Generation of induced pluripotent stem cells (CSSi017-A)(12862) from an ALS patient carrying a repeat expansion in the C9orf72 gene.

Genetic expansions of the hexanucleotide repeats (GGGGCC) in the C9orf72 gene appear in approximately 40% of patients with familial ALS and 7% of patients with sporadic ALS in the European population, making this mutation one of the most prevalent genetic mutations in ALS. Here, we generated a human induced pluripotent stem cell (hiPSC) line from the dermal fibroblasts of a patient carrying a 56-repeat expansion in an ALS disease-causing allele of C9orf72. These iPSCs showed stable amplification in vitro with normal karyotype and high expression of pluripotent markers and differentiated spontaneously in vivo into three germ layers.

GFAP serves as a structural element of tunneling nanotubes between glioblastoma cells and could play a role in the intercellular transfer of mitochondria.

Tunneling nanotubes (TNTs) are long F-actin-positive plasma membrane bridges connecting distant cells, allowing the intercellular transfer of cellular cargoes, and are found to be involved in glioblastoma (GBM) intercellular crosstalk. Glial fibrillary acid protein (GFAP) is a key intermediate filament protein of glial cells involved in cytoskeleton remodeling and linked to GBM progression. Whether GFAP plays a role in TNT structure and function in GBM is unknown. Here, analyzing F-actin and GFAP localization by laser-scan confocal microscopy followed by 3D reconstruction (3D-LSCM) and mitochondria dynamic by live-cell time-lapse fluorescence microscopy, we show the presence of GFAP in TNTs containing functional mitochondria connecting distant human GBM cells. Taking advantage of super-resolution 3D-LSCM, we show the presence of GFAP-positive TNT-like structures in resected human GBM as well. Using H2O2 or the pro-apoptotic toxin staurosporine (STS), we show that GFAP-positive TNTs strongly increase during oxidative stress and apoptosis in the GBM cell line. Culturing GBM cells with STS-treated GBM cells, we show that STS triggers the formation of GFAP-positive TNTs between them. Finally, we provide evidence that mitochondria co-localize with GFAP at the tip of close-ended GFAP-positive TNTs and inside receiving STS-GBM cells. Summarizing, here we found that GFAP is a structural component of TNTs generated by GBM cells, that GFAP-positive TNTs are upregulated in response to oxidative stress and pro-apoptotic stress, and that GFAP interacts with mitochondria during the intercellular transfer. These findings contribute to elucidate the molecular structure of TNTs generated by GBM cells, highlighting the structural role of GFAP in TNTs and suggesting a functional role of this intermediate filament component in the intercellular mitochondria transfer between GBM cells in response to pro-apoptotic stimuli.

Glioma stem cells: turpis omen in nomen? (The evil in the name?).

High-grade gliomas remain incurable and lethal. Through the availability of the stem-like cells responsible for glioblastoma (GB) formation, expansion, resilience and recurrence, the discovery of glioma cancer stem cells (GCSCs) is revolutionizing this field. GCSCs provide an unprecedented opportunity to reproduce and study GB pathophysiology more accurately. This critically emphasizes our ability to unambiguously identify, isolate and investigate cells that do qualify as GCSCs, to use them as a potential model that is truly predictive of GBs and of their regulation and response to therapeutic agents. We review this concept against the background of key findings on somatic, neural and solid tumour stem cells (SCs), also taking into account the emerging phenomenon of phenotypic SC plasticity. We suggest that basic approaches in these areas can be imported into the GCSC field, so that the same functional method used to identify normal somatic SCs becomes the most appropriate to define GCSCs. This, combined with knowledge of the cellular and molecular basis of normal adult neurogenesis, promises to improve the identification of GCSCs and of selective markers, as well as the development of innovative, more specific and efficacious antiglioma strategies.

Murine neural stem cells model Hunter disease in vitro: glial cell-mediated neurodegeneration as a possible mechanism involved.

