CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels

The mammalian central nervous system (CNS) has little capacity for self-repair after injury, and neurons are not capable of proliferating. Therefore, neural tissue engineering that combines neural stem and progenitor cells and biologically derived polymer scaffolds may revolutionize the medical approach to the treatment of damaged CNS tissues. Neural stem and progenitor cells isolated from embryonic rat cortical or subcortical neuroepithelium were dispersed within type I collagen, and the cell-collagen constructs were cultured in serum-free medium containing basic fibroblast growth factor. The collagen-entrapped stem and progenitors actively expanded and efficiently generated neurons, which developed neuronal polarity, neurotransmitters, ion channels/receptors, and excitability. Ca2+ imaging showed that differentiation from BrdU+/TuJ1- to BrdU-/TuJ1+ cells was accompanied by a shift in expression of functional receptors for neurotransmitters from cholinergic and purinergic to predominantly GABAergic and glutamatergic. Spontaneous postsynaptic currents were recorded by patch-clamping from precursor cell-derived neurons and these currents were partially blocked by 10-microM bicuculline, and completely blocked by additional 10 microM of the kainate receptor antagonist CNQX, indicating an appearance of both GABAergic and glutamatergic synaptic activities. Staining with endocytotic marker FM1-43 demonstrated active synaptic vesicle recycling occurring among collagen-entrapped neurons. These results show that neural stem and progenitor cells cultured in 3D collagen gels recapitulate CNS stem cell development; this is the first demonstration of CNS stem and progenitor cell-derived functional synapse and neuronal network formation in a 3D matrix. The proliferative capacity and neuronal differentiating potential of neural progenitors in 3D collagen gels suggest their potential use in attempts to promote neuronal regeneration in vivo.     

Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks

Human astrocytes differ dramatically in cell morphology and gene expression from murine astrocytes. The latter are well known to be of major importance in the formation of neuronal networks by promoting synapse maturation. However, whether human astrocyte lineage cells have a similar role in network formation has not been firmly established. Here, we investigated the impact of human astrocyte lineage cells on the functional maturation of neural networks that were derived from human induced pluripotent stem cells (hiPSCs). Initial in vitro differentiation of hiPSC-derived neural progenitor cells and immature neurons (glia+ cultures) resulted in spontaneously active neural networks as indicated by synchronous neuronal Ca²⁺ transients. Depleting proliferating neural progenitors from these cultures by short-term antimitotic treatment resulted in strongly astrocyte lineage cell-depleted neuronal networks (glia− cultures). Strikingly, in contrast to glia+ cultures, glia− cultures did not exhibit spontaneous network activity. Detailed analysis of the morphological and electrophysiological properties of neurons by patch clamp recordings revealed reduced dendritic arborization in glia− cultures. In addition, a reduced action potential frequency upon current injection in pyramidal-like neurons was observed, whereas the electrical excitability of multipolar neurons was unaltered. Furthermore, we found a reduced dendritic density of PSD95-positive excitatory synapses, and more immature properties of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) miniature excitatory postsynaptic currents (mEPSCs) in glia− cultures, suggesting that the maturation of glutamatergic synapses depends on the presence of hiPSC-derived astrocyte lineage cells. Intriguingly, addition of the astrocyte-derived synapse maturation inducer cholesterol increased the dendritic density of PSD95-positive excitatory synapses in glia− cultures.     

