Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.     

Midkine, a heparin-binding growth factor, is expressed in neural precursor cells and promotes their growth

Midkine, a heparin-binding growth factor, was found to be expressed in neural precursor cells, which consist of neural stem cells and the progenitor cells. When embryonic brain cells were allowed to form neurospheres enriched in neural precursor cells, numbers were significantly smaller from the midkine-deficient brain than from the wild-type brain. Dissociated neurosphere cells yielded nestin-positive neural precursor cells and differentiated neuronal cells upon culture on a substratum. Neural precursor cells from the midkine-deficient brain spread poorly and grew less effectively on a substratum coated with poly-l-lysine than the cells on midkine-coated substratum. Neural precursor cells from the wild-type brain spread and grew well on both the substrata. Differentiation to neurons and glia cells was not affected by the absence of midkine. Heparitinase digestion of dissociated neurosphere cells resulted in poor growth of neural precursor cells, while chondroitinase digestion had no effect. These results indicate that midkine is involved in the growth of neural precursor cells and suggest that the interaction with heparan sulfate proteoglycans is important in midkine action to these cells.     

神经干细胞克隆球中干细胞的比例变化

为了定量研究神经干细胞体外产生的克隆结构“neurospheres”中干细胞的比例变化,利用无血清培养、细胞克隆培养技术及免疫细胞化学染色方法,观察不同代数神经干细胞克隆球中nestin阳性细胞的比例。发现随着传代次数增加,克隆球中nestin阳性细胞的比例也在显著减少(P<0.001)。提示在体外培养体系中,形成的克隆球具有异质性,并且在不同代数间神经干细胞的比例也显著不同。     

Angiogenic potential of CD44+ CD90+ multipotent CNS stem cells in vitro

Although it is now clear that several subpopulations of neural stem cells (NSCs) exist during early development and adulthood, the angiogenic potential of NSCs remains a subject of debate. Here, we report that CD44(+) CD90(+) cells isolated from primary neurospheres can form vascular-tube structures in vitro. NSCs isolated from the mouse embryonic cortex formed neurospheres when cultured in serum-free medium containing 20ng/ml basic fibroblast growth factor (bFGF). CD44(+) CD90(+) cells were enriched from the neurospheres using an EPICS ALTRA flow cytometer, and antibodies against CD44 and CD90. The purified CD44(+) CD90(+) cells generated neurospheres, and differentiated into neurons and astrocytes. When the cells were inoculated into collagen gels and cultured with 20% fetal bovine serum plus bFGF for 7 days, vascular tube-like structures were formed. These results indicate that CD44(+) CD90(+) cells have the ability to generate neurospheres and to form vascular tubes.     

Characterization of aggregation and protein expression of bovine corneal endothelial cells as microcarrier cultures in a rotating-wall vessel

Rotating-wall vessels are beneficial to tissue engineering in that the reconstituted tissue formed in these low-shear bioreactors undergoes extensive three-dimensional growth and differentiation. In the present study, bovine corneal endothelial (BCE) cells were grown in a high-aspect rotating-wall vessel (HARV) attached to collagen-coated Cytodex-3 beads as a representative monolayer culture to investigate factors during HARV cultivation which affect three-dimensional growth and protein expression. A collagen type I substratum in T-flask control cultures increased cell density of BCE cells at confluence by 40% and altered the expression of select proteins (43, 50 and 210 kDa). The low-shear environment in the HARV facilitated cell bridging between microcarrier beads to form aggregates containing upwards of 23 beads each, but it did not promote multilayer growth. A kinetic model of microcarrier aggregation was developed which indicates that the rate of aggregation between a single bead and an aggregate was nearly 10 times faster than between two aggregate and 60 times faster than between two single beads. These differences reflect changes in collision frequency and cell bridge formation. HARV cultivation altered the expression of cellular proteins (43 and 70 kDa) and matrix proteins (50, 73, 89 and 210 kDa) relative to controls perhaps due to hypoxia, fluid flow or distortion of cell shape. In addition to the insight that this work has provided into rotating-wall vessels, it could be useful in modeling aggregation in other cell systems, propagating human corneal endothelial cells for eye surgery and examining the response of endothelial cells to reduced shear.     

An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro

Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.     

Short-Term Withdrawal of Mitogens Prior to Plating Increases Neuronal Differentiation of Human Neural Precursor Cells

Background

Human neural precursor cells (hNPC) are candidates for neural transplantation in a wide range of neurological disorders. Recently, much work has been done to determine how the environment for NPC culture in vitro may alter their plasticity. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) are used to expand NPC; however, it is not clear if continuous exposure to mitogens may abrogate their subsequent differentiation. Here we evaluated if short-term removal of FGF-2 and EGF prior to plating may improve hNPC differentiation into neurons.