Mucopolysaccharidosis type II (MPSII or Hunter Syndrome) is a lysosomal storage disorder caused by the deficit of iduronate 2-sulfatase (IDS) activity and characterized by progressive systemic and neurological impairment. As the early mechanisms leading to neuronal degeneration remain elusive, we chose to examine the properties of neural stem cells (NSCs) isolated from an animal model of the disease in order to evaluate whether their neurogenic potential could be used to recapitulate the early phases of neurogenesis in the brain of Hunter disease patients. Experiments here reported show that NSCs derived from the subventricular zone (SVZ) of early symptomatic IDS-knockout (IDS-ko) mouse retained self-renewal capacity in vitro, but differentiated earlier than wild-type (wt) cells, displaying an evident lysosomal aggregation in oligodendroglial and astroglial cells. Consistently, the SVZ of IDS-ko mice appeared similar to the wt SVZ, whereas the cortex and striatum presented a disorganized neuronal pattern together with a significant increase of glial apoptotic cells, suggesting that glial degeneration likely precedes neuronal demise. Interestingly, a very similar pattern was observed in the brain cortex of a Hunter patient. These observations both in vitro, in our model, and in vivo suggest that IDS deficit seems to affect the late phases of neurogenesis and/or the survival of mature cells rather than NSC self-renewal. In particular, platelet-derived growth factor receptor-α-positive (PDGFR-α+) glial progenitors appeared reduced in both the IDS-ko NSCs and in the IDS-ko mouse and human Hunter brains, compared with the respective healthy controls. Treatment of mutant NSCs with IDS or PDGF throughout differentiation was able to increase the number of PDGFR-α+ cells and to reduce that of apoptotic cells to levels comparable to wt. This evidence supports IDS-ko NSCs as a reliable in vitro model of the disease, and suggests the rescue of PDGFR-α+ glial cells as a therapeutic strategy to prevent neuronal degeneration.

Distinct pools of cancer stem-like cells coexist within human glioblastomas and display different tumorigenicity and independent genomic evolution.

Glioblastomas (GBMs) contain transformed, self-maintaining, multipotent, tumour-initiating cancer stem cells, whose identification has radically changed our perspective on the physiology of these tumours. Currently, it is unknown whether multiple types of transformed precursors, which display alternative sets of the complement of properties of true cancer stem cells, can be found in a GBM. If different subsets of such cancer stem-like cells (CSCs) do exist, they might represent distinct cell targets, with a differential therapeutic importance, also depending on their characteristics and lineage relationship. Here, we report the presence of two types of CSCs within different regions of the same human GBM. Cytogenetic and molecular analysis shows that the two types of CSCs bear quite diverse tumorigenic potential and distinct genetic anomalies, and, yet, derive from common ancestor cells. This provides critical information to unravel the development of CSCs and the key molecular/genetic components underpinning tumorigenicity in human GBMs.

Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.

Transformed, oncogenic precursors, possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours, have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, trigger a significant reduction in the stem-like, tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation, and increased expression of markers of neural differentiation, with no effect on cell viability. The concomitant reduction in clonogenic ability, in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating, stem-like cells from GBMs and the results also identify BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.

Bone morphogenetic proteins regulate tumorigenicity in human glioblastoma stem cells.

Human glioblastomas appear to be established and expanded by cancer stem cells, which are endowed with tumour-initiating and perpetuating ability. We report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, activate their cognate receptors (BMPRs) and trigger the Smad but not the MAP38 kinase signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation and increased expression of differentiated neural markers, without affecting cell viability. The concomitant reduction in the clonogenic ability, both in the size of the CD133+ side population and in the growth kinetics of GBM cells, indicates that BMP4 triggers a reduction in the in vitro cancer stem cell (CSC) pool. Accordingly, transient ex vivo exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most important, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality which occur in 100% of control mice in less than 12 weeks, following intracerebral grafting of human GBM cells. These findings show that the BMP-BMPR signalling system, which controls the activity of normal brain stem cells, may also act as a key inhibitory regulator of cancer-initiating, GBM stem-like cells and identifies BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.

