Pluripotent Stem Cell-derived Neural Precursor Research Articles

Abnormalities in the migration of neural precursor cells in familial bipolar disorder.

ABSTRACTCellular migration is a ubiquitous feature that brings brain cells into appropriate spatial relationships over time; and it helps in the formation of a functional brain. We studied the migration patterns of induced pluripotent stem cell-derived neural precursor cells (NPCs) from individuals with familial bipolar disorder (BD) in comparison with healthy controls. The BD patients also had morphological brain abnormalities evident on magnetic resonance imaging. Time-lapse analysis of migrating cells was performed, through which we were able to identify several parameters that were abnormal in cellular migration, including the speed and directionality of NPCs. We also performed transcriptomic analysis to probe the mechanisms behind the aberrant cellular phenotype identified. Our analysis showed the downregulation of a network of genes, centering on EGF/ERBB proteins. The present findings indicate that collective, systemic dysregulation may produce the aberrant cellular phenotype, which could contribute to the functional and structural changes in the brain reported for bipolar disorder. This article has an associated First Person interview with the first author of the paper.

Conditioned Medium of Human Pluripotent Stem Cell-Derived Neural Precursor Cells Exerts Neurorestorative Effects against Ischemic Stroke Model.

Previous studies have shown that early therapeutic events of neural precursor cells (NPCs) transplantation to animals with acute ischemic stroke readily protected neuronal cell damage and improved behavioral recovery through paracrine mechanisms. In this study, we tested the hypothesis that administration of conditioned medium from NPCs (NPC-CMs) could recapitulate the beneficial effects of cell transplantation. Rats with permanent middle cerebral artery occlusion (pMCAO) were randomly assigned to one of the following groups: PBS control, Vehicle (medium) controls, single (NPC-CM(S)) or multiple injections of NPC-CM(NPC-CM(M)) groups. A single intravenous injection of NPC-CM exhibited strong neuroregenerative potential to induce behavioral recovery, and multiple injections enhanced this activity further by suppressing inflammatory damage and inducing endogenous neurogenesis leading to histopathological and functional recovery. Proteome analysis of NPC-CM identified a number of proteins that are known to be associated with nervous system development, neurogenesis, and angiogenesis. In addition, transcriptome analysis revealed the importance of the inflammatory response during stroke recovery and some of the key hub genes in the interaction network were validated. Thus, our findings demonstrated that NPC-CM promoted functional recovery and reduced cerebral infarct and inflammation with enhanced endogenous neurogenesis, and the results highlighted the potency of NPC-CM in stroke therapy.

SUPT4H1-edited stem cell therapy rescues neuronal dysfunction in a mouse model for Huntington\u2019s disease

Huntington’s disease (HD) is a severe inherited neurological disorder caused by a CAG repeat expansion in the huntingtin gene (HTT), leading to the accumulation of mutant huntingtin with polyglutamine repeats. Despite its severity, there is no cure for this debilitating disease. HTT lowering strategies, including antisense oligonucleotides (ASO) showed promising results very recently. Attempts to develop stem cell-based therapeutics have shown efficacy in preclinical HD models. Using an HD patient’s autologous cells, which have genetic defects, may hamper therapeutic efficacy due to mutant HTT. Pretreating these cells to reduce mutant HTT expression and transcription may improve the transplanted cells’ therapeutic efficacy. To investigate this, we targeted the SUPT4H1 gene that selectively supports the transcription of long trinucleotide repeats. Transplanting SUPT4H1-edited HD-induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) into the YAC128 HD transgenic mouse model improved motor function compared to unedited HD iPSC-NPCs. Immunohistochemical analysis revealed reduced mutant HTT expression without compensating wild-type HTT expression. Further, SUPT4H1 editing increased neuronal and decreased reactive astrocyte differentiation in HD iPSC-NPCs compared to the unedited HD iPSC-NPCs. This suggests that ex vivo editing of SUPT4H1 can reduce mutant HTT expression and provide a therapeutic gene editing strategy for autologous stem cell transplantation in HD.