Principal Findings

We demonstrate that culture of neurospheres in suspension for 2 weeks without EGF-FGF-2 significantly increases neuronal differentiation and neurite extension when compared to cells cultured using standard protocols. In this condition, neurons were preferentially located in the core of the neurospheres instead of the shell. Moreover, after plating, neurons presented radial rather than randomly oriented and longer processes than controls, comprised mostly by neurons with short processes. These changes were followed by alterations in the expression of genes related to cell survival.

Conclusions

These results show that EGF and FGF-2 removal affects NPC fate and plasticity. Taking into account that a three dimensional structure is essential for NPC differentiation, here we evaluated, for the first time, the effects of growth factors removal in whole neurospheres rather than in plated cell culture.     

Tri-dimensional prostate cell cultures in simulated microgravity and induced changes in lipid second messengers and signal transduction

The high aspect rotating-wall vessel (HARV) was designed to cultivate cells in an environment that simulate microgravity. We studied previously the effects of HARV cultivation on DU-145 human prostate carcinoma cells. We determined that HARV cultivation produced a less aggressive, slower growing, less proliferative, more differentiated and less pliant cell than other cell cultivation methods. The result was a 3-dimensional (3D) growth model of prostate cancer which mimics in vivo tissue growth. This work examines the signal transduction-second messenger pathways existing temporarily in these HARV cells and correlates these features with the special properties in growth and 3D spheroid formation. We found an initial very active ceramide, a diacylglycerol increase together with increases in PI-PLC and PLA₂ a central defect in PLD (no phosphatic acid or phosphatidylethanol at any time during 15 days of HARV cultivation). There is a cross-talk between ceramide and PI3K pathways with activation of PI3K, after 6 days of HARV growth concomitant with down-regulation of ceramide. At this time, there is also an increase of cAMP (seen by increases in arachidonic acid). Taken together these results can explain the 3D organoidlike growth. We therefore developed a model for growth in HARV prostate cancer cells which involve temporal "switches" between second messengers, activation and cross-talk between multiplicity of signaling pathways and a central defect in PLD pathways. Essential to the late slow growth, and 3D organotypic formation are the apoptotic, anti-survival, anti-proliferation and differentiation pathways in the first days of HARV, with growth of "new" different types of prostate cancer cells which set-up for later "switch" in ceramide-PI3K to survival and proliferation.     

Cerebellar granule cell neurogenesis is regulated by cell-cell interactions in vitro

When CNS precursor cells purified from the external germinal layer of the early postnatal mouse cerebellum are cultured in cellular reaggregates, DNA synthesis increased 10-fold above that of cells dispersed in a monolayer or embedded in a collagen matrix. Dividing precursor cells gave rise to neurons immunopositive for the neural antigens N-CAM, L1, and TAG-1, but not to astroglial cells immunopositive for glial filament protein. Moreover, proliferating precursor cells did not generate other types of cerebellar neurons, as judged by the lack of expression of glutamic acid decarboxylase, the synthetic enzyme for gamma-amino-n-butyric acid. By contrast, the addition of astroglial cells, or astroglial cell membranes, to cellular reaggregates of granule cell neuroblasts arrested precursor cell DNA synthesis in a dose-dependent manner. These results suggest that homotypic contact interactions among CNS neural progenitors control precursor cell proliferation and fate in generative zones of developing brain.     

人胚不同脑区神经前体细胞的分离培养及分化特性的比较

目的研究人胚不同脑区神经前体细胞(neural progenitor cells,NPCs)培养及增殖分化特性。方法取14-17周人胚脑区组织,分为新皮质、纹状体、间脑、中脑、后脑和延髓组,悬浮培养。鉴定细胞球巢蛋白抗原的表达,分化及自我更新能力。观察各脑区培养细胞的生长、增殖状况。新皮质、纹状体及间脑来源的神经球分化后,运用免疫荧光细胞化学法比较神经元及星形胶质细胞的比例。结果各脑区培养出的悬浮细胞球巢蛋白抗原阳性,可分化为MAP2或GFAP阳性细胞,且BrdU掺入实验阳性。体外培养第3d,纹状体及间脑组均可见大量神经球,且纹状体组明显多于间脑组;新皮质组传代后可见较多神经球;其它组仅见个别神经球。新皮质、纹状体、间脑来源的NPCs诱导分化后,MAP2或GFAP阳性细胞率各组间比较差异无显著性。结论人胚不同脑区均可培养出NPCs,从易到难依次为纹状体、间脑、新皮质及其它脑区。新皮质、纹状体、间脑来源的NPCs体外分化比例一致。     