The neural stem cells and their transdifferentiation capacity.

Stem cells play a critical role during embryo and tissue formation throughout development. Thanks to their multipotentiality - i.e., the ability to give rise to different lineages of mature cells - and to their extensive capacity for self-renewal and expansive growth, stem cells can also contribute to the maintenance of tissue integrity in adulthood. Historically, it has been held that fetal and adult (somatic) stem cells are tissue-specific 'entities' whose differentiation potential is limited to the generation of mature cell types of the tissue/organ in which they reside. Yet, recent years have seen the publication of an impressive sequence of reports dealing with what is now emerging as one of the most striking functional attributes of somatic stem cells, that is, their capacity to undergo transdifferentiation. Thanks to this peculiar characteristic adult stem cells display an unexpected ability to give rise to differentiated cells of tissues and organs different from those in which they reside. This commentary briefly illustrates the characteristics of the neural stem cell and its capacity as a neuroectodermal derivative to undergo transdifferentiation, thus giving rise to differentiated cells that normally originate from the mesoderm, like blood or skeletal muscle cells.

Skeletal myogenic potential of human and mouse neural stem cells.

Distinct cell lineages established early in development are usually maintained throughout adulthood. Thus, adult stem cells have been thought to generate differentiated cells specific to the tissue in which they reside. This view has been challenged; for example, neural stem cells can generate cells that normally originate from a different germ layer. Here we show that acutely isolated and clonally derived neural stem cells from mice and humans could produce skeletal myotubes in vitro and in vivo, the latter following transplantation into adult animals. Myogenic conversion in vitro required direct exposure to myoblasts, and was blocked if neural cells were clustered. Thus, a community effect between neural cells may override such myogenic induction. We conclude that neural stem cells, which generate neurons, glia and blood cells, can also produce skeletal muscle cells, and can undergo various patterns of differentiation depending on exposure to appropriate epigenetic signals in mature tissues.

Excitable properties in astrocytes derived from human embryonic CNS stem cells.

Although it is widely believed that astrocytes lack excitability in adult tissue, primitive action potential-like responses have been elicited from holding potentials negative to -80 mV, in cultured and injury-induced gliotic rodent astrocytes and in human glia under pathological conditions such as glioblastomas and temporal lobe epilepsy. The present study was designed to investigate the properties of astrocytes (identified by immunoreactivity for glial fibrillary acidic protein) derived from multipotent human embryonic CNS stem cells and cultured for 12-25 days in differentiating conditions. We describe here for the first time that brief (1 ms) current pulses elicit spikes from a resting potential (VREST) of approximately -37 mV and, more interestingly, that spontaneous firing can be occasionally recorded in human astrocytes. A voltage-clamp study revealed that in these cells: (i) the half-inactivation of the tetrodotoxin (TTX)-sensitive Na+ channels is around VREST; (ii) the delayed rectifier K+ current is very small; (iii) the ever-present transient outward A-type K+ channels are paradoxically capable of inhibiting the action potentials elicited from a negative membrane potential (-55 to -60 mV); and (iv) inwardly rectifying currents are not present. The responses predicted from a simulation model are in agreement with the experiments. As suggested by recent studies, the decrease of Na+ channel expression and the changes of the electrophysiological properties during the postnatal maturation of the CNS seem to exclude the possibility that astrocytes may play an excitable role in adult tissue. Our data show that excitability and firing should be considered an intrinsic attribute of human astrocytes during CNS development. This is likely to have physiological importance because the role of astrocytes during development is different from the [K+]o-buffering role played in adult CNS, namely the glutamate release and/or the guiding of migrating neurons.

Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells.

