Continuous live imaging of adult neural stem cell division and lineage progression in vitro

Little is known about the intrinsic specification of adult neural stem cells (NSCs) and to what extent they depend on their local niche. To observe adult NSC division and lineage progression independent of their niche, we isolated cells from the adult mouse subependymal zone (SEZ) and cultured them at low density without growth factors. We demonstrate here that SEZ cells in this culture system are primarily neurogenic and that adult NSCs progress through stereotypic lineage trees consisting of asymmetric stem cell divisions, symmetric transit-amplifying divisions and final symmetric neurogenic divisions. Stem cells, identified by their astro/radial glial identity and their slow-dividing nature, were observed to generate asymmetrically and fast-dividing cells that maintained an astro/radial glia identity. These, in turn, gave rise to symmetrically and fast-dividing cells that lost glial hallmarks, but had not yet acquired neuronal features. The number of amplifying divisions was limited to a maximum of five in this system. Moreover, we found that cell growth correlated with the number of subsequent divisions of SEZ cells, with slow-dividing astro/radial glia exhibiting the most substantial growth prior to division. The fact that in the absence both of exogenously supplied growth factors and of signals provided by the local niche neurogenic lineage progression takes place in such stereotypic fashion, suggests that lineage progression is, to a significant degree, cell intrinsic or pre-programmed at the beginning of the lineage.     

An Epigenetic Feedback Regulatory Loop Involving MicroRNA-195 and MBD1 Governs Neural Stem Cell Differentiation

Background

Epigenetic mechanisms, including DNA methylation, histone modification, and microRNAs, play pivotal roles in stem cell biology. Methyl-CpG binding protein 1 (MBD1), an important epigenetic regulator of adult neurogenesis, controls the proliferation and differentiation of adult neural stem/progenitor cells (aNSCs). We recently demonstrated that MBD1 deficiency in aNSCs leads to altered expression of several noncoding microRNAs (miRNAs).

Methodology/Principal Findings

Here we show that one of these miRNAs, miR-195, and MBD1 form a negative feedback loop. While MBD1 directly represses the expression of miR-195 in aNSCs, high levels of miR-195 in turn repress the expression of MBD1. Both gain-of-function and loss-of-function investigations show that alterations of the MBD1–miR-195 feedback loop tip the balance between aNSC proliferation and differentiation.

Conclusions/Significance

Therefore the regulatory loop formed by MBD1 and miR-195 is an important component of the epigenetic network that controls aNSC fate.     

Substrate modulus directs neural stem cell behavior

Although biochemical signals that modulate stem cell self-renewal and differentiation were extensively studied, only recently were the mechanical properties of a stem cell's microenvironment shown to regulate its behavior. It would be desirable to have independent control over biochemical and mechanical cues, to analyze their relative and combined effects on stem-cell function. We developed a synthetic, interfacial hydrogel culture system, termed variable moduli interpenetrating polymer networks (vmIPNs), to assess the effects of soluble signals, adhesion ligand presentation, and material moduli from 10-10,000 Pa on adult neural stem-cell (aNSC) behavior. The aNSCs proliferated when cultured in serum-free growth media on peptide-modified vmIPNs with moduli of ≥100 Pa. In serum-free neuronal differentiation media, a peak level of the neuronal marker, β-tubulin III, was observed on vmIPNs of 500 Pa, near the physiological stiffness of brain tissue. Furthermore, under mixed differentiation conditions with serum, softer gels (∼100-500 Pa) greatly favored neurons, whereas harder gels (∼1,000-10,000 Pa) promoted glial cultures. In contrast, cell spreading, self-renewal, and differentiation were inhibited on substrata with moduli of ∼10 Pa. This work demonstrates that the mechanical and biochemical properties of an aNSC microenvironment can be tuned to regulate the self-renewal and differentiation of aNSCs.     

Prospective isolation of adult neural stem cells from the mouse subependymal zone

Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein-enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP(+)/prominin1(+)), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.     

Topographical analysis of the subependymal zone neurogenic niche

The emerging model for the adult subependymal zone (SEZ) cell population indicates that neuronal diversity is not generated from a uniform pool of stem cells but rather from diverse and spatially confined stem cell populations. Hence, when analysing SEZ proliferation, the topography along the anterior-posterior and dorsal-ventral axes must be taken into account. However, to date, no studies have assessed SEZ proliferation according to topographical specificities and, additionally, SEZ studies in animal models of neurological/psychiatric disorders often fail to clearly specify the SEZ coordinates. This may render difficult the comparison between studies and yield contradictory results. More so, by focusing in a single spatial dimension of the SEZ, relevant findings might pass unnoticed. In this study we characterized the neural stem cell/progenitor population and its proliferation rates throughout the rat SEZ anterior-posterior and dorsal-ventral axes. We found that SEZ proliferation decreases along the anterior-posterior axis and that proliferative rates vary considerably according to the position in the dorsal-ventral axis. These were associated with relevant gradients in the neuroblasts and in the neural stem cell populations throughout the dorsal-ventral axis. In addition, we observed spatially dependent differences in BrdU/Ki67 ratios that suggest a high variability in the proliferation rate and cell cycle length throughout the SEZ; in accordance, estimation of the cell cycle length of the neuroblasts revealed shorter cell cycles at the dorsolateral SEZ. These findings highlight the need to establish standardized procedures of SEZ analysis. Herein we propose an anatomical division of the SEZ that should be considered in future studies addressing proliferation in this neural stem cell niche.     

