Differentiation of insulin-producing cells from human neural progenitor cells

BackgroundSuccess in islet-transplantation-based therapies for type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Islets and neurons share features, including common developmental programs, and in some species brain neurons are the principal source of systemic insulin.Methods and FindingsHere we show that brain-derived human neural progenitor cells, exposed to a series of signals that regulate in vivo pancreatic islet development, form clusters of glucose-responsive insulin-producing cells (IPCs). During in vitro differentiation of neural progenitor cells with this novel method, genes encoding essential known in vivo regulators of pancreatic islet development were expressed. Following transplantation into immunocompromised mice, IPCs released insulin C-peptide upon glucose challenge, remained differentiated, and did not form detectable tumors.ConclusionProduction of IPCs solely through extracellular factor modulation in the absence of genetic manipulations may promote strategies to derive transplantable islet-replacement tissues from human neural progenitor cells and other types of multipotent human stem cells.     

Evidence for neural progenitor cells in the human adult enteric nervous system

Putative neural stem cells have been identified within the enteric nervous system (ENS) of adult rodents and cultured from human myenteric plexus. We conducted studies to identify neural stem cells or progenitor cells within the submucosa of adult human ENS. Jejunum tissue was removed from adult human subjects undergoing gastric bypass surgery. The tissue was immunostained, and confocal images of ganglia in the submucosal plexus were collected to identify protein gene product 9.5 (PGP 9.5) - immunoractive neurons and neuronal progenitor cells that coexpress PGP 9.5 and nestin. In addition to PGP-9.5-positive/nestin-negative neuronal cells within ganglia, we observed two other types of cells: (1) cells in which PGP 9.5 and nestin were co-localized, (2) cells negative for both PGP 9.5 and nestin. These observations suggest that the latter two types of cells are related to a progenitor cell population and are consistent with the concept that the submucosa of human adult ENS contains stem cells capable of maintenance and repair within the peripheral nervous system.     

Identification of white adipocyte progenitor cells in vivo

The increased white adipose tissue (WAT) mass associated with obesity is the result of both hyperplasia and hypertrophy of adipocytes. However, the mechanisms controlling adipocyte number are unknown in part because the identity of the physiological adipocyte progenitor cells has not been defined in vivo. In this report, we employ a variety of approaches, including a noninvasive assay for following fat mass reconstitution in vivo, to identify a subpopulation of early adipocyte progenitor cells (Lin(-):CD29(+):CD34(+):Sca-1(+):CD24(+)) resident in adult WAT. When injected into the residual fat pads of A-Zip lipodystrophic mice, these cells reconstitute a normal WAT depot and rescue the diabetic phenotype that develops in these animals. This report provides the identification of an undifferentiated adipocyte precursor subpopulation resident within the adipose tissue stroma that is capable of proliferating and differentiating into an adipose depot in vivo.     

Transplantation of stem/progenitor cells of human brain into the brain of adult rats

We studied the development of stem/progenitor cells of the human brain transplanted in the adult rat brain after reproduction in an in vitro tissue culture. It was preliminarily shown by the immunological methods that the stem cells grown in a medium with growth factors formed neurospheres, which were heterogenous and contained both stem and progenitor cells of the human brain. The cells were implanted in the hippocampus, striatum, or lateral ventricle of the rat brain as a suspension or aggregates (neurospheres) and their behavior and differentiation were studies within 10, 20, and 30 days using the morphological and immunochemical methods. The cultured cells of the human brain continued their development in the rat brain, migrated, and formed neurons and astrocytes. The white mater fibers, lateral ventricle wall, and perivascular spaces served as the main pathways of migration. The neuronal differentiation was shown by staining with antibodies to beta-tubulin III, neurofilaments-70, and calbindin. Some growing nerve cells had long processes with growth cones. At the same time, some transplanted cells retained the undifferentiated state within one month after the implantation, as shown by the vimentin expression.     

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.     