Sox2 is one of the earliest known transcription factors expressed in the developing neural tube. Although it is expressed throughout the early neuroepithelium, we show that its later expression must depend on the activity of more than one regionally restricted enhancer element. Thus, by using transgenic assays and by homologous recombination-mediated deletion, we identify a region upstream of Sox2 (-5.7 to -3.3 kb) which can not only drive expression of a (beta)-geo transgene to the developing dorsal telencephalon, but which is required to do so in the context of the endogenous gene. The critical enhancer can be further delimited to an 800 bp fragment of DNA surrounding a nuclease hypersensitive site within this region, as this is sufficient to confer telencephalic expression to a 3.3 kb fragment including the Sox2 promoter, which is otherwise inactive in the CNS. Expression of the 5.7 kb Sox2(beta)-geo transgene localizes to the neural plate and later to the telencephalic ventricular zone. We show, by in vitro clonogenic assays, that transgene-expressing (and thus G418-resistant) ventricular zone cells include cells displaying functional properties of stem cells, i.e. self-renewal and multipotentiality. We further show that the majority of telencephalic stem cells express the transgene, and this expression is largely maintained over two months in culture (more than 40 cell divisions) in the absence of G418 selective pressure. In contrast, stem cells grown in parallel from the spinal cord never express the transgene, and die in G418. Expression of endogenous telencephalic genes was similarly observed in long-term cultures derived from the dorsal telencephalon, but not in spinal cord-derived cultures. Thus, neural stem cells of the midgestation embryo are endowed with region-specific gene expression (at least with respect to some networks of transcription factors, such as that driving telencephalic expression of the Sox2 transgene), which can be inherited through multiple divisions outside the embryonic environment.

Regulation of neuronal differentiation in human CNS stem cell progeny by leukemia inhibitory factor.

The generation of diverse types of neural cells during development occurs through the progressive restriction of the fate potential of neuroepithelial progenitor cells. This process is controlled by factors intrinsic and extrinsic to the cell. While the effect of extrinsic cues on multipotent stem cells of the murine central nervous system (CNS) is becoming clearer, little is known of neural stem cells of human origin. We sought to establish the roles played by two cytokines, leukemia inhibitory (LIF) and ciliary neurotrophic factor (CNTF), and by nerve growth factor (NGF) and platelet-derived growth factor (PDGF) in regulating neuronal and astroglial differentiation in cultured embryonic diencephalic human stem cells. While NGF did not influence either neuronal or glial formation, PDGF surprisingly decreased the percentage of stem cell-generated neurons, an effect opposite to that observed in murine progenitors. Furthermore, while we confirmed the known ability of LIF and CNTF to support astroglial differentiation, we also observed that, in contrast with their murine counterparts, the fraction of CNS stem cell-generated neurons in human cultures was enhanced twofold in the presence of both cytokines. These findings highlight important differences between humans and rodents in regard to the way epigenetic cues regulate the function of neural stem cells.

Isolation and intracerebral grafting of nontransformed multipotential embryonic human CNS stem cells.

In this work, we show that the embryonic human brain contains multipotent central nervous system (CNS) stem cells, which may provide a continuous, standardized source of human neurons that could virtually eliminate the use of primary human fetal brain tissue for intracerebral transplantation. Multipotential stem cells can be isolated from the developing human CNS in a reproducible fashion and can be exponentially expanded for longer than 2 years. This allows for the establishment of continuous, nontransformed neural cell lines, which can be frozen and banked. By clonal analysis, reverse transcription polymerase chain reaction, and electrophysiological assay, we found that over such long-term culturing these cells retain both multipotentiality and an unchanged capacity for the generation of neuronal cells, and that they can be induced to differentiate into catechlaminergic neurons. Finally, when transplanted into the brain of adult rodents immunosuppressed by cyclosporin A, human CNS stem cells migrate away from the site of injection and differentiate into neurons and astrocytes. No tumor formation was ever observed. Aside from depending on scarce human neural fetal tissue, the use of human embryonic CNS stem cells for clinical neural transplantation should provide a reliable solution to some of the major problems that pertain to this field, and should allow determination of the safety characteristics of the donor cells in terms of tumorigenicity, viability, sterility, and antigenic compatibility far in advance of the scheduled day of surgery.

