Induced Cognitive Impairments Reversed by Grafts of Neural Precursors: A Longitudinal Study in a Macaque Model of Parkinson's Disease.

Parkinson's disease (PD) evolves over an extended and variable period in humans; years prior to the onset of classical motor symptoms, sleep and biological rhythm disorders develop, significantly impacting the quality-of-life of patients. Circadian-rhythm disorders are accompanied by mild cognitive deficits that progressively worsen with disease progression and can constitute a severe burden for patients at later stages. The gold-standard 6-methyl-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) macaque model of PD recapitulates the progression of motor and nonmotor symptoms over contracted periods of time. Here, this multidisciplinary/multiparametric study follows, in five animals, the steady progression of motor and nonmotor symptoms and describes their reversal following grafts of neural precursors in diverse functional domains of the basal ganglia. Results show unprecedented recovery from cognitive symptoms in addition to a strong clinical motor recuperation. Both motor and cognitive recovery and partial circadian rhythm recovery correlate with the degree of graft integration, and in a subset of animals, with in vivo levels of striatal dopaminergic innervation and function. The present study provides empirical evidence that integration of neural precursors following transplantation efficiently restores function at multiple levels in parkinsonian nonhuman primates and, given interindividuality of disease progression and recovery, underlines the importance of longitudinal multidisciplinary assessments in view of clinical translation.

Determinants of primate neurogenesis and the deployment of top-down generative networks in the cortical hierarchy.

What I cannot create I do not understand - Richard Feynman 1978 Because primate cortical development exhibits numerous specific features, the mouse is an imperfect model for human cortical development. Expansion of supragranular neurons is an evolutionary feature characterizing the primate cortex. Increased production of supragranular neurons is supported by a germinal zone innovation of the primate cortex: the Outer SubVentricular Zone, which along with supragranular neurons constitute privileged targets of primate brain-specific gene evolution. The resulting cell-type diversity of human supragranular neurons link cell and molecular evolutionary changes in progenitors with the emergence of distinctive architectural features in the primate cortex. We propose that these changes are required for the expansion of the primate cortical hierarchy deploying top-down generative networks with potentially important consequences for the neurobiology of human psychiatric disorders.

Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells.

After reprogramming to naive pluripotency, human pluripotent stem cells (PSCs) still exhibit very low ability to make interspecies chimeras. Whether this is because they are inherently devoid of the attributes of chimeric competency or because naive PSCs cannot colonize embryos from distant species remains to be elucidated. Here, we have used different types of mouse, human, and rhesus monkey naive PSCs and analyzed their ability to colonize rabbit and cynomolgus monkey embryos. Mouse embryonic stem cells (ESCs) remained mitotically active and efficiently colonized host embryos. In contrast, primate naive PSCs colonized host embryos with much lower efficiency. Unlike mouse ESCs, they slowed DNA replication after dissociation and, after injection into host embryos, they stalled in the G1 phase and differentiated prematurely, regardless of host species. We conclude that human and non-human primate naive PSCs do not efficiently make chimeras because they are inherently unfit to remain mitotically active during colonization.

Adhesive Sponge Based on Supramolecular Dimer Interactions as Scaffolds for Neural Stem Cells.

Improving cell-material interactions of nonadhesive scaffolds is crucial for the success of biomaterials in tissue engineering. Due to their high surface area and open pore structure, sponges are widely reported as absorbent materials for biomedical engineering. The biocompatibility and biodegradability of polysaccharide sponges, coupled with the chemical functionalities of supramolecular dimers, make them promising combinations for the development of adhesive scaffolds. Here, a supramolecular tactic based on (UPy)-modified polysaccharide associated with three-dimensional structure of sponges was developed to reach enhanced cellular adhesion. For this purpose, three approaches were examined individually in order to accomplish this goal. In the first approach, the backbone polysaccharides with noncell adhesive properties were modified via a modular tactic using UPy-dimers. Hereupon, the physical-chemical characterizations of the supramolecular sponges were performed, showing that the presence of supramolecular dimers improved their mechanical properties and induced different architectures. In addition, small-angle neutron scattering (SANS) measurements and rheology experiments revealed that the UPy-dimers into agarose backbone are able to reorganize in thinning aggregates. It is also demonstrated that the resulted UPy-agarose (AGA-UPy) motifs in surfaces can promote cell adhesion. Finally, the last approach showed the great potential for use of this novel material in bioadhesive scaffolds indicating that neural stem cells show a spreading bias in soft materials and that cell adhesion was enhanced for all UPy-modified sponges compared to the reference, i.e. unmodified sponges. Therefore, by functionalizing sponge surfaces with UPy-dimers, an adhesive supramolecular scaffold is built which opens the opportunity its use neural tissues regeneration.

Bridging the Gap between Mechanics and Genetics in Cortical Folding: ECM as a Major Driving Force.