Effects of simulated microgravity on DU 145 human prostate carcinoma cells

The high aspect rotating-wall vessel (HARV) was recently designed by NASA to cultivate animal cells in an environment that simulates microgravity. This work examines the effects of HARV cultivation on DU 145 human prostate carcinoma cells. In the HARV, these prostate cells grew in suspension on Cytodex-3 microcarrier beads to form bead aggregates with extensive three-dimensional growth between beads and on the aggregate surface. HARV and spinner-flask control cultures of DU 145 cells had similar doubling times, but the former was characterized by a higher percentage of G(1)-phase cells, larger bead aggregates, enhanced development of filopodia and microvilli-like structures on the aggregate surface, and stronger staining for select cytoskeletal proteins (cytokeratins 8 and 18, actin, and vimentin). When compared with static controls grown in a T-flask and Transwell insert, HARV cultures grew more slowly and differences in the cell cycle and immunostaining became more pronounced. These results suggest that HARV cultivation produced a culture that was less aggressive from the perspective of proliferation, more differentiated and less pliant than any of the three control cultures examined in this work. Possible factors effecting this change are discussed including turbulence and three-dimensional growth. (c) 1996 John Wiley & Sons, Inc.     

A clonogenic survival assay of neural stem cells in rat spinal cord after exposure to ionizing radiation

Neural stem cells play an important role in neurogenesis of the adult central nervous system (CNS). Inhibition of neurogenesis has been suggested to be an underlying mechanism of radiation-induced CNS damage. Here we developed an in vivo/ in vitro clonogenic assay to characterize the survival of neural stem cells after exposure to ionizing radiation. Cells were isolated from the rat cervical spinal cord and plated as single cell suspensions in defined medium containing epidermal growth factor and basic fibroblast growth factor. The survival of the proliferating cells was determined by their ability to form neurosphere colonies. The number and size of neurospheres were analyzed quantitatively at day 10, 12, 14 and 16 after plating. Plating cells from 5, 10 and 15 mm of the cervical spinal cord resulted in a linear increase in the number of neurospheres from day 10-16. Compared to the nonirradiated spinal cord, there was a significant decrease in the number and size of neurosphere colonies cultured from a 10-mm length of the rat spinal cord after a single dose of 5 Gy. When dissociated neurospheres derived from a spinal cord that had been irradiated with 5 Gy were allowed to differentiate, the percentages of neurons, oligodendrocytes and astrocytes as determined by immunocytohistochemistry were not altered compared to those from the nonirradiated spinal cord. Secondary neurospheres could be obtained from cells dissociated from primary neurospheres that had been cultured from the irradiated spinal cord. In conclusion, exposure to ionizing radiation reduces the clonogenic survival of neural stem cells cultured from the rat spinal cord. However, neural stem cells retain their pluripotent and self-renewing properties after irradiation. A neurosphere-based assay may provide a quantitative measure of the clonogenic survival of neural stem cells in the adult CNS after irradiation.     

Spontaneous apoptosis in murine free-floating neurospheres

We have shown successful in vitro expansion of rodent and human neural precursor cells (NPC) derived from fetal midbrain and forebrain. Here, we show that mouse neural precursor cells growing in neurospheres proliferate, but also undergo spontaneous apoptosis in vitro. On average, 30.7 +/- 3.4% cells of midbrain-derived neural precursors and 32.1 +/- 2.5% of forebrain-derived neural precursors were found apoptotic within neurospheres. Spontaneous apoptosis involved mitochondrial cytochrome c release and activation of effector caspase-3. Caspase-3 was activated in 26.9 +/- 3.4% of mesencephalic neural precursor cells. Virtually all nuclei with morphological signs of apoptosis belong to caspase-3-positive cells. The great majority of dying cells within neurospheres was positive for CNS precursor cell marker nestin. Pro-apoptotic proteins of the Bcl-2 family, Bax and Bak, exhibited conformational changes in neural precursors expanding in vitro. Key molecules such as executioner caspase-3 may be useful targets to reduce the amount of apoptosis.     

Insulin‐like growth factor binding protein 4 inhibits proliferation of bone marrow mesenchymal stem cells and enhances growth of neurospheres derived from the stem cells

Insulin‐like growth factor binding protein 4 (IGFBP‐4) was reported to trigger cellular senescence and reduce cell growth of bone marrow mesenchymal stem cells (BMSCs), but its contribution to neurogenic differentiation of BMSCs remains unknown. In the present study, BMSCs were isolated from the femur and tibia of young rats to investigate effects of IGFBP‐4 on BMSC proliferation and growth of neurospheres derived from BMSCs. Bone marrow mesenchymal stem cell proliferation was assessed using CCK‐8 after treatment with IGFBP‐4 or blockers of IGF‐IR and β‐catenin. Phosphorylation levels of Akt, Erk, and p38 in BMSCs were analysed by Western blotting. Bone marrow mesenchymal stem cells were induced into neural lineages in NeuroCult medium; the number and the size of BMSC‐derived neurospheres were counted after treatment with IGFBP‐4 or the blockers. It was shown that addition of IGFBP‐4 inhibited BMSC proliferation and immunodepletion of IGFBP‐4 increased the proliferation. The blockade of IGF‐IR with AG1024 increased BMSC proliferation and reversed IGFBP‐4‐induced proliferation inhibition; however, blocking of β‐catenin with FH535 did not. p‐Erk was significantly decreased in IGFBP‐4‐treated BMSCs. IGFBP‐4 promoted the growth of neurospheres derived from BMSCs, as manifested by the increases in the number and the size of the derived neurospheres. Both AG1024 and FH535 inhibited the formation of NeuroCult‐induced neurospheres, but FH535 significantly inhibited the growth of neurospheres in NeuroCult medium with EGF, bFGF, and IGFBP‐4. The data suggested that IGFBP‐4 inhibits BMSC proliferation through IGF‐IR pathway and promotes growth of BMSC‐derived neurospheres via stabilizing β‐catenin.     

