Report of the international conference on manufacturing and testing of pluripotent stem cells.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

Clinical translation of human neural stem cells.

Human neural stem cell transplants have potential as therapeutic candidates to treat a vast number of disorders of the central nervous system (CNS). StemCells, Inc. has purified human neural stem cells and developed culture conditions for expansion and banking that preserve their unique biological properties. The biological activity of these human central nervous system stem cells (HuCNS-SC®) has been analyzed extensively in vitro and in vivo. When formulated for transplantation, the expanded and cryopreserved banked cells maintain their stem cell phenotype, self-renew and generate mature oligodendrocytes, neurons and astrocytes, cells normally found in the CNS. In this overview, the rationale and supporting data for pursuing neuroprotective strategies and clinical translation in the three components of the CNS (brain, spinal cord and eye) are described. A phase I trial for a rare myelin disorder and phase I/II trial for spinal cord injury are providing intriguing data relevant to the biological properties of neural stem cells, and the early clinical outcomes compel further development.

Neural stem cells as tools for drug discovery: novel platforms and approaches.

INTRODUCTION: Neural stem cells catalyze strong interests for the development of systems to screen for effective drugs to treat neurodegenerative conditions and/or improve neurogenesis, fields where the classical approaches have so far failed in discovering successful drugs. AREAS COVERED: The authors review the known biology of NSCs, their normal function in development, the adult brain, and in vitro culture systems. The authors also discuss the scientific and technological progress which will aid wider applications of NSCs for drug screening/development purposes. The authors base this article on literature searches performed through PubMed and Google Scholar. EXPERT OPINION: NSC systems present unique opportunities that are starting to be successfully explored for genetic and chemical screening. These systems provide the possibility of identifying and optimizing molecules/drugs that could lead to the tighter control in self-renewal and lineage specification of NSCs as well as their functional maturation. This could be crucial in moving forward NSC-based therapies. It is expected that recent advances in the method of producing NSCs from patient-specific human induced pluripotent stem (iPS) cells and in the technologies to grow them in vitro, while preserving their full developmental potential, will allow a full exploitation of NSCs both in drug discovery programs and in predictive toxicology studies.

Automated large-scale culture and medium-throughput chemical screen for modulators of proliferation and viability of human induced pluripotent stem cell-derived neuroepithelial-like stem cells.

The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for high-throughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell-derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture ("scale-up") of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated "scale-out" into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dose-dependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system.

Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays.

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133(+)CD24(-/lo) cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca(2+) imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

Restoration of motor control and dopaminergic activity in rats with unilateral 6-hydroxy-dopamine lesions.

AIM: To restore motor function and dopaminergic activity in the nigrostriatum of rats with unilateral 6-hydroxy-dopamine lesions using implants of encapsulated porcine choroid plexus cells. MATERIALS & METHODS: Neonatal porcine choroid plexus cells were prepared and maintained in culture, then encapsulated within alginate-polyornithine capsules (600-660 µm). Rats were unilaterally injected with 6-hydroxy-dopamine into the striatum. Those with lesions verified after 2 and 4 weeks were selected for experiments. Rats were implanted adjacent to the lesion with ten capsules 2-4 days later with (treated, n = 12) or without (control, n = 10) choroid plexus cells. RESULTS: The choroid plexus cells were shown to produce a wide range of neurorestorative proteins. The treated group had a 60% improvement in motor behavior compared with the control group (p < 0.01). The treated group also had a significant improvement in nigrostriatal dopaminergic activity (31%, p < 0.02). CONCLUSION: Capsules containing porcine choroid plexus cells release therapeutic molecules that stimulate regeneration of the lesioned nigrostriatum in rats.

Growth hormone receptor immunoreactivity is increased in the subventricular zone of juvenile rat brain after focal ischemia: a potential role for growth hormone in injury-induced neurogenesis.

BACKGROUND: During recovery from an ischemic brain injury, a cerebral growth hormone (GH) axis is activated. Whilst GH has been demonstrated to be neuroprotective both in vitro and in vivo, a role for GH in neuro-restorative processes after brain injury has yet to be studied. OBJECTIVE: To explore a role for GH in injury-induced neurogenesis by examining GH receptor (GH-R) immunoreactivity within the subventricular zone (SVZ) of juvenile rats after brain injury and by testing the proliferative capacity of GH on embryonic mouse neural stem cells. DESIGN: Twenty-one day old rats were subjected to unilateral hypoxic-ischemia of the brain and sacrificed 1-15days later. Coronal brain sections from these animals and age-matched naïve controls were immunostained for GH-R and cell markers of neurogenesis. The level of GH-R immunoreactivity in the ipsilateral and contralateral SVZ of each animal was semi-quantified both by independent blinded scoring by two examiners and blinded image analysis. To examine the effect of GH on proliferation of embryonic mouse neural stem cells, cells were treated with increasing concentrations of rat pituitary GH for 48h in the presence of 5'-bromo-2'-deoxyuridine. RESULTS: The level of GH-R immunoreactivity in the ipsilateral SVZ was significantly increased 5days after injury vs. the contralateral SVZ, coinciding both spatially and temporally with injury-induced neurogenesis. The population of GH-R immunopositive cells in the ipsilateral SVZ at this time was found to include proliferating cells (Ki67 immunopositive), neural progenitor cells (nestin immunopositive) and post-proliferative migratory neuroblasts (doublecortin immunopositive). Stimulation of embryonic mouse NSCs with physiological concentrations of rat pituitary GH elicited a dose-dependent proliferative response. CONCLUSION: These results indicate a novel role for GH and its receptor in injury-induced neurogenesis, and suggest that GH treatment may potentiate endogenous neuro-restorative processes after brain injury.