GFAP-expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes

Neural stem cells in the adult subventricular zone (SVZ) derive from radial glia and express the astroglial marker glial fibrillary acidic protein (GFAP). Thus, they have been termed astrocytes. However, it remains unknown whether these GFAP-expressing cells express the functional features common to astrocytes. Using immunostaining and patch clamp recordings in acute slices from transgenic mice expressing green fluorescent protein (GFP) driven by the promoter of human GFAP, we show that GFAP-expressing cells in the postnatal SVZ display typical glial properties shared by astrocytes and prenatal radial glia such as lack of action potentials, hyperpolarized resting potentials, gap junction coupling, connexin 43 expression, hemichannels, a passive current profile, and functional glutamate transporters. GFAP-expressing cells express both GLAST and GLT-1 glutamate transporters but lack AMPA-type glutamate receptors as reported for dye-coupled astrocytes. However, they lack 100 microM Ba2+-sensitive inwardly rectifying K+ (K(IR)) currents expressed by astrocytes, but display delayed rectifying K+ currents and 1 mM Ba2+-sensitive K+ currents. These currents contribute to K+ transport at rest and maintain hyperpolarized resting potentials. GFAP-expressing cells stained positive for both K(IR)2.1 and K(IR)4.1 channels, two major K(IR) channels in astrocytes. Ependymal cells, which also derive from radial glia and express GFAP, display typical glial properties and K(IR) currents consistent with their postmitotic nature. Our results suggest that GFAP-expressing cells in concert with ependymal cells can perform typical astrocytic functions such as K+ and glutamate buffering in the postnatal SVZ but display a unique set of functional characteristics intermediate between astrocytes and radial glia.     

Voltage-dependent potassium currents in hypertrophied rat astrocytes after a cortical stab wound

Changes in the membrane properties of reactive astrocytes in gliotic cortex induced by a stab wound were studied in brain slices of 21-28-day-old rats, using the patch-clamp technique and were correlated with changes in resting extracellular K+ concentration ([K+]e) measured in vivo using K+-selective microelectrodes. Based on K+ current expression, three types of astrocytes were identified in gliotic cortex: A1 astrocytes expressing a time- and voltage-independent K+ current component and additional inwardly rectifying K+ currents (K(IR)); A2 astrocytes expressing a time- and voltage-independent K+ current component and additional delayed outwardly rectifying K+ currents (K(DR)); and complex astrocytes expressing K(DR), K(IR), and A-type K+ (K(A)) currents and Na+ currents (I(Na)). Nestin/bromodeoxyuridine (BrdU)-negative A1 astrocytes were found further than approximately 100 microm from the stab wound and showed an upregulation of K(IR) currents within the first day post-injury (PI), correlating with an increased resting [K+]e. Their number declined from 62% of total astrocytes in control rats to 41% in rats at 7 days PI. Nestin/BrdU-positive A2 astrocytes were found only within a distance of approximately 100 microm from the stab wound and, in comparison to those in control rats, showed an upregulation of K(DR) currents. Their number increased from 8% of the total number of astrocytes in control rats to 39% 7 days PI. Both A1 and A2 astrocytes showed hypertrophied processes and increased GFAP staining, but an examination of cell morphology revealed greater changes in the surface/volume ratio in A2 astrocytes than in A1 astrocytes. Complex astrocytes did not display a hypertophied morphology; K(IR) currents in these cells were upregulated within 1 day PI, while the K(DR), K(A), and I(Na) currents were increased only 6 h PI. We conclude that two electrophysiologically, immunohistochemically, and morphologically distinct types of hypertrophied astrocytes are present at the site of a stab wound, depending on the distance from the lesion, and may have different functions in ionic homeostasis and/or regeneration.     

4000/人胚胎干细胞源TH 阳性细胞电压门控性通道的初步研究

目的：检测人胚胎干细胞源TH阳性细胞的神经元性电生理特性。方法：采用我们实验室改良后的“无血清四步法经拟胚体培养体系”的方法,体外诱导人胚胎干细胞源性TH阳性细胞,在对其细胞核型及特异性标志物进行检测的基础上,运用全细胞膜片钳记录的方法,检测其细胞膜上电压门控性离子通道的电生理特性。结果：分化前后细胞核型保持正常;诱导得到的细胞形态一致,大多数细胞（>90%）表达多巴胺能神经元的标志β-tubulion和TH,并仍表达神经前体细胞的标志nestin;膜片钳检测显示诱导分化的TH阳性细胞具有神经元性电压门控钠、钾离子通道。结论：人胚胎干细胞经体外定向诱导分化为TH阳性细胞后具有一定的DA能神经元特性,特别是神经元性电生理特性。     

Two outward potassium current types are expressed during the neural differentiation of neural stem cells

The electrophysiological properties of potassium ion channels are regarded as a basic index for determining the functional differentiation of neural stem cells. In this study, neural stem cells from th...     