Folding of the cerebral cortex results from interrelated biological and mechanical processes that are incompletely understood. In this issue, Long et al. identify the key roles of HAPLN1, lumican, collagen I, and HA in relationship with changes in tissue stiffness.

Effect of polyvinylidene fluoride electrospun fiber orientation on neural stem cell differentiation.

Electrospun polymer piezoelectric fibers can be used in neural tissue engineering (NTE) to mimic the physical, biological, and material properties of the native extracellular matrix. In this work, we have developed scaffolds based on polymer fiber architectures for application in NTE. To study the role of such three-dimensional scaffolds, a rotating drum collector was used for electrospinning poly(vinylidene) fluoride (PVDF) polymer at various rotation speeds. The morphology, orientation, polymorphism, as well as the mechanical behavior of the nonaligned and aligned fiber-based architectures were characterized. We have demonstrated that the jet flow and the electrostatic forces generated by electrospinning of PVDF induced local conformation changes which promote the generation of the ß-phase. Fiber anisotropy could be a critical feature for the design of suitable scaffolds for NTEs. We thus assessed the impact of PVDF fiber alignment on the behavior of monkey neural stem cells (NSCs). NSCs were seeded on nonaligned and aligned scaffolds and their morphology, adhesion, and differentiation capacities into the neuronal and glial pathways were studied using microscopic techniques. Significant changes in the growth and differentiation capacities of NSCs into neuronal and glial cells as a function of the fiber alignment were evidenced. These results demonstrate that PVDF scaffolds may serve as instructive scaffolds for NSC survival and differentiation, and may be valuable tools for the development of cell- and scaffold-based strategies for neural repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2376-2393, 2017.

Transplantation in the nonhuman primate MPTP model of Parkinson's disease: update and perspectives.

In order to calibrate stem cell exploitation for cellular therapy in neurodegenerative diseases, fundamental and preclinical research in NHP (nonhuman primate) models is crucial. Indeed, it is consensually recognized that it is not possible to directly extrapolate results obtained in rodent models to human patients. A large diversity of neurological pathologies should benefit from cellular therapy based on neural differentiation of stem cells. In the context of this special issue of Primate Biology on NHP stem cells, we describe past and recent advances on cell replacement in the NHP model of Parkinson's disease (PD). From the different grafting procedures to the various cell types transplanted, we review here diverse approaches for cell-replacement therapy and their related therapeutic potential on behavior and function in the NHP model of PD.

Development of Bioresorbable Hydrophilic-Hydrophobic Electrospun Scaffolds for Neural Tissue Engineering.

In this study, electrospun fiber scaffolds based on biodegradable and bioabsorbable polymers and showing a similar structure to that of the extracellular matrix (ECM) present in the neural tissues were prepared. The effects of electrospun-based scaffolds processed from poly(lactic acid) (PLA)/poly(lactide-b-ethylene glycol-b-lactide) block copolymer (PELA) and PLA/polyethylene glycol (PEG) (50:50 by wt) blends on the morphology, wettability, and mechanical properties, as well as on neural stem cell (NSC) behavior, were investigated. Thus, PLA/PELA and PLA/PEG fiber mats composed of PEG with different chain lengths were evaluated for optimal use as tissue engineering scaffolds. In both cases, the hydrophilic character of the scaffold surface was increased from the introduction of PEG homopolymer or PEG-based block copolymer compared with neat PLA. A microphase separation and a surface erosion of PLA/PEG blend-based electrospun fibers were highlighted, whereas PLA/PELA blend-based fibers displayed a moderate hydrophilic surface and a tunable balance between surface erosion and bulk degradation. Even if the mechanical properties of PLA fibers containing PEG or PELA decreased slightly, an excellent compromise between stiffness and the ability to sustain large deformation was found for PLA/PELA(2k), which displayed a significant increase in strain at break, that is, up to 500%. Our results suggest that both neat PLA and PLA/PELA blends supplemented with growth factors may mimic neural-like constructs and provide structural stability. Nonetheless, electrospun PLA/PELA blends have a suitable surface property, which may act synergistically in the modulation of biopotential for implantable scaffolding in neural tissue engineering.

Epigenetic status of H19/IGF2 and SNRPN imprinted genes in aborted and successfully derived embryonic stem cell lines in non-human primates.

The imprinted genes of primate embryonic stem cells (ESCs) often show altered DNA methylation. It is unknown whether these alterations emerge while deriving the ESCs. Here we studied the methylation patterns of two differentially methylated regions (DMRs), SNRPN and H19/IGF2 DMRs, during the derivation of monkey ESCs. We show that the SNRPN DMR is characteristically methylated at maternal alleles, whereas the H19/IGF2 DMR is globally highly methylated, with unusual methylation on the maternal alleles. These methylation patterns remain stable from the early stages of ESC derivation to late passages of monkey ESCs and following differentiation. Importantly, the methylation status of H19/IGF2 DMR and the expression levels of IGF2, H19, and DNMT3B mRNAs in early embryo-derived cells were correlated with their capacity to generate genuine ESC lines. Thus, we propose that these markers could be useful to predict the outcomes of establishing an ESC line in primates.

Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in embryonic stem cells.

Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.

Embryonic stem cells in non-human primates: An overview of neural differentiation potential.

Non-human primate (NHP) embryonic stem (ES) cells show unlimited proliferative capacities and a great potential to generate multiple cell lineages. These properties make them an ideal resource both for investigating early developmental processes and for assessing their therapeutic potential in numerous models of degenerative diseases. They share the same markers and the same properties with human ES cells, and thus provide an invaluable transitional model that can be used to address the safety issues related to the clinical use of human ES cells. Here, we review the available information on the derivation and the specific features of monkey ES cells. We comment on the capacity of primate ES cells to differentiate into neural lineages and the current protocols to generate self-renewing neural stem cells. We also highlight the signalling pathways involved in the maintenance of these neural cell types. Finally, we discuss the potential of monkey ES cells for neuronal differentiation.

Novel STAT3 target genes exert distinct roles in the inhibition of mesoderm and endoderm differentiation in cooperation with Nanog.

Leukemia inhibitory factor (LIF) activates the transcription factor signal transducer and activator of transcription 3 (STAT3), which results in the maintenance of mouse embryonic stem cells in the pluripotent state by inhibiting both mesodermal and endodermal differentiation. How the LIF/STAT3 pathway inhibits commitment to both mesoderm and endoderm lineages is presently unknown. Using a hormone-dependent STAT3 and with microarray analysis, we identified 58 targets of STAT3 including 20 unknown genes. Functional analysis showed that 22 among the 23 STAT3 target genes analyzed contribute to the maintenance of the undifferentiated state, as evidenced by an increase in the frequency of differentiated colonies in a self-renewal assay and a concomitant elevation of early differentiation markers upon knockdown. Fourteen of them, including Dact1, Klf4, Klf5, Rgs16, Smad7, Ccrn4l, Cnnm1, Ocln, Ier3, Pim1, Cyr61, and Sgk, were also regulated by Nanog. Analysis of lineage-specific markers showed that the STAT3 target genes fell into three distinct categories, depending on their capacity to inhibit either mesoderm or endoderm differentiation or both. The identification of genes that harness self-renewal and are downstream targets of both STAT3 and Nanog shed light on the mechanisms underlying functional redundancy between STAT3 and Nanog in mouse embryonic stem cells.

Derivation and cloning of a novel rhesus embryonic stem cell line stably expressing tau-green fluorescent protein.

Embryonic stem cells (ESC) have the ability of indefinite self-renewal and multilineage differentiation, and they carry great potential in cell-based therapies. The rhesus macaque is the most relevant preclinical model for assessing the benefit, safety, and efficacy of ESC-based transplantations in the treatment of neurodegenerative diseases. In the case of neural cell grafting, tracing both the neurons and their axonal projections in vivo is essential for studying the integration of the grafted cells in the host brain. Tau-Green fluorescent protein (tau-GFP) is a powerful viable lineage tracer, allowing visualization of cell bodies, dendrites, and axons in exquisite detail. Here, we report the first rhesus monkey ESC line that ubiquitously and stably expresses tau-GFP. First, we derived a new line of rhesus monkey ESC (LYON-ES1) that show marker expression and cell cycle characteristics typical of primate ESCs. LYON-ES1 cells are pluripotent, giving rise to derivatives of the three germ layers in vitro and in vivo through teratoma formation. They retain all their undifferentiated characteristics and a normal karyotype after prolonged culture. Using lentiviral infection, we then generated a monkey ESC line stably expressing tau-GFP that retains all the characteristics of the parental wild-type line and is clonogenic. We show that neural precursors derived from the tau-GFP ESC line are multipotent and that their fate can be precisely mapped in vivo after grafting in the adult rat brain. Disclosure of potential conflicts of interest is found at the end of this article.

Developmental fate of embryonic germ cells (EGCs), in vivo and in vitro.

Embryonic germ cells (EGCs) derived from mouse primordial germ cells (PGCs) are known both to colonize all cell lineages of the fetus and to make tumors in vivo. When aggregated with eight-cell embryos, EGCs from a new EGC line expressing green fluorescent protein (GFP) were found to contribute preferentially to the epiblast but unexpectedly were also capable of colonizing primary endoderm. When injected under the kidney capsule, EGCs derived from 12.5 days post coitum (dpc) PGCs formed differentiated tumors. The ability of EGCs to differentiate in an organ culture system depends upon their partners in cell culture. When EGCs, marked with a LacZ transgene, were mixed with disaggregated and reaggregated mouse fetal lung in an organ culture system, they remained undifferentiated. In urogenital ridge reaggregates on the other hand, some EGCs were capable of differentiating to form small epithelial cysts.