Induced Pluripotent Stem Cell-Derived Neural Precursors Improve Memory, Synaptic and Pathological Abnormalities in a Mouse Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is the most common type of dementia in the elderly population. The disease is characterized by progressive memory loss, cerebral atrophy, extensive neuronal loss, synaptic alterations, brain inflammation, extracellular accumulation of amyloid-β (Aβ) plaques, and intracellular accumulation of hyper-phosphorylated tau (p-tau) protein. Many recent clinical trials have failed to show therapeutic benefit, likely because at the time in which patients exhibit clinical symptoms the brain is irreversibly damaged. In recent years, induced pluripotent stem cells (iPSCs) have been suggested as a promising cell therapy to recover brain functionality in neurodegenerative diseases such as AD. To evaluate the potential benefits of iPSCs on AD progression, we stereotaxically injected mouse iPSC-derived neural precursors (iPSC-NPCs) into the hippocampus of aged triple transgenic (3xTg-AD) mice harboring extensive pathological abnormalities typical of AD. Interestingly, iPSC-NPCs transplanted mice showed improved memory, synaptic plasticity, and reduced AD brain pathology, including a reduction of amyloid and tangles deposits. Our findings suggest that iPSC-NPCs might be a useful therapy that could produce benefit at the advanced clinical and pathological stages of AD.

NFIB induces functional astrocytes from human pluripotent stem cell-derived neural precursor cells mimicking in vivo astrogliogenesis.

The ability to generate astrocytes from human pluripotent stem cells (hPSCs) offers a promising cellular model to study the development and physiology of human astrocytes. The extant methods for generating functional astrocytes required long culture periods and there remained much ambiguityon whether such paradigms follow the innate developmental program. In this report, we provided an efficient and rapid method for generating physiologically functional astrocytes from hPSCs. Overexpressing the nuclear factor IB in hPSC-derived neural precursor cells induced a highly enriched astrocyte population in 2 weeks. RNA sequencing and functional analyses demonstrated progressive transcriptomic and physiological changes in the cells, resembling in vivo astrocyte development. Further analyses substantiated previous results and established the MAPK pathway necessary for astrocyte differentiation. Hence, this differentiation paradigm provides a prospective in vitro model for human astrogliogenesis studies and the pathophysiology of neurological diseases concerning astrocytes.

Engraftable Induced Pluripotent Stem Cell-Derived Neural Precursors for Brain Repair.

Stem cell transplantation has attracted great interest for treatment of neurodegenerative diseases to provide neuroprotection, repair the lesioned neuronal network and restore functionality. Parkinson's disease (PD), in particular, has been a preferred target because motor disability that constitutes a core pathology of the disease is associated with local loss of dopaminergic neurons in a specific brain area, the substantia nigra pars compacta. These cells project to the striatum where they deliver the neurotransmitter dopamine that is involved in control of many aspects of motor behavior. Therefore, cell transplantation approaches in PD aim to replenish dopamine deficiency in the striatum. A major challenge in developing cell therapy approaches is the ability to generate large numbers of transplantable cells in a reliable and reproducible manner. In recent years the technological breakthrough of induced pluripotent stem cells (iPSCs) has demonstrated that this is possible at a preclinical level, accelerating clinical translation. A second important issue is to efficiently differentiate iPSCs into dopaminergic neuronal progenitors with restricted proliferation potential in order to avoid cellular overgrowth in vivo and minimize the risk of tumorigenesis. Here we describe an effective protocol that includes human iPSC differentiation to the dopaminergic lineage and enrichment in neuronal precursor cells expressing the polysialylated form of the neural cell adhesion molecule PSA-NCAM, through magnetically activated cell sorting. The resulting cells are transplanted and shown to survive, differentiate, and integrate within a striatal lesion model generated by unilateral 6-hydroxydopamine administration in mice of the NOD/SCID strain that supports xenografts.

Metabolic Changes in Focal Brain Ischemia in Rats Treated With Human Induced Pluripotent Stem Cell-Derived Neural Precursors Confirm the Beneficial Effect of Transplanted Cells.