Extended serial passaging of mammalian neural stem cells in suspension bioreactors.

Neural stem cells (NSCs) are primitive cells that are the "parent" cells of all the cells in the central nervous system (CNS). Their discovery in 1992 opened the door to a multitude of potential therapies and treatments to cure neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and Huntington's disease, which affect millions of people worldwide and cost billions of dollars in health care each year. This study proposes optimal serial passaging protocols so that mammalian neural stem cells can effectively be grown in suspension culture. We examined stationary culture passaging protocols and developed our own optimal procedure. Also examined was the effect of serially cultivating the neural stem cells in suspension culture for an extended period of time. The cells were grown for over 35 days in suspension with an overall multiplication ratio of over 10(7) with no decrease in growth rate, maximum cell density, or viability. The cells also remained karyotypically normal through 25 doublings and retained their ability to be differentiated into all the major cell types of the CNS-neurons, astrocytes, and oligodendrocytes. For the first time, mammalian neural stem cells were grown on a larger scale in suspension culture and maintained their stem cell characteristics. A semicontinuous scheme for large-scale production is also presented.

Lineage restriction of neuroepithelial precursor cells from fetal human spinal cord.

In the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF2), neuroepithelial precursor cells from dissociated fetal human spinal cord are mitotically active and form free-floating spheres of undifferentiated cells. Proliferating cells were obtained in approximately 40% of preparations with each mitogen, were immunoreactive for the intermediate filament nestin, and did not express neuronal- or glial-specific markers. Early passage neuroepithelial precursor cells were pluripotent and differentiated into neurons expressing MAP2a,b, NF-M, and TuJ1, and GFAP-positive astrocytes; however, oligodendrocytes were never seen. As the cells were passaged from P0 to P4, the percentage of differentiating neurons significantly decreased and the prevalence of astrocytes significantly increased. While the majority of cell populations from individual preparations stopped proliferating between 3 and 6 passages, two expanding cell lines have been successfully expanded in EGF and FGF2 for over 25 passages and have been maintained in culture for over one year. These cells express nestin and not other cell-specific lineage markers. When differentiated, these neuroepithelial cell lines differentiate only into astrocytes, showing no expression of any neuronal marker. These data suggest that continued passage under these conditions preferentially selects for spinal cord neural precursors that are restricted to the astrocytic lineage. Despite the lineage restriction of later passage cell populations, these results provide a rationale for future investigation into the lineage potential of these cells in vivo following transplantation into the adult CNS, potentially as a therapeutic approach for traumatic injury and neurodegenerative disease.

Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo.

The study of the basic physiology of the neural precursors generated during brain development is driven by two inextricably linked goals. First, such knowledge is instrumental to our understanding of how the high degree of cellular complexity of the mature central nervous system (CNS) is generated, and how to dissect the steps of proliferation, fate commitment, and differentiation that lead early pluripotent neural progenitors to give rise to mature CNS cells. Second, it is hoped that the isolation, propagation, and manipulation of brain precursors and, particularly, of multipotent neural stem cells (NSCs), will lead to therapeutic applications in neurological disorders. The debate is still open concerning the most appropriate definition of a stem cell and on how it is best identified, characterized, and manipulated. By adopting an operational definition of NSCs, we review some of the basic findings in this area and elaborate on their potential therapeutic applications. Further, we discuss recent evidence from our two groups that describe, based on that rigorous definition, the isolation and propagation of clones of NSCs from the human fetal brain and illustrate how they have begun to show promise for neural cell replacement and molecular support therapy in models of degenerative CNS diseases. The extensive propagation and engraftment potential of human CNS stem cells may, in the not-too-distant-future, be directed towards genuine clinical therapeutic ends, and may open novel and multifaceted strategies for redressing a variety of heretofore untreatable CNS dysfunctions.

Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation.

Stem cells that can give rise to neurons, astroglia, and oligodendroglia have been found in the developing and adult central nervous system (CNS) of rodents. Yet, their existence within the human brain has not been documented, and the isolation and characterization of multipotent embryonic human neural stem cells have proven difficult to accomplish. We show that the developing human CNS embodies multipotent precursors that differ from their murine counterpart in that they require simultaneous, synergistic stimulation by both epidermal and fibroblast growth factor-2 to exhibit critical stem cell characteristics. Clonal analysis demonstrates that human C NS stem cells are multipotent and differentiate spontaneously into neurons, astrocytes, and oligodendrocytes when growth factors are removed. Subcloning and population analysis show their extensive self-renewal capacity and functional stability, their ability to maintain a steady growth profile, their multipotency, and a constant potential for neuronal differentiation for more than 2 years. The neurons generated by human stem cells over this period of time are electrophysiologically active. These cells are also cryopreservable. Finally, we demonstrate that the neuronal and glial progeny of long-term cultured human CNS stem cells can effectively survive transplantation into the lesioned striatum of adult rats. Tumor formation is not observed, even in immunodeficient hosts. Hence, as a consequence of their inherent biology, human CNS stem cells can establish stable, transplantable cell lines by epigenetic stimulation. These lines represent a renewable source of neurons and glia and may significantly facilitate research on human neurogenesis and the development of clinical neural transplantation.

Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain.

The subventricular zone (SVZ) of the adult mammalian forebrain contains kinetically distinct precursor populations that contribute new neurons to the olfactory bulb. Because among forebrain precursors there are stem-like cells that can be cultured in the presence of mitogens such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), we asked whether distinct subsets of stem-like cells coexist within the SVZ or whether the proliferation of a single type of SVZ stem-like cell is controlled by several GFs. We show that the latter is the case. Thus cells isolated from the SVZ coexpress the EGF and FGF receptors; by quantitative analysis, the number of stem-like cells isolated from the SVZ by either FGF2 or EGF is the same, whereas no additive effect occurs when these factors are used together. Furthermore, short-term administration of high-dose [3H]thymidine in vivo depletes both the EGF- and FGF2-responsive stem-like cell populations equally, showing they possess closely similar proliferation kinetics and likely belong to the constitutively proliferating SVZ compartment. By subcloning and population analysis, we demonstrate that responsiveness to more than one GF endows SVZ cells with an essential stem cell feature, the ability to vary self-renewal, that was until now undocumented in CNS stem-like cells. The multipotent stem cell-like population that expands slowly in the presence of FGF2 in culture switches to a faster growth mode when exposed to EGF alone and expands even faster when exposed to both GFs together. Analogous responses are observed when the GFs are used in the reverse order, and furthermore, these growth rate modifications are fully reversible.

Expression and modulation of matrix metalloproteinase-2 and tissue inhibitors of metalloproteinases in human embryonic CNS stem cells.

The expression and regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in neuroectodermal precursor cells is undocumented. We report the presence of MMP-2, but no MMP-9, and of all the four known TIMPs in neuroepithelial stem cells isolated from the human CNS. The expression of TIMP-1, TIMP-2 and TIMP-3 was unchanged following stem cells differentiation into neurons and glia. In contrast, while MMP-2 and TIMP-4 were localized to both stem and mature CNS cells, their levels of expression were substantially reduced in the latter. TIMP-4 showed a 23-fold reduction in media conditioned by differentiated cells compared with stem cell-conditioned media, reflecting a 6-fold decrease in mRNA expression. Interestingly, TIMP-4 also differed from the other TIMPs in that it was cell-associated in the stem cells, where this fraction remained unchanged upon differentiation. Hence, regulation of selective MMPs and TIMPs occurs during differentiation of human neural precursors suggesting that MMP-2 and TIMP-4 in particular may perform regulatory roles in the developing CNS.