of embryonic development. Thus, in this work, we studied the origin and migration of oligodendrocyte precursos in the diencephalon of quail-chick chimeras. Homotopic and homochronic grafts demonstrated that, during embryonic development, diencephalic oligodendrocytes emerge from a common neuroepithelial domain in the basal plate of prosomere 1 and migrate tangentially, invading the dorsal regions of the diencephalic prosomeres and the telencephalon.

Encapsulated living choroid plexus cells: potential long-term treatments for central nervous system disease and trauma.

In neurodegenerative disease and in acute brain injury, there is often local up-regulation of neurotrophin production close to the site of the lesion. Treatment by direct injection of neurotrophins and growth factors close to these lesion sites has repeatedly been demonstrated to improve recovery. It has therefore been proposed that transplanting viable neurotrophin-producing cells close to the trauma lesion, or site of degenerative disease, might provide a novel means for continuous delivery of these molecules directly to the site of injury or to a degenerative region. The aim of this paper is to summarize recent published information and present new experimental data that indicate that long-lasting therapeutic implants of choroid plexus (CP) neuroepithelium may be used to treat brain disease. CP produces and secretes numerous biologically active neurotrophic factors (NT). New gene microarray and proteomics data presented here indicate that many other anti-oxidant, anti-toxin and neuronal support proteins are also produced and secreted by CP cells. In the healthy brain, these circulate in the cerebrospinal fluid through the brain and spinal cord, maintaining neuronal networks and associated cells. Recent publications describe how transplanted CP cells and tissue, either free or in an immunoprotected encapsulated form, can effectively deliver therapeutic molecules when placed near the lesion or site of degenerative disease in animal models. Using simple techniques, CP neuroepithelial cell clusters in suspension culture were very durable, remaining viable for 6 months or more in vitro. The cell culture conditions had little effect on the wide range and activity of genes expressed and proteins secreted. Recently, completed experiments show that implanting CP within alginate-poly-ornithine capsules effectively protected these xenogeneic cells from the host immune system and allowed their survival for 6 months or more in the brains of rats, causing no adverse effects. Previously reported evidence demonstrated that CP cells support the survival and differentiation of neuronal cells in vitro and effectively treat acute brain injury and disease in rodents and non-human primates in vivo. The accumulated preclinical data together with the long-term survival of implanted encapsulated cells in vivo provide a sound base for the investigation of these treatments for chronic inherited and established neurodegenerative conditions.

Neuroepithelial stem cells differentiate into neuronal phenotypes and improve intestinal motility recovery after transplantation in the aganglionic colon of the rat.

The purpose of this study was to elucidate the possibility and the biological significance of intracolonic grafting of neuroepithelial stem cells (NESCs) as a therapeutic strategy for neuronal replacement in disorders of the enteric nervous system (ENS) such as aganglionosis. The enteric plexus of rat colon were eliminated by serosal application of the cationic surfactant benzalkonium chloride. NESCs were harvested from the neural tube of embryonic rat, labelled with bromodeoxyuridine (BrdU), and transplanted into the denervated colon. After 2, 4 and 8 weeks, grafted cells were visualized in colon sections by fluorescent double-staining for BrdU and neuronal, astrocytic, neurochemical or stem cell markers. Eight weeks post-transplantation, the intestinal motility was assessed by measuring the changes of intraluminal pressure responding to inflating stimulation and the responses to electrical field stimulation (EFS). Our results indicate that when transplanted into the denervated gut, NESCs survived and could differentiate into neurons and glial cells in vivo. Furthermore, inflation stimulated contraction and EFS-induced response were observed in NESCs grafted group compared with no reaction in denervated group. Therefore, NESCs can survive and function in the denervated rat colon in vivo, which indicates that NESCs provide a promising cellular replacement candidate for ENS.

Neural-tube-derived neuroepithelial stem cells: a new transplant resource for Parkinson's disease.

To assess the feasibility of using neuroepithelial stem cells as a transplant source for Parkinson's disease, neuroepithelial cells were harvested from the neural tube, cultured and stereotactically transplanted into the striatum of a rat model of Parkinson's disease. In culture, neuroepithelial cells generated abundant neurospheres and differentiated into both neurons and glia. After transplantation, tyrosine-hydroxylase-positive cells were detected in the graft. Furthermore, an apomorphine-induced rotation test showed that the implanted cells successfully promoted functional recovery in animals that underwent this transplantation procedure. Our results demonstrate that neuroepithelial cells may be a new source of donor material for Parkinson's disease.

Human embryonic stem cell-derived neural precursors develop into neurons and integrate into the host brain.

Whether and how in-vitro-produced human neural precursors mature and integrate into the brain are crucial to the utility of human embryonic stem (hES) cells in treating neurological disorders. After transplantation into the ventricles of neonatal immune-deficient mice, hES-cell-derived neural precursors stopped expressing the cell division marker Ki67, except in neurogenic areas, and differentiated into neurons and then glia in a temporal course intrinsic to that of human cells regardless of location. The human cells located in the gray matter became neurons in the olfactory bulb and striatum, whereas those in the white matter produced exclusively glia. Importantly, the grafted human cells formed synapses. Thus, the in-vitro-produced human neural precursors follow their intrinsic temporal program to produce neurons and glia and, in response to environmental signals, generate cells appropriate to their target regions and integrate into the brain.