OBJECTIVETo find a better method for harvesting highly purified neurons by comparing three methods used for co-culture of neurons and astrocytes.

METHODSThe co-culture models of neurons and astrocytes were established by primary culture, Banker's co-culture method or Transwell cell-culture inserts. The neurons and astrocytes cultured in vitro were from neonatal rats.

RESULTSThe highly purified neurons were not harvested by primary culture because the neurons and astrocytes grew on the same cover slip and it was difficult to control the growth velocity of astrocytes. The highly purified neurons were harvested by Banker's co-culture method or the method using Transwell cell-culture inserts, but the procedure of the former was more complicated than that of the later.

CONCLUSIONSThe culture method using Transwell cell-culture inserts is recommended for the establishment of the co-culture system of neurons and astrocytes.

Animals ; Astrocytes ; Cells, Cultured ; Coculture Techniques ; Neurons ; cytology

Active migration of immature neurons occurs when fragments of human foetal cerebral tissues are explanted as organotypic cultures. The sequence of events during neuronal migration is orderly and consistent under different cultural conditions as evidenced by continuous time-lapse cinematographic studies. Migrating neurons utilize astrocytes to anchor neurites, and move in clusters on or along the processes of astrocytes or other neurons. Translocation of neuronal soma is accomplished by nuclear movement within extended neurites. A unique junction develops between neurites and astrocytic membrane during early phases in culture to suggest a special affinity of neurons to astrocytes. It is concluded from these observations that immature neurons have inherent capacity for active migration in-vitro; preferentially utilize astrocytes and astrocytic processes for anchoring as well as for directional guidance during migration; and translocate their soma by nuclear movement within extended neurites. It is suggested that similar mechanisms may be at play during migration of postmitotic neurons in developing cerebral cortex in human.
Astrocytes/cytology ; Brain/cytology ; Brain/embryology* ; Cell Movement ; Fetus ; Human ; Neural Conduction ; Neurons/cytology* ; Tissue Culture

Altitude ; Animals ; Astrocytes ; cytology ; Brain ; cytology ; Hypoxia ; Male ; Microglia ; cytology ; Rats ; Rats, Wistar

To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes, including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio in the cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5% among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.
Astrocytes/*cytology ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; Neurons/*cytology ; Stem Cells/*cytology ; Tretinoin/pharmacology

Human pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine as they are an important source of functional cells for potential cell replacement. These human PSCs, similar to their counterparts of mouse, have the full potential to give rise to any type of cells in the body. However, for the promise to be fulfilled, it is necessary to convert these PSCs into functional specialized cells. Using the developmental principles of neural lineage specification, human ESCs and iPSCs have been effectively differentiated to regional and functional specific neurons and glia, such as striatal gama-aminobutyric acid (GABA)-ergic neurons, spinal motor neurons and myelin sheath forming oligodendrocytes. The human PSCs, in general differentiate after the similar developmental program as that of the mouse: they use the same set of cell signaling to tune the cell fate and they share a conserved transcriptional program that directs the cell fate transition. However, the human PSCs, unlike their counterparts of mouse, tend to respond divergently to the same set of extracellular signals at certain stages of differentiation, which will be a critical consideration to translate the animal model based studies to clinical application.
Astrocytes ; cytology ; Cell Differentiation ; Embryonic Stem Cells ; cytology ; Humans ; Neuroglia ; cytology ; Neurons ; cytology ; Pluripotent Stem Cells ; cytology

Cultured primary hippocampal neurons are ideal tool for investigating the subcellular localization and trafficking of neuronal proteins. The aim of the present study was to establish a method to co-culture hippocampal neurons and cortical astrocytes, which would guarantee well conditions of neurons. Newborn Sprague-Dawley (SD) rats were sacrificed by decapitation. The cortex of cerebrum was cut into pieces, and the cortical tissue was digested with trypsin. The liquid suspension of single cells was planted onto a 25 cm² culture flask. On the fourth day of culture, the tissue cells except astrocytes were removed by intensive agitation of culture flask. Purified astrocytes were allowed to grow continuously until they reached most area of flask. At this time point, we replaced the culture media with neuronal cell media containing cytarabine, and planted the primary culture of rat hippocampal neurons onto the feed layer of cortical astrocytes. The microscopic observation results showed that, the astrocytes evenly grew without obvious boundaries between each other, and exhibited good purity. The co-cultured hippocampal neurons were in good condition, developed intertwined network of axons and dendrites, lived for a long time, and could tolerate gene transfection. Above all, this method is relatively simple from a technical point of view, yet provides healthy and reliable neuronal culture.
Animals ; Astrocytes ; cytology ; Cells, Cultured ; Cerebral Cortex ; cytology ; Coculture Techniques ; Culture Media ; Hippocampus ; cytology ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley

The aim of this study is to establish a method to culture and purify cerebral astrocyte of tree shrew (Tupaia belangeri), a kind of new laboratorial animal which is a relative of primates. Newborn tree shrews were used in this experiment. The cortex of cerebrum was isolated and placed in 4°C for 20 min to injure neurons. The cortical tissue was disaggregated by trypsin digestion. Differential attachment method was used to remove fibroblasts. The mixed culture was rinsed by trypsin (0.005%) solution to remove neurons. Upon reaching 70% confluence, the culture was subjected to static trypsin digestion until a white slice film exfoliated from the bottom of culture bottle. This film, i.e. astrocyte layer, was taken out and cultured, and the third passage was identified by immunocytochemical staining and immunofluorescence with anti-glial fibrillary acidic protein (GFAP) antibody. The result showed the purity of tree shrew astrocytes was more than 98%. Thus the method to culture highly purified astrocyte of tree shrew was successfully established, which would contribute to further study in central nervous system physiology and diseases in this new laboratorial animal.
Animals ; Astrocytes ; cytology ; Brain ; cytology ; Cell Separation ; methods ; Primary Cell Culture ; methods ; Tupaiidae

BACKGROUNDThe production of neural stem cells (NSCs) derived from embryonic stem (ES) cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro.

METHODSMouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells' morphology and formation of teratoma in severe combined immunodeficiency disease (SCID) mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 (Oct-4) and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription-polymerase chain reaction (RT-PCR) method to monitor the process of cell differentiation.

RESULTSThe ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2 +/- 3.5 neurospheres per plate formed in astrocyte-induced group and only 0.8 +/- 0.3 per plate in the control group (clonogenic assay, P < 0.01), and the ratio of nestin positive cells was (50.2 +/- 2.8)% in astrocyte-induced group and only (1.4 +/- 0.5)% in the control group (flow cytometry, P < 0.01). With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into neurons, astrocytes, and oligodendrocytes in differentiating medium supplemented with fetal calf serum. The results of differentiating test showed that the ratio of NF-200 and NSE positive cells was (42.7 +/- 2.6)% in astrocyte-induced group and only (11.2 +/- 1.8)% in the control group (P < 0.01).

CONCLUSIONSAstrocytes can not only increase the production of NSCs derived from ES cells but also promote the differentiation of NSCs toward neuronal lineage.

Animals ; Astrocytes ; physiology ; Cell Differentiation ; Cell Lineage ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Mice ; Neurons ; cytology ; Stem Cells ; cytology

OBJECTIVETo investigate the effects of inhibiting gap junctional intercellular communication on hypoxia/reoxygenation injury in astrocytes.

METHODSPrimary cultured cerebral cortical astrocytes of neonate rats were divided into normal control group, hypoxia reoxygenation injury group and 18-α-glycyrrhetinic acid and oleamide (gap junctional intercellular channel inhibitors) group. The gap junction intercellular communication was determined by Parachute assay. The viability of astrocyes was detected by MTT assay. The apoptosis of astrocytes were detected with annexin V/PI and Hoechst 33258 staining.

RESULTSCompared with the normal control group, the gap junctional function of astrocytes was increased significantly in ischemia/reperfusion group (P<0.01), the surviving fraction of astrocytes decreased significantly (P<0.01) and its cell apoptosis ratio increased significantly (P<0.01). Compared with the ischemia/reperfusion group, the gap junctional function of astrocytes in18-α-glycyrrhetinic acid and oleamide group decreased significantly (P<0.01), the viability of astrocytes increased significantly (P<0.01), while cell apoptosis decreased significantly (P<0.01).

CONCLUSIONInhibition of intercellular gap junction has protective effect against hypoxia/reoxygenation injury in astrocytes.

Animals ; Apoptosis ; Astrocytes ; cytology ; pathology ; Cell Communication ; Cell Hypoxia ; Cells, Cultured ; Gap Junctions ; Oxygen ; Rats

OBJECTIVETo evaluate the impact of the topographic features of electrospun polymethylmethacrylate (PMMA) nanofibers on growth pattern of primary rat astrocytes.

METHODSRat astrocytes were cultured on fabricated random and aligned electrospun nanofibers. Using PMMA film as the control, the cells were transfected with a lentivirus harboring GFP gene to reveal the cell growth pattern on different substrates.

RESULTSoth random and aligned electrospun PMMA nanofibers could support the growth of rat astrocytes, but the topographic features of the fibers significantly affected their growth pattern. On aligned nanofibers, astrocytes extended long cell processes along the direction of the substrate fibers, and on random fibers the astrocytes formed shorter processes. On merged images of GFP expressions and substrate fibers, the cell processes of the astrocytes were shown to adhere to and elongate along the fibers both on random and aligned nanofibers.

CONCLUSIONThe topographic features of PMMA nanofibers can significantly influence the growth pattern of primary rat astrocytes. Aligned electrospun nanofibers has the potential to serve as scaffold material for reducing glia scar formation after spinal cord injury.

Animals ; Astrocytes ; cytology ; Cells, Cultured ; Nanofibers ; Polymethyl Methacrylate ; Rats ; Tissue Engineering ; Tissue Scaffolds