Prospero distinguishes sibling cell fate without asymmetric localization in the Drosophila adult external sense organ lineage

The adult external sense organ precursor (SOP) lineage is a model system for studying asymmetric cell division. Adult SOPs divide asymmetrically to produce IIa and IIb daughter cells; IIa generates the external socket (tormogen) and hair (trichogen) cells, while IIb generates the internal neuron and sheath (thecogen) cells. Here we investigate the expression and function of prospero in the adult SOP lineage. Although Prospero is asymmetrically localized in embryonic SOP lineage, this is not observed in the adult SOP lineage: Prospero is first detected in the IIb nucleus and, during IIb division, it is cytoplasmic and inherited by both neuron and sheath cells. Subsequently, Prospero is downregulated in the neuron but maintained in the sheath cell. Loss of prospero function leads to 'double bristle' sense organs (reflecting a IIb-to-IIa transformation) or 'single bristle' sense organs with abnormal neuronal differentiation (reflecting defective IIb development). Conversely, ectopic prospero expression results in duplicate neurons and sheath cells and a complete absence of hair/socket cells (reflecting a IIa-to-IIb transformation). We conclude that (1) despite the absence of asymmetric protein localization, prospero expression is restricted to the IIb cell but not its IIa sibling, (2) prospero promotes IIb cell fate and inhibits IIa cell fate, and (3) prospero is required for proper axon and dendrite morphology of the neuron derived from the IIb cell. Thus, prospero plays a fundamental role in establishing binary IIa/IIb sibling cell fates without being asymmetrically localized during SOP division. Finally, in contrast to previous studies, we find that the IIb cell divides prior to the IIa cell in the SOP lineage.     

Adult human neural stem cell therapeutics: Current developmental status and prospect

Over the past two decades, regenerative therapies using stem cell technologies have been developed for various neurological diseases. Although stem cell therapy is an attractive option to reverse neural tissue damage and to recover neurological deficits, it is still under development so as not to show significant treatment effects in clinical settings. In this review, we discuss the scientific and clinical basics of adult neural stem cells (aNSCs), and their current developmental status as cell therapeutics for neurological disease. Compared with other types of stem cells, aNSCs have clinical advantages, such as limited proliferation, inborn differentiation potential into functional neural cells, and no ethical issues. In spite of the merits of aNSCs, difficulties in the isolation from the normal brain, and in the in vitro expansion, have blocked preclinical and clinical study using aNSCs. However, several groups have recently developed novel techniques to isolate and expand aNSCs from normal adult brains, and showed successful applications of aNSCs to neurological diseases. With new technologies for aNSCs and their clinical strengths, previous hurdles in stem cell therapies for neurological diseases could be overcome, to realize clinically efficacious regenerative stem cell therapeutics.     

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

Stromal cell-dependent growth of B-1 B cell progenitors in the absence of direct contact

This protocol describes a transwell culture system in which stromal cells support the growth and differentiation of B cell progenitors in the absence of direct contact. In this system, a confluent layer of S17 stromal cells pre-established in 0.4 microm transwells is placed over wells seeded with purified B cell progenitors. The stromal cell-derived factors and additional cytokines added to the culture medium support the differentiation of the progenitors in the lower chamber. B-1 B cell progenitors seeded in the presence of thymic stromal lymphopoietin undergo significant expansion and differentiation in this culture system. Since the expanded B-1 B lineage cells are not contaminated with stromal cells, no additional purification steps are required before subsequent phenotypic, functional or genetic analyses of these lymphoid cells are performed. Once the transwell cultures and B cell progenitors are available, cultures can be initiated in less than an hour. The overall procedure, however, takes approximately 10 h when the initiation of the S17 transwell cultures and the isolation of the B cell progenitors steps are included.     

A Specialized Microvascular Domain in the Mouse Neural Stem Cell Niche

The microenvironment of the subependymal zone (SEZ) neural stem cell niche is necessary for regulating adult neurogenesis. In particular, signaling from the microvasculature is essential for adult neural stem cell maintenance, but microvascular structure and blood flow dynamics in the SEZ are not well understood. In this work, we show that the mouse SEZ constitutes a specialized microvascular domain defined by unique vessel architecture and reduced rates of blood flow. Additionally, we demonstrate that hypoxic conditions are detectable in the ependymal layer that lines the ventricle, and in a subpopulation of neurons throughout the SEZ and striatum. Together, these data highlight previously unidentified features of the SEZ neural stem cell niche, and further demonstrate the extent of microvascular specialization in the SEZ microenvironment.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