There is currently no treatment for restoring lost neurological function after stroke. A growing number of studies have highlighted the potential of stem cells. However, the mechanisms underlying their beneficial effect have yet to be explored in sufficient detail. In this study, we transplanted human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) in rat temporary middle cerebral artery occlusion (MCAO) model. Using magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) we monitored the effect of cells and assessed lesion volume and metabolite changes in the brain. We monitored concentration changes of myo-inositol (Ins), Taurine (Tau), Glycerophosphocholine+Phosphocholine (GPC+PCh), N-acetyl-aspartate+N-acetyl-aspartyl-glutamate (NAA+NAAG), Creatine+Phosphocreatine (Cr+PCr), and Glutamate+Glutamine (Glu+Gln) in the brains of control and iPSC-NP-transplanted rats. Based on initial lesion size, animals were divided into small lesion and big lesion groups. In the small lesion control group (SCL), lesion size after 4 months was three times smaller than initial measurements. In the small lesion iPSC-NP-treated group, lesion volume decreased after 1 month and then increased after 4 months. Although animals with small lesions significantly improved their motor skills after iPSC-NP transplantation, animals with big lesions showed no improvement. However, our MRI data demonstrate that in the big lesion iPSC-NP-treated (BTL) group, lesion size increased only up until 1 month after MCAO induction and then decreased. In contrast, in the big lesion control group, lesion size increased throughout the whole experiment. Significantly higher concentrations of Ins, Tau, GPC+PCh, NAA+NAAG, Cr+PCr, and Glu+Gln were found in in contralateral hemisphere in BTL animals 4 months after cell injection. Lesion volume decreased at this time point. Spectroscopic results of metabolite concentrations in lesion correlated with volumetric measurements of lesion, with the highest negative correlation observed for NAA+NAAG. Altogether, our results suggest that iPSC-NP transplantation decreases lesion volume and regulates metabolite concentrations within the normal range expected in healthy tissue. Further research into the ability of iPSC-NPs to differentiate into tissue-specific neurons and its effect on the long-term restoration of lesioned tissue is necessary.

Three-dimensional differentiation of human pluripotent stem cell-derived neural precursor cells using tailored porous polymer scaffolds

This study investigates the utility of a tailored poly(ethylene glycol) diacrylate-crosslinked porous polymeric tissue engineering scaffold, with mechanical properties specifically optimised to be comparable to that of mammalian brain tissue for 3D human neural cell culture. Results obtained here demonstrate the attachment, proliferation and terminal differentiation of both human induced pluripotent stem cell- and embryonic stem cell-derived neural precursor cells (hPSC-NPCs) throughout the interconnected porous network within laminin-coated scaffolds. Phenotypic data and functional analyses are presented demonstrating that this material supports terminal in vitro neural differentiation of hPSC-NPCs to a mixed population of viable neuronal and glial cells for periods of up to 49 days. This is evidenced by the upregulation of TUBB3, MAP2, SYP and GFAP gene expression, as well as the presence of the proteins βIII-TUBULIN, NEUN, MAP2 and GFAP. Functional maturity of neural cells following 49 days 3D differentiation culture was tested via measurement of intracellular calcium. These analyses revealed spontaneously active, synchronous and rhythmic calcium flux, as well as response to the neurotransmitter glutamate. This tailored construct has potential application as an improved in vitro human neurogenesis model with utility in platform drug discovery programs. Statement of significanceThe interconnected porosity of polyHIPE scaffolds exhibits the ability to support three-dimensional neural cell network formation due to limited resistance to cellular migration and re-organisation. The previously developed scaffold material displays mechanical properties similar to that of the mammalian brain. This research also employs the utility of pluripotent stem cell-derived neural cells which are of greater clinical relevance than primary neural cell lines. This scaffold material has future potential in better mimicking three-dimensional neural networks found in the human brain and may result in improved in vitro models for disease modelling and drug screening applications.

