Direct isolation of committed neuronal progenitor cells from transgenic mice coexpressing spectrally distinct fluorescent proteins regulated by stage-specific neural promoters

Many tissues arise from pluripotent stem cells through cell-type specification and maturation. In the bone marrow, primitive stem cells generate all the different types of blood cells via the sequential differentiation of increasingly committed progenitor cells. Cell-surface markers that clearly distinguish stem cells, restricted progenitors, and differentiated progeny have enabled researchers to isolate these cells and to study the regulatory mechanisms of hematopoiesis. Neuronal differentiation appears to involve similar mechanisms. However, neural progenitor cells that are restricted to a neuronal fate have not been characterized in vivo, because specific cell-surface markers are not available. We have developed an alternative strategy to identify and isolate neuronal progenitor cells based on dual-color fluorescent proteins. To identify and isolate directly progenitor cells from brain tissue without the need for either transfection or intervening cell culture, we established lines of transgenic mice bearing fluorescent transgenes regulated by neural promoters. One set of transgenic lines expressed enhanced yellow fluorescent protein (EYFP) in neuronal progenitor cells and neurons under the control of the Talpha1 alpha-tubulin promoter. Another line expressed enhanced green fluorescent protein (EGFP) in immature neural cells under the control of the enhancer/promoter elements of the nestin gene. By crossing these lines we obtained mice expressing both transgenes. To isolate neuronal progenitor cells directly from the developing brain, we used flow cytometry, selecting cells that expressed EGFP and EYFP simultaneously. We expect this strategy to provide valuable material with which to study the mechanisms of neurogenesis and to develop cell-based therapies for neurological disorders.     

Neurons derived from human-induced pluripotent stem cells express mu and kappa opioid receptors

Neuroprotection studies have shown that induced pluripotent stem(iPS) cells have the possibility to transform neuroprotection research. In the present study, iPS cells were generated from human renal epithelial cells and were then differentiated into neurons. Cells in the iPScell group were maintained in stem cell medium. In contrast, cells in the iPS-neuron group were first maintained in neural induction medium and expansion medium containing ROCK inhibitors, and then cultivated in neuronal differentiation medium and neuronal maturation medium to induce the neural stem cells to differentiate into neurons. The expression of relevant markers was compared at different stages of differentiation. Immunofluorescence staining revealed that cells in the iPS-neuron group expressed the neural stem cell markers SOX1 and nestin on day 11 of induction, and neuronal markers TUBB3 and NeuN on day 21 of induction. Polymerase chain reaction results demonstrated that, compared with the iPS-cell group, TUBB3 gene expression in the iPS-neuron group was increased 15.6-fold. Further research revealed that, compared with the iPS-cell group, the gene expression and immunoreactivity of mu opioid receptor in the iPS-neuron group were significantly increased(38.3-fold and 5.7-fold, respectively), but those of kappa opioid receptor had only a slight change(1.33-fold and 1.57-fold increases, respectively). Together, these data indicate that human iPS cells can be induced into mu opioid receptor-and kappa opioid receptor-expressing neurons, and that they may be useful to simulate human opioid receptor function in vitro and explore the underlying mechanisms of human conditions.     

Induced pluripotent stem (iPS) cells as <Emphasis Type="Italic">in vitro</Emphasis> models of human neurogenetic disorders

The recent discovery of genomic reprogramming of human somatic cells into induced pluripotent stem cells offers an innovative and relevant approach to the study of human genetic and neurogenetic diseases. By reprogramming somatic cells from patient samples, cell lines can be isolated that self-renew indefinitely and have the potential to develop into multiple different tissue lineages. Additionally, the rapid progress of research on human embryonic stem cells has led to the development of sophisticated in vitro differentiation protocols that closely mimic mammalian development. In particular, there have been significant advances in differentiating human pluripotent stem cells into defined neuronal types. Here, we summarize the experimental approaches employed in the rapidly evolving area of somatic cell reprogramming and the methodologies for differentiating human pluripotent cells into neurons. We also discuss how the availability of patient-specific fibroblasts offers a unique opportunity for studying and modeling the effects of specific gene defects on human neuronal development in vitro and for testing small molecules or other potential therapies for the relevant neurogenetic disorders.     

Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell‐derived neurons in culture

Human induced pluripotent stem (iPS) cell‐derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell‐derived neural stem cells (NSCs). We first demonstrate human iPS cell‐derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co‐culture on feeder layers of isogenic human iPS cell‐derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor‐channel function and occurred without changes in I_Na, I_K, V_m, and AP properties of iPS cell‐derived neurons. These data demonstrate co‐culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell‐derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell‐derived neurons in culture. We present RNA‐sequencing data for an immature, fetal‐like status of our human iPS cell‐derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co‐culture system.     

Induced pluripotent stem cells and promises of neuroregenerative medicine

First created in 2006 from adult somatic cells by a simple molecular genetic trick, induced pluripotent stem cells (iPS) system is the latest platform in stem cell research. Induced pluripotent stem cells are produced by nuclear reprogramming technology and they resemble embryonic stem cells (ES) in key elements; they possess the potentiality to differentiate into any type of cell in the body. More importantly, the iPS platform has distinct advantage over ES system in the sense that iPS-derived cells are autologous and therefore the iPS-derived transplantation does not require immunosuppressive therapy. In addition, iPS research obviates the political and ethical quandary associated with embryo destruction and ES research. This remarkable discovery of cellular plasticity has important medical implications. This brief review summarizes currently available stem cell platforms, with emphasis on cellular reprogramming and iPS technology and its application in disease modeling and cell replacement therapy in neurodegenerative diseases.     

Isolation of neuronal precursors from differentiating P19 embryonal carcinoma cells by neuronal T alpha 1-promoter-driven GFP.

The induction of pluripotent P19 embryonal carcinoma (EC) cells with retinoic acid results in their differentiation into cells that resemble neurons, glia, and fibroblasts. To isolate and enrich the developing neurons from heterogeneously differentiating P19 EC cells, we used a recently introduced protocol combining the expression of green fluorescent protein (GFP) driven by a tissue-specific promoter and fluorescence-activated cell sorting. Cells were transfected with the gene for GFP, which is under the control of the neuronal T alpha 1 tubulin promoter. After four days of retinoic acid treatment, GFP was specifically detected in cells undergoing neuronal differentiation. Sorting of fluorescent differentiating P19 EC transfectants yielded populations highly enriched in neuronal precursors and neurons. Immunoreactivity for nestin and neurofilament was observed in 80 and 25% of the sorted cell population, respectively. These results demonstrate that differentiated neuronal precursor cells can be efficiently isolated from differentiating pluripotent embryonic cells in vitro, suggesting that this method can reproducibly provide homogeneous materials for further studies on neurogenesis.     

Slower cycling of nestin-positive cells in neurosphere culture

Neural stem cells are multipotent and self-renewing cells with important potential application in cell replacement therapy in brain damage. Many studies have shown that nestin-positive cells represent neural stem and progenitor cells in the central neural system. Here, we derived neural stem cells from the subventricular zone of a newborn nestin-promoter-driven green fluorescent protein mouse, and found that the percentage of nestin-positive cells decreased continuously at each passage in neurosphere culture. Using the relative proliferation ratio and relative division ratio analysis, we concluded that the slower cycling of nestin-positive cells was responsible for this decrease.     

Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast‐derived iPS cells using zinc‐finger nuclease technology

Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast‐derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human‐derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc‐finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte‐specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno‐associated virus integration site 1 (AAVS1) locus in disease and control‐derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP‐iPS astrocytes exhibited a well‐described functional astrocytic activity in vitro characterized by neuron‐dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP‐iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP‐iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte‐driven disease pathogenesis and therapy. GLIA 2016;64:63–75     

Intraocular injection of folate antagonist methotrexate induces neuronal differentiation of embryonic stem cells transplanted in the adult mouse retina

Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuronal cells. However, the integrated ES cells also have a tumorigenic effect just because they have the ability for multipotential differentiation to various types of tissues. Thus, control of neoplastic potentials of ES cells is very important for the treatment of degenerative or injured diseases. Mouse ES cells carrying the sequence for the green fluorescent protein (GFP) gene were transplanted into adult mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-d-aspartate (NMDA) administration. One week after the ES cell injection, folate antagonist methotrexate (MTX) was injected intravitreally. Eyes were retrieved 4 weeks after ES cell transplantation for histologic analyses. Conventional histological analysis was performed by hematoxylin and eosin staining with the use of paraffin-embedded sections. Neuronal differentiation and teratogenic potential of ES cells were demonstrated by immunohistochemistry. The proliferative activity of transplanted cells was detected by mitotic index, proliferating cell nuclear antigen index and AgNOR count. The incorporation of transplanted ES cells in MTX-treated and non-treated retinas at 4 weeks after transplantation was observed in 8/16 eyes (50%) and 8/16 eyes (50%), respectively. Transplanted ES cells in MTX-treated retina showed increased neuronal differentiation and decreased expression of teratogenic markers, compared with ES cells in non-treated retina. The proliferative activity of transplanted ES cells in MTX-treated retina was lower than that in non-treated retina. These results suggest that intravitreal MTX treatment following transplantation can induce neuronal differentiation in the transplanted ES cells and decrease their proliferative activity.     

Generation of Human-Induced Pluripotent Stem Cells to Model Spinocerebellar Ataxia Type 2 In vitro

Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotide repeat (CAG) expansion in the coding region of the ATAXN2 gene on chromosome 12, which produces an elongated, toxic polyglutamine tract, leading to Purkinje cell loss. There is currently no effective therapy. One of the main obstacles that hampers therapeutic development is lack of an ideal disease model. In this study, we have generated and characterized SCA2-induced pluripotent stem (iPS) cell lines as an in vitro cell model. Dermal fibroblasts (FBs) were harvested from primary cultures of skin explants obtained from a SCA2 subject and a healthy subject. For reprogramming, hOct4, hSox2, hKlf4, and hc-Myc were transduced to passage-3 FBs by retroviral infection. Both SCA2 iPS and control iPS cells were successfully generated and showed typical stem cell growth patterns with normal karyotype. All iPS cell lines expressed stem cell markers and differentiated in vitro into cells from three embryonic germ layers. Upon in vitro neural differentiation, SCA2 iPS cells showed abnormality in neural rosette formation but successfully differentiated into neural stem cells (NSCs) and subsequent neural cells. SCA2 and normal FBs showed a comparable level of ataxin-2 expression; whereas SCA2 NSCs showed less ataxin-2 expression than normal NSCs and SCA2 FBs. Within the neural lineage, neurons had the most abundant expression of ataxin-2. Time-lapsed neural growth assay indicated terminally differentiated SCA2 neural cells were short-lived compared with control neural cells. The expanded CAG repeats of SCA2 were stable throughout reprogramming and neural differentiation. In conclusion, we have established the first disease-specific human SCA2 iPS cell line. These mutant iPS cells have the potential for neural differentiation. These differentiated neural cells harboring mutations are invaluable for the study of SCA2 pathogenesis and therapeutic drug development.     

Differentiation of induced pluripotent stem cells into functional oligodendrocytes

The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells.     

