Single‐cell study of neural stem cells derived from human iPSCs reveals distinct progenitor populations with neurogenic and gliogenic potential

We used single‐cell RNA sequencing (seq) on several human induced pluripotent stem (iPS) cell‐derived neural stem cell (NSC) lines and one fetal brain‐derived NSC line to study inherent cell type heterogeneity at proliferating neural stem cell stage and uncovered predisposed presence of neurogenic and gliogenic progenitors. We observed heterogeneity in neurogenic progenitors that differed between the iPS cell‐derived NSC lines and the fetal‐derived NSC line, and we also observed differences in spontaneous differentiation potential for inhibitory and excitatory neurons between the iPS cell‐derived NSC lines and the fetal‐derived NSC line. In addition, using a recently published glia patterning protocol we enriched for gliogenic progenitors and generated glial cells from an iPS cell‐derived NSC line.     

Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle

Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.     

Different effects of anti-sonic hedgehog antibodies and the hedgehog pathway inhibitor cyclopamine on generation of dopaminergic neurons from neurospheres of mesencephalic precursors.

Production of dopaminergic neurons from stem/precursor cells for transplantation in Parkinson's disease has become a major focus of research. However, the inductive signals mediating this process have not been clarified. Reported data on the effects of Sonic hedgehog on differentiation of dopaminergic and serotonergic neurons from cultures of neural precursors are controversial. In the present study, cultures of proliferating neurospheres of mesencephalic precursors treated with anti-sonic hedgehog antibodies showed significantly less serotonergic and GABAergic cells and a markedly higher number of dopaminergic neurons generated from the neurospheres than control cultures. Treatment of the neurospheres with cyclopamine, which selectively inhibits sonic hedgehog signaling by preventing Smoothened activation, did not induce significant changes in generation of serotonergic and dopaminergic neurons. This suggests that Smoothened activation is not significantly involved in the above-mentioned effects and that sonic hedgehog may exert effects on the mesencephalic precursors that do not involve the canonical Patched-Smoothened-Gli signaling.     

Brain injury activates microglia that induce neural stem cell proliferation ex vivo and promote differentiation of neurosphere-derived cells into neurons and oligodendrocytes

Brain damage, such as ischemic stroke, enhances proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ). To date, no reliable in vitro systems, which can be used to unravel the potential mechanisms underlying this lesion-induced effect, have been established. Here, we developed an ex vivo method to investigate how the proliferation of NSPCs changes over time after experimental stroke or excitotoxic striatal lesion in the adult rat brain by studying the effects of microglial cells derived from an injured brain on NSPCs. We isolated NSPCs from the SVZ of brains with lesions and analyzed their growth and differentiation when cultured as neurospheres. We found that NSPCs isolated from the brains 1-2 weeks following injury consistently generated more and larger neurospheres than those harvested from naive brains. We attributed these effects to the presence of microglial cells in NSPC cultures that originated from injured brains. We suggest that the effects are due to released factors because we observed increased proliferation of NSPCs isolated from non-injured brains when they were exposed to conditioned medium from cultures containing microglial cells derived from injured brains. Furthermore, we found that NSPCs derived from injured brains were more likely to differentiate into neurons and oligodendrocytes than astrocytes. Our ex vivo system reliably mimics what is observed in vivo following brain injury. It constitutes a powerful tool that could be used to identify factors that promote NSPC proliferation and differentiation in response to injury-induced activation of microglial cells, by using tools such as proteomics and gene array technology.     

来源于人胚室下区神经干/祖细胞的贴壁法培养和鉴定

目的建立有效的人胚胎神经干/祖细胞分离及纯化方法以达到临床需要。方法无菌取胎脑室管膜下区组织,经反复机械吹打制备细胞混悬液,采用贴壁法进行体外培养、传代。传代后用Nestin进行细胞鉴定。结果应用贴壁法进行胎脑室管膜下区神经干/祖细胞的培养能够稳定传代20代以上;共聚焦显微镜下可见Nestin阳性细胞表达呈递增趋势,传至P3(passage3)阳性率为35%,P7为79%,P12为90%,P15为99%。传到前七代时分别做细胞存活率鉴定,可达(82.57±1.38)%。结论成功建立了来源于人胚室下区的神经干/祖细胞的贴壁法培养,为该细胞移植治疗神经系统疾病提供了技术方法支持。     