Pax6 directly modulate Sox2 expression in the neural progenitor cells

Pax6 is a key regulator in the neuronal fate determination as well as the proliferation of neural stem cells, but the mechanisms are still unknown. Our study shows that Pax6 regulate the proliferation of neural progenitor cells of cortical subventricular zone, through direct modulation of the Sox2 expression during the late developmental stage in mice. We found a dramatic decrease in the number of Sox2+ neural progenitor cells in the subventricular zone of E18.5 Pax6(-/-) mice. We confirmed that Pax6 could bind to the Sox2 promoter by chromatin immunoprecipitation assay and activate Sox2 expression by a luciferase reporter gene assay. Moreover, neural progenitors isolated from the Pax6(-/-) embryos showed a decreased neurosphere formation as well as proliferation.     

Moderate traumatic brain injury promotes proliferation of quiescent neural progenitors in the adult hippocampus

Recent evidence shows that traumatic brain injury (TBI) regulates proliferation of neural stem/progenitor cells in the dentate gyrus (DG) of adult hippocampus. There are distinct classes of neural stem/progenitor cells in the adult DG, including quiescent neural progenitors (QNPs), which carry stem cell properties, and their progeny, amplifying neural progenitors (ANPs). The response of each class of progenitors to TBI is not clear. We here used a transgenic reporter Nestin-GFP mouse line, in which QNP and ANP cells are easily visualized and quantified, to determine the targets of the TBI in the DG. We examined changes in proliferation of QNPs and ANPs in the acute phase following TBI and found that QNPs were induced by TBI insult to enter the cell cycle whereas proliferation of ANPs was not significantly affected. These results indicate that different subtypes of neural stem/progenitor cells respond differently to TBI insult. Stem cell activation by the TBI may reflect the induction of innate repair and plasticity mechanisms by the injured brain.     

Activity-dependent long-term plasticity of afferent synapses on grafted stem/progenitor cell-derived neurons

Stem cell-based cell replacement therapies aiming at restoring injured or diseased brain function ultimately rely on the capability of transplanted cells to promote functional recovery. The mechanisms by which stem cell-based therapies for neurological conditions can lead to functional recovery are uncertain, but structural and functional repair appears to depend on integration of transplanted cell-derived neurons into neuronal circuitries. The nature by which stem/progenitor cell-derived neurons synaptically integrate into neuronal circuitries is largely unexplored. Here we show that transplanted GFP-labeled neuronal progenitor cells into the rat hippocampus exhibit mature neuronal morphology following 4–10 weeks. GFP-positive cells were preferentially integrated into the principal cell layers of hippocampus, particularly CA3. Patch-clamp recordings from GFP-expressing cells revealed that they generated fast action potentials, and their intrinsic membrane properties were overall similar to endogenous host neurons recorded in same areas. As judged by occurrence of spontaneous excitatory postsynaptic currents (EPSCs), transplanted GFP-positive cells were synaptically integrated into the host circuitry. Comparable to host neurons, both paired-pulse depression and facilitation of afferent fiber stimulation-evoked EPSCs were observed in GFP-positive cells. Upon high-frequency stimulation, GFP-positive cells displayed post-tetanic potentiation of EPSCs, in some cases followed by long-term potentiation (LTP) lasting for more than 30 min. Our data show for the first time that transplanted neuronal progenitor cells can become functional neurons and their afferent synapses are capable of expressing activity-dependent short and long-term plasticity. These synaptic properties may facilitate host-to-graft interactions and regulate activity of the grafted cells promoting functional recovery of the diseased brain.     

Skeletal muscle-derived progenitor cells exhibit neural competence

Skeletal muscle contains heterogenous progenitor cells that give rise to muscle, hematopoietic cells and bone. The exact phenotypic definition of skeletal muscle progenitor cells has not been fully elucidated nor the potential of these cells to differentiate into neurons. Here, we demonstrate that phenotypically homogenous skeletal muscle progenitor cells defined as Lin-CD45-CD117-CD90+ cells express neural stem cell markers and are responsive to neural induction signals. When exposed to neural induction medium containing basic fibroblast growth factor and brain-derived neurotrophic factor, skeletal muscle progenitor cells dramatically changed their cell morphology, became postmitotic and began expressing neuronal markers. These results reveal unexpected potentials of muscle progenitor cells and suggest that these cells may potentially be used in cell-based therapies to replace damaged neurons.     

