Time-sensitive effects of hypoxia on differentiation of neural stem cells derived from mouse embryonic stem cells in vitro

Abstract

Objectives:

Oxygen tension is an important component of microenvironment for the differentiation of embryonic stem cells including neural lineage. However, the comprehensive influence of hypoxia on neural differentiation during embryonic neural development has not yet been examined.

Methods:

In this study, we investigated the effect of low oxygen levels (5% O₂), or hypoxia, in two stages of neural differentiation in vitro: (1) inducing mouse embryonic stem cells into neural stem cells (NSCs); and then (2) inducing NSCs into neural progenitor cells in neurospheres.

Results:

In the first stage, NSCs generation was reduced under hypoxia. Less mature morphological changes (including neural marker) of NSCs were observed, suggesting the prevention of early differentiation under hypoxic conditions. Thus undifferentiated stem cells were maintained in this stage. However, in the second stage, hypoxia induced neural differentiation in neurospheres. Nevertheless, non-neural progenitor cell formation, such as mesoderm progenitor cell lines or epithelial cell lines, was restricted by low oxygen tension.

Discussions:

Our results demonstrate that hypoxia is essential for regulating neural differentiation and show the different effects on NSC differentiation dependent on the time-course of NSC development. In the early stage of NSCs induction, hypoxia inhibits neural differentiation and maintains the undifferentiated state; in the later stage of NSCs induction, hypoxia induces neural differentiation. Our study may contribute to the development of new insights for expansion and control of neural differentiation.     

Potential role of KCNQ/M-channels in regulating neuronal differentiation in mouse hippocampal and embryonic stem cell-derived neuronal cultures

Voltage-gated K⁺ channels are key regulators of neuronal excitability, playing major roles in setting resting membrane potential, repolarizing the cell membrane after action potentials and affecting transmitter release. The M-type channel or M-channel is a unique voltage- and ligand-regulated K⁺ channel. It is composed of the molecular counterparts KCNQ2 and KCNQ3 (also named Kv7.2 and Kv7.3) channels and expressed in the soma and dendrites of neurons. The present investigation examined the hypothesis that KCNQ2/3 channels played a regulatory role in neuronal differentiation and maturation. In cultured mouse embryonic stem (ES) cells undergoing neuronal differentiation and primary embryonic (E15–17) hippocampal cultures, KCNQ2 and KCNQ3 channels and underlying M-currents were identified. Blocking of KCNQ channels in these cells for 5 days using the specific channel blocker XE991 (10 μM) or linopirdine (30 μM) significantly decreased synaptophysin and syntaxin expression without affecting cell viability. Chronic KCNQ2/3 channel block reduced the expression of vesicular GABA transporter (v-GAT), but not vesicular glutamate transporter (v-GluT). Enhanced ERK1/2 phosphorylation was observed in XE991- and linopirdine-treated neural progenitor cells. In electrophysiological recordings, cells undergoing chronic block of KCNQ2/3 channels showed normal amplitude of mPSCs while the frequency of mPSCs was reduced. On the other hand, KCNQ channel opener N-Ethylmaleimide (NEM, 2 μM) increased mPSC frequency. Fluorescent imaging using fluorescent styryl-dye FM4-64 revealed that chronic blockade of KCNQ2/3 channels decreased endocytosis but facilitated exocytosis. These data indicate that KCNQ2/3 channels participate in the regulation of neuronal differentiation and show a tonic regulation on pre-synaptic transmitter release and recycling in developing neuronal cells.     

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neurocyte in vitro. Methods Isolate the blastula of 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (inner cell mass, ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem cells were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunochemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 (stage specific embryonic antigen 1). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated cells presented the characters of ESCs. Then the isolated cells were able to differentiate into neurocytes in vitro. Conclusion Mouse embryonic stem cells isolation, culture and differentiation system has been established.     

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass （inner cell mass, ICM） which were isolated by mechanical method on the mouse embryonic fibroblaste cell （MEF） feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 （ stage specific embryonic antigen 1 ）. Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.     

Effects of lead exposure on proliferation and differentiation of neural stem cells derived from different regions of embryonic rat brain

Lead is a potent neurotoxin, causing brain damage and cognitive deficits in children even at low exposure levels. Although lead neurotoxicity can occur after prenatal or postnatal exposure, little is known of the effects of lead on embryonic neural stem cells (NSCs) or the extent to which NSCs originating in different brain regions may be differentially sensitive to the effects of lead exposure. The present study examined the effects of lead on proliferation and differentiation of neural stem cells (NSCs) originating from E15 rat cortex (CX), striatum (ST) or ventral mesencephalon (VM). Free-floating neurospheres were grown under standard conditions or in lead (0.01-100 microM)-containing conditioned media for 5 days and proliferation assessed by 3H-thymidine uptake. In other studies, control and lead-exposed neurospheres were collected, dissociated and re-plated in control or lead-containing differentiation media for 7 days. Cells were immunostained for visualization of mature neural and glial markers and counted. Lead exposure (0.01-10 microM) had no effect on neurosphere viability but caused a significant dose-dependent inhibition of proliferation in VM and ST but not CX neurospheres. The number of MAP2 positive neurons differentiated from lead-exposed neurospheres of VM and ST origin (but not CX) was significantly decreased from control as were the number of oligodendrocytes obtained, regardless of their region of origin. In contrast, lead exposure significantly increased the number of astrocytes obtained regardless of site of origin. These data suggest that even low levels of lead can differentially affect proliferation and differentiation of embryonic NSCs originating from different brain regions and supports the need for prevention of prenatal lead exposure.     

Secretion of nerve growth factor in cultures of glial cells and neurons derived from different regions of the mouse brain

The regional ability of central neurons and glial cells to produce nerve growth factor (NGF) was studied in vitro. NGF secretion was compared in cultures of perinatal astrocytes or embryonic neurons that were derived from various mouse brain structures. No regional differences were detected among cultures of post-natal day 2 glial cells of hippocampal, cortical, striatal, or mesencephalic origin. In all cases, levels of NGF released by the cells were very similar. They were closely correlated to the growth rate as shown by the fact that exponentially growing cells produced relatively more factor than did confluent cells, a finding in agreement with previous observations. Unlike growth-phase cells, primary astrocytes immediately plated at high cell density did not secrete any assayable factor before the 7th day of culture. Levels of NGF found during the following days remained low. In contrast, striking differences were observed among cultures of embryonic neurons. NGF was found in relatively large amounts in cultures of embryonic day 17 or 19 striatal neurons, whereas media conditioned by neurons from the mesencephalon, cortex, or septum contained much less factor. Amounts of NGF assayed in cultures of hippocampal neurons varied with the time of sampling of this brain structure. Levels of factor were significantly higher in media conditioned by embryonic day 19 neurons than in media of embryonic day 17 neurons. However, amounts of NGF found in supernatants of hippocampal neurons remained smaller than those present in cultures of striatal nerve cells. Altogether, the results suggest that, in addition to astrocytes, central neurons may also synthesize and secrete NGF in vitro and that this phenomenum is dependent on both the origin and the developmental stage of the neuronal population.