Proteomic studies on the white matter of human brain

Limited information on the protein expression profiles of the different components of mammalian brain is available to date. In the present study, proteomic analysis was performed on 32 white matter samples obtained from 8 different regions of brains of four post mortem cases. Proteins were separated by 2D gel electrophoresis and identified by mass spectrometry. Most of the protein spots (98%) are reproducibly present in all the samples analyzed. A total of 64 different proteins were identified and divided into seven functional groups. These include metabolic proteins (33%), structural proteins (9%), proteins involved in signal transduction (9%), blood proteins (8%), stress related proteins (23%), and proteins involved in the ubiquitin mediated proteolysis (6%). This protein database obtained from the white matter of human brain contributes to deepen our knowledge on the molecular mechanisms that control several pathologies affecting this key component of the brain.     

Promoter-defined isolation and identification of hepatic progenitor cells from the human fetal liver

Hepatoblasts, which are considered one type of hepatic progenitor cell, reside in the fetal liver. To selectively identify these cells, we transfected primary cultured human fetal liver cells (FLCs) with a pGL3 vector bearing the gene for the enhanced green fluorescence protein (EGFP) under the control of the alpha-fetoprotein (AFP) promoter expressed in hepatoblasts. The FLCs were then sorted by fluorescence-activated cell sorting (FACS) on the basis of AFP promoter-driven EGFP expression. The EGFP-positive cells expressed AFP, albumin, and cytokeratin 19, and could be expanded in vitro. Thus, the AFP promoter-EGFP reporter system is highly useful for identification and isolation of hepatic progenitor cells.     

Neurogenesis and progenitor cells in the adult human brain: a comparison between hippocampal and subventricular progenitor proliferation

For more than a decade, we have known that the human brain harbors progenitor cells capable of becoming mature neurons in the adult human brain. Since the original landmark article by Eriksson et al. in 1998 (Nat Med 4:1313-1317), there have been many studies investigating the effect that depression, epilepsy, Alzheimer's disease, Huntington's disease, and Parkinson's disease have on the germinal zones in the adult human brain. Of particular interest is the demonstration that there are far fewer progenitor cells in the hippocampal subgranular zone (SGZ) compared with the subventricular zone (SVZ) in the human brain. Furthermore, the quantity of progenitor cell proliferation in human neurodegenerative diseases differs from that of animal models of neurodegenerative diseases; there is minimal progenitor proliferation in the SGZ and extensive proliferation in the SVZ in the human. In this review, we will present the data from a range of human and rodent studies from which we can compare the amount of proliferation of cells in the SVZ and SGZ in different neurodegenerative diseases.     

Differential development of human brain white matter tracts

Neuroscience is increasingly focusing on developmental factors related to human structural and functional connectivity. Unfortunately, to date, diffusion-based imaging approaches have only contributed modestly to these broad objectives, despite the promise of diffusion-based tractography. Here, we report a novel data-driven approach to detect similarities and differences among white matter tracts with respect to their developmental trajectories, using 64-direction diffusion tensor imaging. Specifically, using a cross-sectional sample comprising 144 healthy individuals (7 to 48 years old), we applied k-means cluster analysis to separate white matter voxels based on their age-related trajectories of fractional anisotropy. Optimal solutions included 5-, 9- and 14-clusters. Our results recapitulate well-established tracts (e.g., internal and external capsule, optic radiations, corpus callosum, cingulum bundle, cerebral peduncles) and subdivisions within tracts (e.g., corpus callosum, internal capsule). For all but one tract identified, age-related trajectories were curvilinear (i.e., inverted 'U-shape'), with age-related increases during childhood and adolescence followed by decreases in middle adulthood. Identification of peaks in the trajectories suggests that age-related losses in fractional anisotropy occur as early as 23 years of age, with mean onset at 30 years of age. Our findings demonstrate that data-driven analytic techniques may be fruitfully applied to extant diffusion tensor imaging datasets in normative and neuropsychiatric samples.     