Three-Dimensional Differentiation of Human Pluripotent Stem Cell-Derived Neural Precursor Cells Using Tailored Porous Polymer Scaffolds

This study investigates the utility of a novel poly(ethylene glycol) diacrylate-crosslinked porous polymeric tissue engineering scaffold, with mechanical properties specifically optimised to be comparable to that of mammalian brain tissue for 3D human neural cell culture. Results obtained here demonstrate the attachment, proliferation and terminal differentiation of both human induced pluripotent stem cell- and embryonic stem cell-derived neural precursor cells (hPSC-NPCs) throughout the interconnected porous network within laminin-coated scaffolds. Phenotypic data and functional analyses are presented demonstrating that this material supports terminal in vitro neural differentiation of hPSC-NPCs to a mixed population of viable neuronal and glial cells for periods of up to 49 days. This is evidenced by the upregulation of TUBB3, MAP2, SYP and GFAP gene expression, as well as the presence of the proteins βIII-TUBULIN, NEUN, MAP2 and GFAP. Functional maturity of neural cells following 49 days 3D differentiation culture was tested via measurement of intracellular calcium. These analyses revealed spontaneously active, synchronous and rhythmic calcium flux, as well as response to the neurotransmitter glutamate. This novel construct has potential application as an improved in vitro human neurogenesis model with utility in platform drug discovery programs.

Transplantation of Human-Induced Pluripotent Stem Cell-Derived Neural Precursors into Early-Stage Zebrafish Embryos.

Induced pluripotent stem cells (iPS cells) generated from somatic cells through reprogramming hold great promises for regenerative medicine. However, how reprogrammed cells survive, behave in vivo, and interact with host cells after transplantation still remains to be addressed. There is a significant need for animal models that allow in vivo tracking of transplanted cells in real time. In this regard, the zebrafish, a tropical freshwater fish, provides significant advantage as it is optically transparent and can be imaged in high resolution using confocal microscopy. The principal goal of this study was to optimize the protocol for successful short-term and immunosuppression-free transplantation of human iPS cell-derived neural progenitor cells into zebrafish and to test their ability to differentiate in this animal model. To address this aim, we isolated human iPS cell-derived neural progenitor cells from human fibroblasts and grafted them into (a) early (blastocyst)-stage wild-type AB zebrafish embryos or (b) 3-day-old Tg(gfap:GFP) zebrafish embryos (intracranial injection). We found that transplanted human neuronal progenitor cells can be effectively grafted and that they differentiate and survive in zebrafish for more than 2weeks, validating the model as an ideal platform for in vivo screening experiments. We conclude that zebrafish provides an excellent model for studying iPS cell-derived cells in vivo.

Effect of leukocyte inhibitory factor on neuron differentiation from human induced pluripotent stem cell-derived neural precursor cells.

Direct derivation of human induced pluripotent stem cells into neural precursor cells and differentiation of these into neurons holds great promise in the cell therapy of neuro-degenerative diseases. However, the availability and survival rate of neurons requires improvement. In the present study, it was found that the addition of 5 ng/ml leukocyte inhibitory factor (LIF) during the process of differentiation significantly improved the expression of neuron-specific class III β-tubulin (TUJ1) and microtubule-associated protein 2 (MAP2), as detected by immunofluorescence and western blotting. In addition, LIF improved the cell viability, increased the expression of phosphorylated-protein kinase B (AKT), downregulated the expression of proinflammatory cytokines, including interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), and upregulated the expression of anti-inflammatory cytokines, including interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). After adding the phosphatidylinositol 3-kinase (PI3K)/AKT signaling inhibitor LY294002 or wortmannin to the LIF differentiation group, LIF-induced changes in the protein expression of TUJ1 and MAP2 were reversed, but this effect could not be prevented by rapamycin, a mechanistic target of rapamycin signaling inhibitor. The expression of cytokines associated with inflammation and cell viability was reversed by LY294002 and wortmannin, but not by rapamycin. In conclusion, LIF could improve neuronal differentiation and survival through the activation of PI3K/AKT signaling and the anti-inflammatory effect. The anti-inflammatory effect may be mediated by the activation of PI3K/AKT.