NGF protects Dorsal Root Ganglion neurons from oxaliplatin by modulating JNK/Sapk and ERK1/2

The involvement of the Mitogen-Activated Protein Kinases (MAPKs) family in platinum derivative-induced peripheral neuropathy has already been demonstrated. In particular, it has been evidenced that in Dorsal Root Ganglion (DRG) neurons prolonged exposure to oxaliplatin (OHP) induces early activation of p38 and ERK1/2, which mediate neuronal apoptosis, while the neuroprotective action of JNK/Sapk is downregulated by the drug treatment. In this study, the exposure of OHP-treated neurons to a neuroprotective stimulus, represented by a high dose of NGF, counteracts OHP-induced neuronal mortality. This effect was achieved by restoring the MAPK activation existing in untreated control cells. Increased viability occurred also after the administration of retinoic acid (RA), a pro-differentiative agent able to activate both JNK/Sapk and ERK1/2. The use of specific chemical inhibitors of MAPKs confirms the importance of this class of proteins for the neuroprotective pathway, since they reverse the protective effect. In summary, our findings assess the validity of MAPKs as the target of neuroprotective therapies during chemotherapeutic treatment. Moreover they also describe a double role for ERK1/2, depending on cellular stimulation, since it mediates neuronal apoptosis after OHP exposure. However, it is also important, as is JNK/Sapk, in preserving the correct cellular differentiation that is pivotal for neuronal survival.     

Axonal branching of medullary swallowing neurons projecting on the trigeminal and hypoglossal motor nuclei: demonstration by electrophysiological and fluorescent double labeling techniques

Summary The projections of ventral medullary reticular neurons on both trigeminal (Vth) and hypoglossal (XIIth) motor nucleus were studied in sheep anesthetized with halothane. In a first series of experiments, extracellular microelectrodes were used to record the activity of medullary swallowing interneurons (SINs) located in the ventral region (around the nucleus ambiguus) of the swallowing center. Antidromic activation after electrical stimulation of the Vth and XIIth nuclei was tested in 83 SINs. For 38 SINs a clear antidromic activation was observed and for 8 of them the response was triggered by stimulation of either nucleus. As confirmed by the reciprocal collision test, these 8 SINs had branched axons sending information to both nuclei tested. Average latencies for antidromic activation of branched SINs after stimulation of the XIIth and the Vth motor nucleus were 2.2±0.6 ms and 2.7±0.8 ms respectively. The axonal conduction velocity of these neurons was 4.4±1.3 m/s for the collateral to the Vth motor nucleus and 2.7±0.7 m/s for axons projecting to the XIIth motor nucleus. In a second series of experiments the double retrograde labeling technique was used to confirm the existence of neurons with branched axons in the medullary regions corresponding to the swallowing center. Small and well localized injections of Fast Blue (FB) and Diamidino Yellow (DY) fluorescent tracers were made in the Vth and in the XIIth motor nucleus respectively. A relatively large number of double-labeled cells was found in the ventral region of swallowing center (reticular formation around the nucleus ambiguus, 2–4 mm in front of obex). Such neurons (supplying both the XIIth and the Vth motor nucleus) appeared mixed with those innervating only either the XIIth or the Vth motor nucleus. Each type of neuron, i.e. single or double labeled, was shown to have bilateral distribution with an ipsilateral predominance.     