Expression of nestin by neural cells in the adult rat and human brain

Neurons and glial cells in the developing brain arise from neural progenitor cells (NPCs). Nestin, an intermediate filament protein, is thought to be expressed exclusively by NPCs in the normal brain, and is replaced by the expression of proteins specific for neurons or glia in differentiated cells. Nestin expressing NPCs are found in the adult brain in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. While significant attention has been paid to studying NPCs in the SVZ and SGZ in the adult brain, relatively little attention has been paid to determining whether nestin-expressing neural cells (NECs) exist outside of the SVZ and SGZ. We therefore stained sections immunocytochemically from the adult rat and human brain for NECs, observed four distinct classes of these cells, and present here the first comprehensive report on these cells. Class I cells are among the smallest neural cells in the brain and are widely distributed. Class II cells are located in the walls of the aqueduct and third ventricle. Class IV cells are found throughout the forebrain and typically reside immediately adjacent to a neuron. Class III cells are observed only in the basal forebrain and closely related areas such as the hippocampus and corpus striatum. Class III cells resemble neurons structurally and co-express markers associated exclusively with neurons. Cell proliferation experiments demonstrate that Class III cells are not recently born. Instead, these cells appear to be mature neurons in the adult brain that express nestin. Neurons that express nestin are not supposed to exist in the brain at any stage of development. That these unique neurons are found only in brain regions involved in higher order cognitive function suggests that they may be remodeling their cytoskeleton in supporting the neural plasticity required for these functions.     

Differing lectin binding profiles among human embryonic stem cells and derivatives aid in the isolation of neural progenitor cells

Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells.     

Heparan sulfates isolated from adult neural progenitor cells can direct phenotypic maturation

Multipotent progenitor stem cells that generate both neurons and glia are components of the hippocampus, subventricular zone and olfactory system of adult mammalian nervous system. The lineage choices any stem cell makes are known to be greatly dependent on the constitution of the extracellular matrix to which they are exposed during their development. Here, the adult rat hippocampus was used as a source of cells for clonal culture in order to investigate the effects of the extracellular glycosaminoglycan heparan sulfate (HS). Neurospheres were readily generated from adult tissue and could be used as a source of cells for further experiments. HS species that promote the actions of fibroblast growth factor-2 (FGF2) for embryonic neural progenitors were found to inhibit the actions of this mitogen for adult progenitors. Only HS fractions that promoted the actions of FGF1 had mitogenic effects on these adult cells. The adult cells proved difficult to clone from single cells. However, when endogenous HS was purified from these cells and added back at high concentration to single cells, the clones were capable of generating plentiful neuronal and glial progeny. The adult hippocampal progenitor (AHP) HS is composed of 32 kDa chains bearing 3 sulfated domains. A proportion of primary osteoblast stem cells exposed to the hippocampal HS adopt neuronal phenotypes. Hence, there appears to be a combination of HS-binding extracellular molecules that predispose cells to particular lineages.     

Excitation-neurogenesis coupling in adult neural stem/progenitor cells

A wide variety of in vivo manipulations influence neurogenesis in the adult hippocampus. It is not known, however, if adult neural stem/progenitor cells (NPCs) can intrinsically sense excitatory neural activity and thereby implement a direct coupling between excitation and neurogenesis. Moreover, the theoretical significance of activity-dependent neurogenesis in hippocampal-type memory processing networks has not been explored. Here we demonstrate that excitatory stimuli act directly on adult hippocampal NPCs to favor neuron production. The excitation is sensed via Ca(v)1.2/1.3 (L-type) Ca(2+) channels and NMDA receptors on the proliferating precursors. Excitation through this pathway acts to inhibit expression of the glial fate genes Hes1 and Id2 and increase expression of NeuroD, a positive regulator of neuronal differentiation. These activity-sensing properties of the adult NPCs, when applied as an "excitation-neurogenesis coupling rule" within a Hebbian neural network, predict significant advantages for both the temporary storage and the clearance of memories.     

Transplantation of adult neural progenitor cells transfected with vascular endothelial growth factor rescues grafted cells in the rat brain

Growth factors are currently evaluated as therapeutics in stroke and neurodegeneration. Besides direct neurotrophic effects, they promote proliferation, survival, and differentiation of both transplanted and endogenous neural precursor cells (NPCs). In the current study, we investigated whether NPCs expressing Vascular Endothelial Growth Factor VEGF-A165 are a useful vehicle for growth factor delivery after transplantation into the caudate putamen of the rat brain. We found an increased survival of adenovirally transfected NPCs after 11 days, but not after 24 hours or 4 days. Additional brain immunohistochemistry revealed increased expression of the endothelial cell marker PECAM-1 (CD31) after 24 hours, 4 day, and 11 days after transplantation. In conclusion, we show that the graft itself is a useful vehicle for growth factor delivery, promoting the survival of NPCs. Moreover, transplantation of VEGF-expressing NPCs supports angiogenesis in the brain, which may contribute to potential brain repair.     