Changes of KCl sensitivity of proliferating neural progenitors during in vitro neurogenesis

The effects of KCl-treatment on the survival and proliferation of NE-4C self-renewing neural progenitor cells were investigated during early phases of in vitro induced neurogenesis. NE-4C cells, derived from the anterior brain vesicles of embryonic mouse (E9), divided continuously under non-inducing conditions, but acquired neuronal features within 6 days, if induced by all-trans retinoic acid (RA). During the first 2 days of induction, the cells went on proliferating and did not show signs of morphological differentiation. In this stage, the resting membrane potential of RA-induced cells adopted more negative values in comparison to non-induced ones. Despite the increased membrane polarity and K+ conductance, addition of 20-50 mM KCl failed to elicit inward Na+ currents and did not induce an increase in the intracellular Ca+ level. Long-term treatment with 25 mM KCl, on the other hand, resulted in a selective loss of cells committed to neuronal fate by both decreasing the rate of cell proliferation and increasing the rate of cell death. The data indicate that the viability and proliferation of neural progenitors are influenced by extracellular K+-level in a differentiation stage-dependent manner.     

Effects of doxapram on ionic currents recorded in isolated type I cells of the neonatal rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of the respiratory stimulant doxapram on K+ and Ca2+ currents in isolated type I cells of the neonatal rat carotid body. Doxapram (1-100 microM) caused rapid, reversible and dose-dependent inhibitions of K+ currents recorded in type I cells (IC50 approximately 13 microM). Inhibition was voltage-dependent, in that the effects of doxapram were maximal at test potentials where a shoulder in the current-voltage relationship was maximal. These K+ currents were composed of both Ca(2+)-activated and Ca(2+)-independent components. Using high [Mg2+], low [Ca2+] solutions to inhibit Ca(2+)-activated K+ currents, doxapram was also seen to directly inhibit Ca(2+)-independent K+ currents. This effect was voltage-independent and was less potent (IC50 approximately 20 microM) than under control conditions, suggesting that doxapram was a more potent inhibitor of the Ca(2+)-activated K+ currents recorded under control conditions. Doxapram (10 microM) was without effect on L-type Ca2+ channel currents recorded under conditions where K+ channel activity was minimized and was also without significant effect on K+ currents recorded in the neuronal cell line NG-108 15, suggesting a selective effect on carotid body type I cells. The effects of doxapram on type I cells show similarities to those of the physiological stimuli of the carotid body, suggesting that doxapram may share a similar mechanism of action in stimulating the intact organ.     

Human NT2/D1 cells differentiate into functional astrocytes

As the most numerous cell type in the brain, astrocytes are coupled via gap junction channels. It is believed that communication among astrocytes is normally regulated by extracellular ions, neurotransmitters and neuromodulators. However, the level of astrocytic coupling is altered in abnormal conditions such as stroke, brain trauma and Alzheimer's disease. A well established human progenitor cell line, NT2/D1, has been previously differentiated into pure neuronal cultures. In the current study, for the first time, we report the differentiation of NT2/D1 cells into astrocytes, which express connexin43 and are coupled via gap junctions. Thus, human NT2/D1 cells are not merely committed neuronal progenitors but, similar to the embryonal stem cells, they can give rise to both lineages.     

