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Eric D Laywell

Publications and source records attributed to Eric D Laywell.

14 recordsLinked to original sources
PubMed2006

Fusion of neural stem cells in culture.

An important issue in stem cell biology relates to mechanisms of cellular plasticity. Specifically, could any observed multipotency of, e.g., adult stem cells arise from true transdifferentiation or as a result of cell-cell fusion? We studied this issue using a culture paradigm of astrocyte monolayers and multipotent neurospheres generated from neonatal cerebellar cortex and the subventricular zone (SVZ). Based on fluorescence in situ hybridization (FISH), cells from these cultures were found to contain an abnormal number of sex chromosomes, suggesting that cellular fusion is a common in vitro occurrence. A Cre/lox recombination method was also exploited to further confirm the evidence of fusion. Next, we assessed the potential of fusogenic microglial involvement by combining CD11b immunolabeling with FISH sex chromosome analysis. Differentiating neurospheres were also studied from the PU.1 knockout mouse that lacks cells of myeloid origin, presumed to be a source of central nervous system microglia. Very few cells immunopositive for the microglial marker CD11b were found to be aneuploid, and there was no difference in fusion frequency between PU.1+/+ and PU.1-/- neurospheres. These results, together, suggest that stem and/or progenitor cells that generate neurons and glia in culture possess the ability to generate fused polyploidal cells, but microglial participation is not a requirement for fusion to occur. In addition to caution that should be exerted during the interpretation of in vitro neural cell plasticity, the data also suggest that novel therapeutic treatments could be designed that exploit cellular fusion in rescue paradigms for degenerating neuronal populations.

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PubMed2006

Microglia instruct subventricular zone neurogenesis.

Microglia are increasingly implicated as a source of non-neural regulation of postnatal neurogenesis and neuronal development. To evaluate better the contributions of microglia to neural stem cells (NSCs) of the subventricular neuraxis, we employed an adherent culture system that models the continuing proliferation and differentiation of the dissociated neuropoietic subventricular tissues. In this model, neuropoietic cells retain the ability to self-renew and form multipotent neurospheres, but progressively lose the ability to generate committed neuroblasts with continued culture. Neurogenesis in highly expanded NSCs can be rescued by coculture with microglial cells or microglia-conditioned medium, indicating that microglia provide secreted factor(s) essential for neurogenesis, but not NSC maintenance, self-renewal, or propagation. Our findings suggest an instructive role for microglial cells in contributing to postnatal neurogenesis in the largest neurogenic niche of the mammalian brain.

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PubMed2005

Neuron-to-astrocyte transition: phenotypic fluidity and the formation of hybrid asterons in differentiating neurospheres.

To the extent that their fate choice and differentiation processes can be understood and manipulated, neural stem cells represent a promising therapeutic tool for a variety of neuropathologies. We have previously shown that mature astrocytes possess neural stem cell attributes, and can give rise to neurons through the formation of multipotent neurosphere clones. Here we show that relatively mature neurons generated from neurospheres derived from postnatal subependymal zone or cerebellar cortex undergo a phenotypic transformation into astrocytes that coincides with the appearance of a nonfused, hybrid cell type that shares the morphology, antigenicity, and physiology of both neurons and astrocytes. We refer to this astrocyte/neuron hybrid as an "asteron," and hypothesize that it represents an intermediate step in the trans- or dedifferentiation of neurons into astrocytes. The present finding suggests that seemingly terminally differentiated neural cells may in fact represent points along a bidirectionally fluid continuum of differentiation, with intermediate points represented by "hybrid" cells coexpressing phenotypic markers of more than one lineage.

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PubMed2005

In vitro-derived "neural stem cells" function as neural progenitors without the capacity for self-renewal.

Hematopoietic stem cells have been defined by their ability to self-renew and successfully reconstitute hematopoiesis throughout the life of a transplant recipient. Neural stem cells (NSCs) are believed to exist in the regenerating regions of the brain in adult mice: the subependymal zone (SEZ) of the lateral ventricles (LVs) and the hippocampal dentate gyrus. Cells from the SEZ can be cultured to generate neurospheres or multipotent astrocytic stem cells (MASCs), both of which demonstrate the stem cell qualities of multipotency and self-renewal in vitro. Whether neurospheres and MASCs possess the true stem cell quality of functional self-renewal in vivo is unknown. The definitive tests for this unique capability are long-term engraftment and serial transplantation. Both neurospheres and MASCs transplanted into the LVs of C57BL/6 mice resulted in short-term engraftment into the recipient brain, with donor-derived migratory neuroblasts visible in the rostral migratory stream and olfactory bulb after transplantation. To test in vivo expansion/self-renewal of the transplanted cells, we attempted to reisolate donor-derived neurospheres and MASCs. Even when rigorous drug selection was used to select for rare events, no donor-derived neurospheres or MASCs could be reisolated. Furthermore, donor-derived migratory neuroblasts were not observed in the rostral migratory stream (RMS) for more than 1 month after transplantation, indicating a transient rather than long-term engraftment. Therefore, in vitro-derived neurospheres and MASCs do not function as NSCs with long-term, self-renewal capabilities in vivo but instead represent short-term neural progenitor cells as defined by an in vivo functional assay.