Functional differentiation of human brain progenitor cells

Stem cells and progenitor cells derived from the developing human brain have been shown to differentiate into neurons and astrocytes. However, few studies have examined the functional, physiological properties of these differentiated neurons and astrocytes. In this study we have used immunocytochemistry in combination with electrophysiology to examine protein machinery and functional properties of neurons and astrocytes differentiated from human brain progenitor cells (hBPCs).Our results show that serum induces mainly astrocytic phenotype cells that express GFAP and have physiological properties that are typical of astrocytes. hBPCs differentiated with BDNF and PDGF develop mainly into neurons expressing mature neuronal proteins MAP-2, synaptobrevin II and vesicular glutamate transporter I in the process, plus a small population of GFAP-positive radial cells. Based on electrophysiology of BDNF/PDGF-treated cells two classes of cell were identified. Class I cells have functional neuronal properties, including functional voltage-gated Na(+) and K(+) currents, functional AMPA receptors and the ability to generate action potentials. A smaller subpopulation of cells (Class II cells) expresses GFAP and exhibit functional properties of astrocytes, including linear current-voltage relationship and dye-coupling.     

Mesodermal cell types induce neurogenesis from adult human hippocampal progenitor cells

Neurogenesis in the adult human brain occurs within two principle neurogenic regions, the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. Recent reports demonstrated the isolation of human neuroprogenitor cells (NPCs) from these regions, but due to limited tissue availability the knowledge of their phenotype and differentiation behavior is restricted. Here we characterize the phenotype and differentiation capacity of human adult hippocampal NPCs (hNPCs), derived from patients who underwent epilepsy surgery, on various feeder cells including fetal mixed cortical cultures, mouse embryonic fibroblasts (MEFs) and PA6 stromal cells. Isolated hNPCs were cultured in clonal density by transferring the cells to serum-free media supplemented with FGF-2 and EGF in 3% atmospheric oxygen. These hNPCs showed neurosphere formation, expressed high levels of early neuroectodermal markers, such as the proneural genes NeuroD1 and Olig2, the NSC markers Nestin and Musashi1, the proliferation marker Ki67 and significant activity of telomerase. The phenotype was CD15low/-, CD34-, CD45- and CD133-. After removal of mitogens and plating them on poly D-lysine, they spontaneously differentiated into a neuronal (MAP2ab+), astroglial (GFAP+), and oligodendroglial (GalC+) phenotype. Differentiated hNPCs showed functional properties of neurons, such as sodium channels, action potentials and production of the neurotransmitters glutamate and GABA. Co-culture of hNPCs with fetal cortical cultures, MEFs and PA6 cells increased neurogenesis of hNPCs in vitro, while only MEFs and PA6 cells also led to a morphological and functional neurogenic maturation. Together we provide a first detailed characterization of the phenotype and differentiation potential of human adult hNPCs in vitro. Our findings reinforce the emerging view that the differentiation capacity of adult hNPCs is critically influenced by non-neuronal mesodermal feeder cells.     

IGF-I instructs multipotent adult neural progenitor cells to become oligodendrocytes

Adult multipotent neural progenitor cells can differentiate into neurons, astrocytes, and oligodendrocytes in the mammalian central nervous system, but the molecular mechanisms that control their differentiation are not yet well understood. Insulin-like growth factor I (IGF-I) can promote the differentiation of cells already committed to an oligodendroglial lineage during development. However, it is unclear whether IGF-I affects multipotent neural progenitor cells. Here, we show that IGF-I stimulates the differentiation of multipotent adult rat hippocampus-derived neural progenitor cells into oligodendrocytes. Modeling analysis indicates that the actions of IGF-I are instructive. Oligodendrocyte differentiation by IGF-I appears to be mediated through an inhibition of bone morphogenetic protein signaling. Furthermore, overexpression of IGF-I in the hippocampus leads to an increase in oligodendrocyte markers. These data demonstrate the existence of a single molecule, IGF-I, that can influence the fate choice of multipotent adult neural progenitor cells to an oligodendroglial lineage.