Daisy Shum - Academia.edu (original) (raw)

Papers by Daisy Shum

Figure S4. Flow cytometry analysis of rat BMSCs. Rat BMSCs (A) between passage number 5 and 8 wer... more Figure S4. Flow cytometry analysis of rat BMSCs. Rat BMSCs (A) between passage number 5 and 8 were analyzed by flow cytometry. In all panels, isotype controls are represented by red lines, while immunopositivity for respective cell surface markers are represented by blue lines. Percentages of positive cells as shown within individual panels are representative of one sample. Immunopositivity for CD90, STRO-1, and CD73 in isolated rat BMSCs were 95.70 ± 1.21 %, 95.91 ± 1.86 %, and 92.80 ± 1.46 %, respectively (B–D). Immunopositivity for nestin was 11.31 ± 1.25 % (E). Immunopositivity towards the hematopoietic progenitor marker CD45 was negligible (F). Mean ± SD, n = 4. (PDF 288 kb)

Figure S2. Isotype controls for immunocytochemistry. (A, B) Mouse and rabbit isotype control anti... more Figure S2. Isotype controls for immunocytochemistry. (A, B) Mouse and rabbit isotype control antibody-stained neurospheres derived following normoxic and hypoxic treatment, respectively. (C, D) Mouse and rabbit isotype control antibody-stained SCLCs derived from normoxia- and hypoxia-treated BMSCs, respectively. (E, F) Mouse and rabbit isotype control antibody-stained fate-committed Schwann cells generated from normoxia- and hypoxia-treated BMSCs, respectively. (PDF 211 kb)

Figure S1. Work flow for the derivation of fate-committed Schwann cells from BMSCs. BMSCs isolate... more Figure S1. Work flow for the derivation of fate-committed Schwann cells from BMSCs. BMSCs isolated by means of adherence to tissue culture plastic were dissociated and maintained for 12 days in the presence of EGF/bFGF on low-attachment culture plates. Thereafter, resultant neurospheres were plated onto PDL/laminin-coated culture plates and differentiated into Schwann cell-like cells (SCLCs) in glial differentiation medium containing β-heregulin, bFGF, and PDGF for 7 days. In order to direct SCLCs to fate commitment, they were co-cultured with purified and partially dissociated dorsal root ganglia (DRG) neurons for a further 15 days. (PDF 159 kb)

Sheng li xue bao : [Acta physiologica Sinica], 2014

The capability of the central vestibular system in utilizing cues arising from the inner ear dete... more The capability of the central vestibular system in utilizing cues arising from the inner ear determines the ability of animals to acquire the sense of head orientations in the three-dimensional space and to shape postural movements. During development, neurons in the vestibular nucleus (VN) show significant changes in their electrophysiological properties. An age-dependent enhancement of membrane excitability is accompanied by a progressive increase in firing rate and discharge regularity. The coding of horizontal and vertical linear motions also exhibits developmental refinement in VN neurons. Further, modification of cell surface receptors, such as glutamate receptors, of developing VN neurons are well-orchestrated in the course of maturation, thereby regulating synaptic efficacy and spatial coding capacity of these neurons in local circuits. Taken together, these characteristic features of VN neurons contribute to developmental establishment of space-centered coordinates within t...

Sheng li xue bao : [Acta physiologica Sinica], 2014

The availability of human stem cells heralds a new era for in vitro cell-based modeling of neurod... more The availability of human stem cells heralds a new era for in vitro cell-based modeling of neurodevelopmental and neurodegenerative diseases. Adding to the excitement is the discovery that somatic cells of patients can be reprogrammed to a pluripotent state from which neural lineage cells that carry the disease genotype can be derived. These in vitro cell-based models of neurological diseases hold promise for monitoring of disease initiation and progression, and for testing of new drug treatments on the patient-derived cells. In this review, we focus on the prospective applications of different stem cell types for disease modeling and drug screening. We also highlight how the availability of patient-specific induced pluripotent stem cells (iPS cells) offers a unique opportunity for studying and modeling human neurodevelopmental and neurodegenerative diseases in vitro and for testing small molecules or other potential therapies for these disorders. Finally, the limitations of this te...

Development, 2000

The effects of removing chondroitin sulfate from chondroitin sulfate proteoglycan molecules on gu... more The effects of removing chondroitin sulfate from chondroitin sulfate proteoglycan molecules on guidance of retinal ganglion cell axons at the optic chiasm were investigated in a brain slice preparation of mouse embryos of embryonic day 13 to 15. Slices were grown for 5 hours and growth of dye-labeled axons was traced through the chiasm. After continuous enzymatic digestion of the chondroitin sulfate proteoglycans with chondroitinase ABC, which removes the glycosaminoglycan chains, navigation of retinal axons was disrupted. At embryonic day 13, before the uncrossed projection forms in normal development, many axons deviated from their normal course, crossing the midline at aberrant positions and invading the ventral diencephalon. In slices from embryonic day 14 embryos, axons that would normally form the uncrossed projection at this stage failed to turn into the ipsilateral optic tract. In embryonic day 15 slices, enzyme treatment caused a reduction of the uncrossed projection that d...