切割穹窿海马伞海马提取液和银杏叶提取物联合诱导海马NSCs向胆碱能神经元的分化

为探讨切割穹窿海马伞海马提取液和银杏叶提取物(extract of ginkgo bilobo,EGb)在海马NSCs向胆碱能神经元定向分化中的作用,分别制备大鼠穹窿海马伞切割侧和正常侧海马提取液;将从鼠胚海马中分离扩增的NSCs球分成4组,应用不同的培养液促其分化:(1)联合组:含切割侧海马提取液和银杏内酯的DMEM/F12培养基;(2)提取液组:含切割侧海马提取液的DMEM/F12培养基;(3)EGb组:含银杏内酯的DMEM/F12培养基;(4)对照组:含正常侧海马提取液的DMEM/F12培养基。培养14d后行ChAT免疫荧光检测,计算ChAT阳性神经元的分化率,图像处理细胞面积和周长。结果显示联合组各项指标均明显优于其它各组(P<0.01);提取液组、EGb组各项指标也均优于对照组(P<0.05);细胞面积提取液组优于EGb组(P<0.05),细胞周长EGb组优于提取液组(P<0.05),两组ChAT阳性神经元分化率无明显差异(P>0.05)。上述提示切割穹窿海马伞的海马提取液和EGb联合应用可诱导海马NSCs分化为更多、更为成熟的胆碱能神经元。     

Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ

Current classification systems in proliferative mammary gland pathology are based on a two-cell system, recognizing only glandular and myoepithelial lines of differentiation. A third cell type has recently been characterized in normal breast tissue by double-immunofluorescence analysis to express cytokeratin 5 (Ck5) only. These cells were shown to represent progenitor or adult stem cells that give rise to the glandular and myoepithelial cell lineage. The double-labelling technique has been applied to characterize a spectrum of intraductal epithelial proliferations, namely benign usual ductal hyperplasia, atypical ductal hyperplasia, and ductal carcinoma in situ, all of which are thought to represent the gradual steps of a sequence in the development of breast cancer. Immunofluorescence studies with specific antibodies against Ck5, Ck8/18/19, and smooth muscle actin were complemented by western blotting analysis of Ck5 and Ck8/18/19 expression in normal breast tissue and in proliferative lesions. Usual ductal hyperplasia appears to be a Ck5-positive committed stem (progenitor) cell lesion with the same differentiation potential as seen in the normal breast. This is in sharp contrast to atypical ductal hyperplasia/ductal carcinoma in situ, which display the differentiated glandular immunophenotype (Ck8/18/19-positive, but Ck5-negative). These data require the abandonment of the idea of an obligate biological continuum of intraductal proliferations from benign to malignant. This study provides evidence that cells undergoing malignant transformation tend to be fairly advanced in the glandular lineage of differentiation. The committed stem (progenitor) cell model may contribute to a better understanding of both benign proliferative breast disease and breast cancer development.     

Retinoic acids acting through retinoid receptors protect hippocampal neurons from oxygen-glucose deprivation-mediated cell death by inhibition of c-jun-N-terminal kinase and p38 mitogen-activated protein kinase

Retinoic acids (RAs), including all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), play fundamental roles in a variety of physiological events in vertebrates, through their specific nuclear receptors: retinoic acid receptor (RAR) and retinoid X receptor (RXR). Despite the physiological importance of RA, their functional significance under pathological conditions is not well understood. We examined the effect of ATRA on oxygen/glucose-deprivation/reperfusion (OGD/Rep)-induced neuronal damage in cultured rat hippocampal slices, and found that ATRA significantly reduced neuronal death. The cytoprotective effect of ATRA was observed not only in cornu ammonis (CA) 1 but also in CA2 and dentate gyrus (DG), and was attenuated by selective antagonists for RAR or RXR. By contrast, in the CA3 region, no protective effects of ATRA were observed. The OGD/Rep also increased phosphorylated forms of c-jun-N-terminal kinase (P-JNK) and p38 (P-p38) in hippocampus, and specific inhibitors for these kinases protected neurons. ATRA prevented the increases in P-JNK and P-p38 after OGD/Rep, as well as the decrease in NeuN and its shrinkage, all of which were inhibited by antagonists for RAR or RXR. These findings suggest that the ATRA signaling via retinoid receptors results in the inhibition of JNK and p38 activation, leading to the protection of neurons against OGD/Rep-induced damage in the rat hippocampus.     

“The developmental and functional logic of neuronal circuits”: commentary on the Kavli Prize in Neuroscience

The first Kavli Prize in Neuroscience recognizes a confluence of career achievements that together provide a fundamental understanding of how brain and spinal cord circuits are assembled during development and function in the adult. The members of the Kavli Neuroscience Prize Committee have decided to reward three scientists (Sten Grillner, Thomas Jessell, and Pasko Rakic) jointly “for discoveries on the developmental and functional logic of neuronal circuits”.