Isolation of neuronal precursors from differentiating P19 embryonal carcinoma cells by neuronal Tα1-promoter-driven GFP

The induction of pluripotent P19 embryonal carcinoma (EC) cells with retinoic acid results in their differentiation into cells that resemble neurons, glia, and fibroblasts. To isolate and enrich the developing neurons from heterogeneously differentiating P19 EC cells, we used a recently introduced protocol combining the expression of green fluorescent protein (GFP) driven by a tissue-specific promoter and fluorescence-activated cell sorting. Cells were transfected with the gene for GFP, which is under the control of the neuronal Tα1 tubulin promoter. After four days of retinoic acid treatment, GFP was specifically detected in cells undergoing neuronal differentiation. Sorting of fluorescent differentiating P19 EC transfectants yielded populations highly enriched in neuronal precursors and neurons. Immunoreactivity for nestin and neurofilament was observed in 80 and 25% of the sorted cell population, respectively. These results demonstrate that differentiated neuronal precursor cells can be efficiently isolated from differentiating pluripotent embryonic cells in vitro, suggesting that this method can reproducibly provide homogeneous materials for further studies on neurogenesis.     

2-DE proteomic profiling of neuronal stem cells

Proteomics has become a powerful tool in neuroscience studies. Although numerous human neural stem cells are available for research purposes since many years, there exists only limited information on proteomic data from stable neural stem cell lines. Profiling and functional proteome studies of neuronal stem cells will help to describe the protein inventory as well as protein activity and interactions, subcellular localization and posttranslational modifications. The proteomic analysis of neuronal differentiation processes will elucidate the complex events leading to the generation of different phenotypes via distinctive developmental programs that control self-renewal, differentiation, and plasticity. Using the ReNcell VM197 model, a cell line derived from human fetal ventral mesencephalon stem cells, we studied the protein inventory of the stem cells by 2-DE gel electrophoresis and mass spectrometric protein identification and constructed a 2-DE protein map consisting of more than 400 identified protein spots. This proteome reference database constitutes the basis for further investigations of differential protein expression during differentiation. A profiling of the neuronal differentiation-associated changes displayed the large rearrangement of the proteome during this process, and the proteomic techniques proved to be a valuable tool for the elucidation of neuronal differentiation process and for target protein screening.     

Isolation of a glial-restricted tripotential cell line from embryonic spinal cord cultures

Neuroepithelial stem cells (NEPs), glial-restricted precursors (GRPs), and neuron-restricted precursors (NRPs) are present during early differentiation of the spinal cord and can be identified by cell surface markers. In this article, we describe the properties of GRP cells that have been immortalized using a regulatable v-myc retrovirus construct. Immortalized GRP cells can be maintained in an undifferentiated dividing state for long periods and can be induced to differentiate into two types of astrocytes and into oligodendrocytes in culture. A clonal cell line prepared from immortalized GRP cells, termed GRIP-1, was also shown to retain the properties of a glial-restricted tripotential precursor. Transplantation of green fluorescent protein (GFP)-labeled subclones of the immortalized cells into the adult CNS demonstrates that this cell line can also participate in the in vivo development of astrocytes and oligodendrocytes. Late passages of the immortalized cells undergo limited transdifferentiation into neurons as assessed by expression of multiple neuronal markers. The availability of a conditionally immortalized cell line obviates the difficulties of obtaining a large and homogeneous population of GRPs that can be used for studying the mechanism and signals for glial cell differentiation as well as their application in transplantation protocols.     

Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice

We compared the effects of hematopoietic stem cell and marrow stromal cell transplantation for spinal cord injury in mice. From green fluorescent protein transgenic mouse bone marrow, lineage-negative, c-kit- and Sca-1-positive cells were sorted as hematopoietic stem cells and plastic-adherent cells were cultured as marrow stromal cells. One week after injury, hematopoietic stem cells or marrow stromal cells were injected into the lesioned site. Functional recovery was assessed and immunohistochemistry was performed. In the hematopoietic stem cell group, a portion of green fluorescent protein-positive cells expressed glial marker. In the marrow stem cell group, a number of green fluorescent protein and fibronectin-double positive cells were observed. No significant difference was observed in the recovery between both groups. Both hematopoietic stem cells and marrow stromal cells have the potential to restore the injured spinal cord and to promote functional recovery.