In vitro characterization of fetal hematopoietic stem cells: range and kinetics of cell production from individual stem cells

We have developed methods for detailed characterization of the proliferation kinetics and lineage potential of single human hematopoietic progenitor cells in an in vitro culture system. Fetal bone marrow CD34(hi)/lin(-) cells were cultured at one cell per well in the presence of c-kit ligand (KL), interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) on a murine stroma cell monolayer. Individual wells were scored for growth between 1 and 10 weeks of culture and analyzed by flow cytometry for lineage composition. A wide variation in time (1 to 8 weeks) was observed before initial cell division, even in the presence of cytokines promoting cell division in primitive progenitors. Eleven percent of the plated cells eventually produced a confluent culture well of approximately 20,000 progeny. Confluent wells were harvested and individually analyzed by flow cytometry for cell surface phenotype. Forty-eight percent of confluent wells contained primitive progenitors (CD34(+)lin(-)), 16% contained B-lymphoid cells (CD19(+) or CD10(+)), and 100% contained cells committed to the myelo-erythroid lineage (CD33(+)). CD34(+)/lin(-) cells from confluent wells were replated at one cell per well in secondary culture and the analysis repeated. One of 216 original single cells plated produced populations of B-lymphoid cells, myeloid cells, and primitive progenitors (CD34(+)/lin(-)) which persisted through two expansion cycles. We estimate that more than 36 million cells can be produced from a single cell under these culture conditions. A very small percentage of the CD34(hi)/lin(-) population (about 1%) was responsible for the majority of subsequent cell production. Our estimate of stem cell content in fetal bone marrow, defined by self-renewal as well as both B-lymphoid and myeloid differentiation from one cell, is approximately 1/13,000. This assay system provides direct in vitro measurements of the expected characteristics of hematopoietic stem cells (high proliferation potential, multilineage potential, self-renewal, and quiescence), and is therefore well suited to assessment of stem cell activity within various cell populations. (c) 1996 John Wiley & Sons, Inc.     

Ablation of Fmrp in adult neural stem cells disrupts hippocampus-dependent learning

Deficiency in fragile X mental retardation protein (FMRP) results in fragile X syndrome (FXS), an inherited form of intellectual disability. Despite extensive research, it is unclear how FMRP deficiency contributes to the cognitive deficits in FXS. Fmrp-null mice show reduced adult hippocampal neurogenesis. As Fmrp is also enriched in mature neurons, we investigated the function of Fmrp expression in neural stem and progenitor cells (aNSCs) and its role in adult neurogenesis. Here we show that ablation of Fmrp in aNSCs by inducible gene recombination leads to reduced hippocampal neurogenesis in vitro and in vivo, as well as markedly impairing hippocampus-dependent learning in mice. Conversely, restoration of Fmrp expression specifically in aNSCs rescues these learning deficits in Fmrp-deficient mice. These data suggest that defective adult neurogenesis may contribute to the learning impairment seen in FXS, and these learning deficits can be rectified by delayed restoration of Fmrp specifically in aNSCs.     

Development of adult sensilla on the wing and notum of Drosophila melanogaster

We have investigated the temporal pattern of appearance, cell lineage, and cytodifferentiation of selected sensory organs (sensilla) of adult Drosophila. This analysis was facilitated by the discovery that the monoclonal antibody 22C10 labels not only the neuron of the developing sensillum organ, but the accessory cells as well. The precursors of the macrochaetes and the recurved (chemosensory) bristles of the wing margin divide around and shortly after puparium formation, while those of the microchaetes and the stout and slender (mechanosensory) bristles of the wing margin divide between 9 h and 18 h after puparium formation (apf). The onset of sensillum differentiation follows the terminal precursor division within a few hours. Four of the cells in an individual microchaete organ are clonally related: A single first-order precursor cell divides to produce two second-order precursors; one of these divides into the neuron and thecogen cell, the other into the trichogen cell and tormogen cell. Along the anterior wing margin, two rounds of division generate the cells of the mechanosensory sensilla; here, no strict clonal relationship seems to exist between the cells of an individual sensillum. At the time of sensillum precursor division, many other, non-sensillum-producing cells within the notum and wing proliferate as well. This mitotic activity follows a spatially non-random pattern.     

A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging

This protocol describes a method for the dissection of egg chambers from intact Drosophila females and culture conditions that permit live imaging of them, with a particular emphasis on stage 9. This stage of development is characterized by oocyte growth and patterning, outer follicle cell rearrangement and migration of border cells. Although in vitro culture of egg chambers of later developmental stages has long been possible, until recently stage 9 egg chambers could only be kept alive for short periods, did not develop normally, and border cell migration failed entirely. We have established culture conditions that support overall egg chamber development including border cell migration in vitro. This protocol makes possible direct observation of molecular and cellular dynamics in both wild-type and mutant egg chambers, and opens the door to testing of pharmacological inhibitors and the use of biosensors. The entire protocol takes approximately 24 h while the preparation of egg chambers for live imaging requires only 15-20 min.