Neural regeneration protein is a novel chemoattractive and neuronal survival-promoting factor.

Neurogenesis and neuronal migration are the prerequisites for the development of the central nervous system. We have identified a novel rodent gene encoding for a neural regeneration protein (NRP) with an activity spectrum similar to the chemokine stromal-derived factor (SDF)-1, but with much greater potency. The Nrp gene is encoded as a forward frameshift to the hypothetical alkylated DNA repair protein AlkB. The predicted protein sequence of NRP contains domains with homology to survival-promoting peptide (SPP) and the trefoil protein TFF-1. The Nrp gene is first expressed in neural stem cells and expression continues in glial lineages. Recombinant NRP and NRP-derived peptides possess biological activities including induction of neural migration and proliferation, promotion of neuronal survival, enhancement of neurite outgrowth and promotion of neuronal differentiation from neural stem cells. NRP exerts its effect on neuronal survival by phosphorylation of the ERK1/2 and Akt kinases, whereas NRP stimulation of neural migration depends solely on p44/42 MAP kinase activity. Taken together, the expression profile of Nrp, the existence in its predicted protein structure of domains with similarities to known neuroprotective and migration-inducing factors and the high potency of NRP-derived synthetic peptides acting in femtomolar concentrations suggest it to be a novel gene of relevance in cellular and developmental neurobiology.

Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.

Parvalbumin expression in visual cortical interneurons depends on neuronal activity and TrkB ligands during an Early period of postnatal development.

The differentiation of cortical interneurons is controlled by environmental factors. Here, we describe the role of activity and neurotrophins in regulating parvalbumin (PARV) expression using organotypic cultures (OTC) of rat visual cortex as model system. In OTC, PARV expression was dramatically delayed. The organotypic proportion of approximately 6% PARV neurons was not established before 50-70 DIV, whereas in vivo all neurons are present until P20. Thalamic afferents increased cortical PARV mRNA in OTC, but not to the age-matched in vivo level. During the first 10 DIV, BDNF and NT-4 accelerated PARV mRNA expression in a Trk receptor and MEK2 dependent manner. The BDNF action required PI3 kinase signalling. PARV expression required activity. The proportion of neurons which managed to up-regulate PARV was inversely related to the duration of early transient periods of activity deprivation. Long-term activity-deprived OTC completely failed to up-regulate PARV mRNA. Both TrkB ligands failed to promote PARV expression in activity-deprived OTC. However, a few basket and chandelier neurons were observed, suggesting that the development of class-specific morphological features is activity-independent. Once established, PARV expression became resistant to late-onset activity deprivation. In conclusion, PARV expression depended on activity and TrkB ligands which appear to prime the PARV expression already before its developmental onset.

Neuronal activity and neurotrophic factors regulate GAD-65/67 mRNA and protein expression in organotypic cultures of rat visual cortex.

Environmental factors are known to regulate the molecular differentiation of neocortical interneurons. Their class-defining transmitter synthetic enzymes are the glutamic acid decarboxylases (GAD); yet, fairly little is known about the developmental regulation of transcription and translation of the GAD-65/67 isoforms. We have characterized the role of neuronal activity, neurotrophins and afferent systems for GAD-65/67 expression in visual cortex in organotypic cultures (OTC) compared with in vivo in order to identify cortex-intrinsic regulatory mechanisms. Spontaneously active OTC prepared at postnatal day 0 displayed from 10 days in vitro (DIV) onwards 12-14% GAD-65/GAD-67 neurons similar to in vivo. However, GAD-65 mRNA was higher, whereas GAD-67 protein was lower, than in vivo. During the first week neurotrophins increased whereas the Trk receptor inhibitor K252a and MEK inhibitors decreased both GAD mRNAs and proteins. After 10 DIV GAD expression no longer depended on neurotrophin signalling. Activity-deprived OTC revealed only 6% GAD-67 neurons and mRNA and protein were reduced by 50%. GAD-65 mRNA was less reduced, but protein was reduced by half, suggesting translational regulation. Upon recovery of activity GAD mRNAs, cell numbers, and both proteins quickly returned to normal and these 'adult' levels were resistant to late-onset deprivation. In 20 DIV activity-deprived OTC, only neurotrophin 4 increased GAD-65/67 mRNAs, rescued the percentage of GAD-67 neurons and increased both proteins in a TrkB-dependent manner. Activity deprivation had thus shifted the period of neurotrophin sensitivity to older ages. The results suggested neuronal activity as a major regulator differentially affecting transcription and translation of the GAD isoforms. The early presence of neuronal activity promoted the GAD expression in OTC to a neurotrophin-independent state suggesting that neurotrophins play a context-dependent role.

Pharmacological potential of embryonic stem cells.

Established embryonic stem (ES) cell lines have been at the forefront of approaches to understand gene function during embryogenesis and in adult vertebrate organisms, principally due to exploitation of two essential attributes; their pluripotency or ability to contribute to all three germinal layers and germ line in mice and their ease of genetic modification. Endeavours to routinely establish ES cells from species other than mice have met with limited success, although with rapid progress being made in our understanding of their basic cell biology, the regular derivation of lines from pre-implantation embryos will become easier for many species including humans. With a recent growing awareness of how these cells can be made to grow in an unlimited, but regulated manner plus how their fate can be directed or manipulated into diverse, mature phenotypes in culture, it has become clear that the biological resource offers additional attractive features applicable for future biomedical research and therapy. Advanced mouse ES-based technologies are being used in the industry for pharmaceutical discovery and development, while it is also anticipated that human ES cell reagents will revolutionise aspects of regenerative medicine. This review will summarise the advantages, potential and great hope for ES cell based systems in these contexts.