Skeletal muscle satellite cells cultured in simulated microgravity

Summary Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (∼ 200 gm) were used for all studies and were composed of greater than 75% satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture. Plating efficiency (cells attached ÷ cells plated ×100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods.     

Neural precursors express multiple chondroitin sulfate proteoglycans, including the lectican family

Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid (CSF) of both human neonates with preterm hydrocephalus, and P8 hydrocephalic mice. We hypothesized CSF CSPGs are synthesized by neural precursors, separated from ventricular CSF by ependyma, which is often disrupted in hydrocephalus. Western blotting demonstrates that neural precursors cultured as neurospheres secrete CSPGs (> 30 microg/ml) into their media which appear to be very similar to these CSF CSPGs. Some CSPGs bear the stage-specific embryonic antigen-1 (ssea-1), associated with embryonic/neural stem cells. Neurospheres transcribe many CSPG genes, including the entire aggrecan/lectican family, phosphacan, and tenascin. Phosphacan can be detected in media by Western blotting. Aggrecan can be detected in media after purification using hyaluronic acid affinity chromatography. During differentiation, neurospheres downregulate CSPGs. This is the first report to show that proliferating neural precursors synthesize lecticans, including aggrecan, which are downregulated with differentiation. These observations suggest novel links between CSPGs and CNS precursor biology.     

Ionizing Radiation Perturbs Cell Cycle Progression of Neural Precursors in the Subventricular Zone Without Affecting Their Long-Term Self-Renewal

Damage to normal human brain cells from exposure to ionizing radiation may occur during the course of radiotherapy or from accidental exposure. Delayed effects may complicate the immediate effects resulting in neurodegeneration and cognitive decline. We examined cellular and molecular changes associated with exposure of neural stem/progenitor cells (NSPs) to ¹³⁷Cs γ-ray doses in the range of 0 to 8 Gy. Subventricular zone NSPs isolated from newborn mouse pups were analyzed for proliferation, self-renewal, and differentiation, shortly after irradiation. Strikingly, there was no apparent increase in the fraction of dying cells after irradiation, and the number of single cells that formed neurospheres showed no significant change from control. Upon differentiation, irradiated neural precursors did not differ in their ability to generate neurons, astrocytes, and oligodendrocytes. By contrast, progression of NSPs through the cell cycle decreased dramatically after exposure to 8 Gy (p < .001). Mice at postnatal day 10 were exposed to 8 Gy of γ rays delivered to the whole body and NSPs of the subventricular zone were analyzed using a four-color flow cytometry panel combined with ethynyl deoxyuridine incorporation. Similar flow cytometric analyses were performed on NSPs cultured as neurospheres. These studies revealed that neither the percentage of neural stem cells nor their proliferation was affected. By contrast, γ-irradiation decreased the proliferation of two classes of multipotent cells and increased the proliferation of a specific glial-restricted precursor. Altogether, these results support the conclusion that primitive neural precursors are radioresistant, but their proliferation is slowed down as a consequence of γ-ray exposure.     

Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology

For more than a decade the 'neurosphere assay' has been used to define and measure neural stem cell (NSC) behavior, with similar assays now used in other organ systems and in cancer. We asked whether neurospheres are clonal structures whose diameter, number and composition accurately reflect the proliferation, self-renewal and multipotency of a single founding NSC. Using time-lapse video microscopy, coculture experiments with genetically labeled cells, and analysis of the volume of spheres, we observed that neurospheres are highly motile structures prone to fuse even under ostensibly 'clonal' culture conditions. Chimeric neurospheres were prevalent independent of ages, species and neural structures. Thus, the intrinsic dynamic of neurospheres, as conventionally assayed, introduces confounders. More accurate conditions (for example, plating a single cell per miniwell) will be crucial for assessing clonality, number and fate of stem cells. These cautions probably have implications for the use of 'cytospheres' as an assay in other organ systems and with other cell types, both normal and neoplastic.