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PubMed2005

Mesenchymal stem cells spontaneously express neural proteins in culture and are neurogenic after transplantation.

Reports of neural transdifferentiation of mesenchymal stem cells (MSCs) suggest the possibility that these cells may serve as a source for stem cell-based regenerative medicine to treat neurological disorders. However, some recent studies controvert previous reports of MSC neurogenecity. In the current study, we evaluate the neural differentiation potential of mouse bone marrow-derived MSCs. Surprisingly, we found that MSCs spontaneously express certain neuronal phenotype markers in culture, in the absence of specialized induction reagents. A previously published neural induction protocol that elevates cytoplasmic cyclic AMP does not upregulate neuron-specific protein expression significantly in MSCs but does significantly increase expression of the astrocyte-specific glial fibrillary acidic protein. Finally, when grafted into the lateral ventricles of neonatal mouse brain, MSCs migrate extensively and differentiate into olfactory bulb granule cells and periventricular astrocytes, without evidence of cell fusion. These results indicate that MSCs may be "primed" toward a neural fate by the constitutive expression of neuronal antigens and that they seem to respond with an appropriate neural pattern of differentiation when exposed to the environment of the developing brain.

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PubMed2005

Ionizing radiation enhances the engraftment of transplanted in vitro-derived multipotent astrocytic stem cells.

The subependymal zone (SEZ) is a region of persistent neurogenesis in the adult mammalian brain containing a neural stem cell (NSC) pool that continuously generates migratory neuroblasts that travel in chains through the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate and functionally integrate into existing neural circuitry. NSCs can be isolated from the SEZ and cultured to generate either neurospheres (NSs) or multipotent astrocytic stem cells (MASCs), with both possessing the stem cell characteristics of multipotency and self-renewal. NSs and MASCs home to the SEZ after transplantation into the lateral ventricle (LV) and contribute to neuroblast migration, with minimal engraftment into the OB observed in the adult mouse. Recent studies have compared the relatively uncharacterized NSC with the more established hematopoietic stem cell (HSC) in an effort to determine the level of stemness possessed by the NSC. Depletion of native HSCs in the bone marrow by lethal irradiation (LI) is necessary to maximize functional engraftment of donor HSCs. Our data show that the NSC pool and neuroblasts in the SEZ can be significantly and permanently depleted by exposure to LI. Attenuation of donor-derived migratory neuroblast engraftment into the OB is observed after transplantation of gfp+ MASCs into the LV of LI animals, whereas engraftment is significantly enhanced after transplantation into animals exposed to sublethal levels of ionizing radiation. By increasing receptiveness of the NSC niche through depletion of indigenous cells, the adult SEZ-RMS-OB can be used as a model to further characterize the NSC.

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PubMed2004

GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis.

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis. GM1 loading of wild-type neurospheres recapitulated the phenotype of beta-gal-/- cells and activated this pathway by depleting ER calcium stores, which ultimately culminated in apoptosis. Activation of UPR pathways did not occur in mice double deficient for beta-gal and ganglioside synthase, beta-gal-/-/GalNAcT-/-, which do not accumulate GM1. These findings suggest that the UPR can be induced by accumulation of the sialoglycolipid GM1 and this causes a novel mechanism of neuronal apoptosis.

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PubMed2004

Bone marrow transdifferentiation in brain after transplantation: a retrospective study.

BACKGROUND: End-organ repair by adult haemopoietic stem cells is under great scrutiny with investigators challenging the notion of these cells' plasticity. Some investigations of animals and short-term human bone marrow transplants suggest that bone marrow can repair brain. We looked for evidence of clinically relevant marrow-derived restorative neurogenesis: long-term, multilineage, neural engraftment that is not the result of cell-fusion events. METHODS: We examined autopsy brain specimens from three sex-mismatched female bone-marrow-transplantation patients, a female control, and a male control. We did immunohistochemistry, fluorescence in-situ hybridisation, and tissue analysis to look for multilineage, donor-derived neurogenesis. FINDINGS: Hippocampal cells containing a Y chromosome were present up to 6 years post-transplant in all three patients. Transgender neurons accounted for 1% of all neurons; there was no evidence of fusion events since only one X chromosome was present. Moreover, transgender astrocytes and microglia made up 1-2% of all glial cells. INTERPRETATION: Postnatal human neuropoiesis happens, and human haemopoietic cells can transdifferentiate into neurons, astrocytes, and microglia in a long-term setting without fusing. Transplantable human haemopoietic cells could serve as a therapeutic source for long-term regenerative neuropoiesis.