Modification of the brain-derived neurotrophic factor gene: a portal to transform mesenchymal stem cells into advantageous engineering cells for neuroregeneration and neuroprotection

Multipotential mesenchymal stem cells (MSCs) are ideal seed cells for recruiting the loss of neural cells due to their strong proliferative capacity, easy acquisition, and considerable tolerance of genetic modifications. After transduction of brain-derived neurotrophic factor (BDNF) gene via recombinant retroviral vectors into the human MSCs, nearly 100% of cells expressed BDNF (which were therefore transformed into BNDF-MSCs) as detected by immunocytochemistry, and the quantity of BDNF in the culture medium was increased by approximately 20,000-fold. In spite of the genomic integration of an exogenous gene, BDNF-MSCs did not present any structural aberration in the chromosomes. All-trans-retinoic acid (RA) induction caused the BDNF-MSCs to differentiate into neural cells with significantly increased expressions of such neural-specific proteins as nestin, NeuN, O4, and glial fibrillary acidic protein (GFAP). The voltage-dependent K+/Ca2+ currents were recorded from the induced BDNF-MSCs using patch-clamp technique. Compared with the MSCs induced by both RA and BDNF, BDNF-MSCs survived in significantly greater number in the induction medium, and also more cells were induced into neuron-like cells (NeuN, P < 0.01) and oligodendrocyte-like cells (O4, P < 0.05). We suppose that, once engrafted into human central nervous system, the BDNF-MSCs would not only recruit the neuronal losses, but also provide, by way of paracrine, large quantities of BDNF that effectively perform the functions of neuroprotection and neuroregeneration, promoting the activation of endogenous neural stem/progenitor cells and their chemotactic migration. On the other hand, the BDNF-MSCs that can survive in the host environment and differentiate subsequently into functional mature cells may also serve as specifically targeting vectors for ex vivo gene therapy.     

The atypical homeoprotein Pbx1a participates in the axonal pathfinding of mesencephalic dopaminergic neurons

Background

Pannexin 1 forms ion and metabolite permeable hexameric channels and is abundantly expressed in the brain. After discovering pannexin 1 expression in postnatal neural stem and progenitor cells we sought to elucidate its functional role in neuronal development.

Results

We detected pannexin 1 in neural stem and progenitor cells in vitro and in vivo. We manipulated pannexin 1 expression and activity in Neuro2a neuroblastoma cells and primary postnatal neurosphere cultures to demonstrate that pannexin 1 regulates neural stem and progenitor cell proliferation likely through the release of adenosine triphosphate (ATP).

Conclusions

Permeable to ATP, a potent autocrine/paracine signaling metabolite, pannexin 1 channels are ideally suited to influence the behavior of neural stem and progenitor cells. Here we demonstrate they play a robust role in the regulation of neural stem and progenitor cell proliferation. Endogenous postnatal neural stem and progenitor cells are crucial for normal brain health, and their numbers decline with age. Furthermore, these special cells are highly responsive to neurological injury and disease, and are gaining attention as putative targets for brain repair. Therefore, understanding the fundamental role of pannexin 1 channels in neural stem and progenitor cells is of critical importance for brain health and disease.     

Utilizing X-irradiation to selectively eliminate neural stem/progenitor cells from neurogenic regions of the mammalian brain

Neural stem/progenitor cells residing in the mammalian CNS provide a potential endogenous source for replenishing neurons that are lost due to aging, trauma or disease. However, little is known about their functional potential due to the lack of methodologies that allow for the reproducible alteration of stem cell numbers in vivo. Accordingly, we describe a methodology that utilizes targeted X-irradiation to experimentally generate neural stem/progenitor cell-depleted rat models. We show that, by virtue of their mitotic activity, proliferating neural stem/progenitor cells can be selectively eliminated from either the subventricular zone (SVZ) or dentate gyrus of a rat by treating it to an (unilateral or bilateral) exposure of X-irradiation. Utilizing BrdU incorporation, it was found that a single 15 gray (Gy) exposure to the SVZ resulted in the elimination of 85% of the proliferating cell population for up to 3 months. Immunohistochemistry, ultrastructural analysis and proteomics were employed to confirm that the cells eliminated following X-irradiation were neural stem/progenitor cells. Similar depletions of the stem/progenitor cell population in the dentate gyrus were achieved by targeting the hippocampus with a single 15Gy exposure. The reproducibility, versatility and ease of generation make these experimental animal models a valuable tool to aid in our understanding of the properties and functions of neural stem/progenitor cells.     

Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke

In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.