Neural precursor cells derived from induced pluripotent stem cells exhibit reduced susceptibility to infection with a neurotropic coronavirus

The present study examines the susceptibility of mouse induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) to infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Similar to NPCs derived from striatum of day 1 postnatal GFP-transgenic mice (GFP-NPCs), iPSC-derived NPCs (iPSC-NPCs) are able to differentiate into terminal neural cell types and express MHC class I and II in response to IFN-γ treatment. However, in contrast to postnatally-derived NPCs, iPSC-NPCs express low levels of carcinoembryonic antigen-cell adhesion molecule 1a (CEACAM1a), the surface receptor for JHMV, and are less susceptible to infection and virus-induced cytopathic effects. The relevance of this in terms of therapeutic application of NPCs resistant to viral infection is discussed.

The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors.

IntroductionMagnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation.Materials and methodsWe investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR.ResultsCell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles.ConclusionOur results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders.

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

Beneficial Effect of Human Induced Pluripotent Stem Cell-Derived Neural Precursors in Spinal Cord Injury Repair.

Despite advances in our understanding and research of induced pluripotent stem cells (iPSCs), their use in clinical practice is still limited due to lack of preclinical experiments. Neural precursors (NPs) derived from a clone of human iPSCs (IMR90) were used to treat a rat spinal cord lesion 1 week after induction. Functional recovery was evaluated using the BBB, beam walking, rotarod, and plantar tests. Lesion morphology, endogenous axonal sprouting, graft survival, and iPSC-NP differentiation were analyzed immunohistochemically. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted iPSC-NPs on endogenous regenerative processes and also to monitor their behavior after transplantation. Human iPSC-NPs robustly survived in the lesion, migrated, and partially filled the lesion cavity during the entire period of observation. Transplanted animals displayed significant motor improvement already from the second week after the transplantation of iPSC-NPs. qPCR revealed the increased expression of human neurotrophins 8 weeks after transplantation. Simultaneously, the white and gray matter were spared in the host tissue. The grafted cells were immunohistochemically positive for doublecortin, MAP2, βIII-tubulin, GFAP, and CNPase 8 weeks after transplantation. Human iPSC-NPs further matured, and 17 weeks after transplantation differentiated toward interneurons, dopaminergic neurons, serotoninergic neurons, and ChAT-positive motoneurons. Human iPSC-NPs possess neurotrophic properties that are associated with significant early functional improvement and the sparing of spinal cord tissue. Their ability to differentiate into tissue-specific neurons leads to the long-term restoration of the lesioned tissue, making the cells a promising candidate for future cell-based therapy of SCI.

Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.

Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs), and their neuronal progeny, will play an important role in disease modeling, drug screening tests, central nervous system development studies, and may even become valuable for regenerative medicine treatments. Nonetheless, it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs, and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here, we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days, and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS), leaving the NP population nearly free of PSCs.

Integrated Platform for Production and Purification of Human Pluripotent Stem Cell-Derived Neural Precursors

Human pluripotent stem cells (hPSCs) are a promising source of cells for clinical applications, such as transplantation of clinically engineered tissues and organs, and drug discovery programs due to their ability to self-renew and to be differentiated into cells from the three embryonic germ layers. In this study, the differentiation of two hPSC-lines into neural precursors (NPs) was accomplished with more than 80% efficiency, by means of the dual-SMAD inhibition protocol, based on the use of two small molecules (SB431542 and LDN193189) to generate Pax6 and Nestin-positive neural entities. One of the major hurdles related to the in vitro generation of PSC-derived populations is the tumorigenic potential of cells that remain undifferentiated. These remaining hPSCs have the potential to generate teratomas after being transplanted, and may interfere with the outcome of in vitro differentiation protocols. One strategy to tackle this problem is to deplete these "contaminating" cells during the differentiation process. Magnetic activated cell sorting (MACS) was used for the first time for purification of hPSC-derived NPs after the neural commitment stage using anti-Tra-1-60 micro beads for negative selection of the unwanted hPSCs. The depletion had an average efficiency of 80.4 ± 5% and less than 1.5% of Tra-1-60 positive cells were present in the purified populations. After re-plating, the purified neural precursors maintained their phenotype, and the success of the preparative purification with MACS was further confirmed with a decrease of 94.3% in the number of Oct4-positive proliferating hPSC colonies. Thus, the integration of the MACS depletion step with the neural commitment protocol paves the way towards the establishment of a novel bioprocess for production of purified populations of hPSC-derived neural cells for different applications.