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PubMed2003

Astrocytes as stem cells: nomenclature, phenotype, and translation.

Recently discovered multipotent astrocytic stem cells are discussed in light of current nomenclature for glial precursor and lineage-associated cells in the developing, postnatal, and adult mammalian brain. Defining the phenotype of any immature cell in the nervous system is a challenge, and a position is stated that includes the need for categorizing cells within a continuum of differentiation potential. The possibility for dedifferentiating glial cells into clonogenic stem-like cells offers numerous possibilities for translating knowledge and technology from this subfield of stem cell biology to regenerative medicine. Along with the need for developing a new lexicon for defining the cellular players that contribute to the generation of glia and neurons in the developing and mature central nervous system, the relationships also need to be established among potency, repopulation attempts, and tumorigenesis of cells meeting the criteria of glial stem cells. Finally, it is possible that understanding the normal differentiation, de- and transdifferentiation potential of glial stem-like cells in the mature central nervous system will provide insights into the possible use of these cells, or biogenic factors associated with their growth and differentiation, in therapeutic approaches for a variety of neurological disorders.

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PubMed2003

Neural trans-differentiation potential of hepatic oval cells in the neonatal mouse brain.

Although the existence of the hepatic oval cell (HOC), the liver stem cell has been known for almost 70 years, little is known about the potential for this adult stem cell to trans-differentiate into cells of other tissues. While their origin remains enigmatic, HOCs share many similarities with hematopoietic stem cells. Recent studies have revealed that a small percentage of HOCs can arise from a bone marrow-derived stem cell source. Here we report that, like bone marrow stem cells, HOCs can survive transplantation to the neonatal mouse brain and show signs of trans-differentiation by adopting the morphology and antigenic phenotype of both macro- and microglia cells. Trans-differentiated microglia cells were functional, showing active phagocytosis when cotransplanted with latex microbeads in vivo. In addition to glial markers, a small number of transplanted HOCs were immunopositive for neuronal markers, but displayed ambiguous phenotype, making their characterization difficult.

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PubMed2002

Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro.

Neural stem cells from neurogenic regions of mammalian CNS are clonogenic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cellulose and the pleiotropic growth factors EGF, FGF2, and insulin. The aim of this study was to test whether cortical glial tumors contain stem-like cells capable, under this culture system, of forming clones showing intraclonal heterogeneity in the expression of neural lineage-specific proteins. The high frequencies of clone-forming cells (about 0.1-10 x 10(-3)) in clinical tumor specimens with mutated p53, and in neurogenic regions of normal human CNS, suggest that the ability to form clones in this culture system is induced epigenetically. RT-PCR analyses of populations of normal brain- and tumor-derived sister clones revealed transcripts for nestin, neuron-specific enolase, and glial fibrillary acidic protein (GFAP). However, the tumor-derived clones were different from clones derived from neurogenic regions of normal brain in the expression of transcripts specific for genes associated with neural cell fate determination via the Notch-signaling pathway (Delta and Jagged), and cell survival at G2 or mitotic phases (Survivin). Moreover, the individual glioma-derived clones contain cells immunopositive separately for GFAP or neuronal beta-III tubulin, as well as single cells coexpressing both glial and neuronal markers. The data suggest that the latent critical stem cell characteristics can be epigenetically induced by growth conditions not only in cells from neurogenic regions of normal CNS but also in cells from cortical glial tumors. Moreover, tumor stem-like cells with genetically defective responses to epigenetic stimuli may contribute to gliomagenesis and the developmental pathological heterogeneity of glial tumors.

Astrocytes↗
PubMed2002

Transplantation of an indigenous neural stem cell population leading to hyperplasia and atypical integration.

Astrocytes exhibit neural stem cell characteristics in vitro by generating multipotent clones of cells. In order to see if normal cues are present in vivo that can direct these astrocytes to generate cells of neuronal lineage, the astrocytes were transplanted into the persistently neurogenic mouse subependymal zone/rostral migratory stream. Grafted astrocytes assumed migratory profiles, joined chains of indigenous neuroblasts, and migrated into the olfactory bulb. Additionally, however, some grafted astrocytes "homed" to the lateral ventricle where they became hyperplastic, forming spherical structures composed of cells of mixed phenotype that attached to the ventricular wall, and eventually penetrated and dispersed within surrounding brain parenchyma. It is proposed that, with an interest in the use of stem cell transplants for neurological disease, findings of hyperplasia and apparent atypical integration of a native population of multipotent astrocytic stem cells suggest the need for caution before beginning even autologous neural stem cell transplants.

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