Neuroembryology and Aging, 2009

The expression patterns of the two subunits of the metabotropic GABAB receptor, GABABR1 and GABAB... more The expression patterns of the two subunits of the metabotropic GABAB receptor, GABABR1 and GABABR2, vary among various neural regions and their relative expression in the rat central vestibular system remains unknown. To identify the expression patterns of these two subunits of the metabotropic GABAB receptor in the vestibular nucleus of adult rats, double immunofluorescence experiments were performed. GABABR1 or GABABR2 immunoreactivity was observed in neurons and neuropilar elements of the vestibular nuclear complex and its subgroups x and y. These neurons were oval, multipolar, fusiform or triangular in shape. In contrast to other brain regions, individual neurons or neuropil within the various vestibular subnuclei and subgroups displayed comparable levels of GABABR1 and GABABR2 immunoreactivity. Double immunofluorescence experiments further demonstrated the coexpression of GABABR1 and GABABR2 receptor subunits in individual central vestibular neurons. The present findings provi...

Stem Cell Research & Therapy, 2016

Background Bone marrow stromal cells (BMSCs) are attractive as a source of neural progenitors for... more Background Bone marrow stromal cells (BMSCs) are attractive as a source of neural progenitors for ex vivo generation of neurons and glia. Limited numbers of this subpopulation, however, hinder translation into autologous cell-based therapy. Here, we demonstrate rapid and efficient conditioning with hypoxia to enrich for these neural progenitor cells prior to further expansion in neurosphere culture. Method Adherent cultures of BMSCs (rat/human) were subjected to 1 % oxygen for 24 h and then subcultured as neurospheres with epidermal growth factor (EGF) and basic fibroblast growth factor supplementation. Neurospheres and cell progeny were monitored immunocytochemically for marker expression. To generate Schwann cell-like cells, neurospheres were plated out and exposed to gliogenic medium. The resulting cells were co-cultured with purified dorsal root ganglia (rat) neurons and then tested for commitment to the Schwann cell fate. Fate-committed Schwann cells were subjected to in vitro ...

Urological Research, 1987

The inhibitory activity of urine on calcium oxalate crystallization was measured as an inhibitory... more The inhibitory activity of urine on calcium oxalate crystallization was measured as an inhibitory index using a gel model modified from Schneider et al. [8]. Urine sampies from 36 recurrent stone formers and 21 controls in 3 separate periods (morning, afternoon and evening) were tested. Mean inhibitory indices of > 0 were observed among normal controls in the 3 sampled periods and among stone formers in the morning and afternoon samples. A significantly lower (negative) value was observed in the evening samples of stone formers, indicating a higher tendency towards crystallization than normal. The gel method may be applied to identify individuals at risk of stone formation if sufficient numbers of period-defined urine samples from the individual are tested for a statistically significant mean inhibitory index but whether this is practical in a clinical laboratory will need further evaluations.

Tissue Engineering Part A, 2011

Mesenchymal stem cell (MSC)-based engineering is promising for cartilage repair. However, the com... more Mesenchymal stem cell (MSC)-based engineering is promising for cartilage repair. However, the compositional mechanical relationship of the engineered structures has not been extensively studied, given the importance of such relationship in native cartilage tissues. In this study, a novel human MSC-collagen microsphere system was used to study the compositional mechanical relationship during in vitro chondrogenic differentiation using histological and biochemical methods and a microplate compression assay. The mechanical property was found positively correlating with newly deposited cartilage-relevant matrices, glycosaminoglycan, and type II collagen, and with the collagen crosslinker density, in agreement with the presence of thick collagen bundles upon structural characterization. On the other hand, the mechanical property negatively correlates with type I collagen and total collagen, suggesting that the initial collagen matrix scaffold of the microsphere system was being remodeled by the differentiating human MSCs. This study also demonstrated the application of a simple, sensitive, and nondestructive tool for monitoring the progression of chondrogenic differentiation of MSCs in tissue-engineered constructs and therefore contributes to future development of novel cartilage repair strategies.

The FASEB Journal, 2003

Grafting of Schwann cell-seeded channels into hemisected adult rat thoracic spinal cords has been... more Grafting of Schwann cell-seeded channels into hemisected adult rat thoracic spinal cords has been tested as a strategy to bridge the injured cord. Despite success in guiding axonal growth into the graft, regeneration across the distal graft−host interface into the host spinal cord was limited. We hypothesized that chondroitin sulfate (CS) glycoforms deposited at the gliotic front of the interface constitute a molecular barrier to axonal growth into the host cord. Because CS glycoforms deposited by purified astrocytes in vitro were removable by digestion with chondroitinase ABC, we attempted to achieve likewise by infusion of the enzyme to the host side of the interface. By 1 month post-treatment, significant numbers of regenerating axons crossed an interface that was subdued in macrophage/microglia reaction and decreased in CSimmunopositivity. The axons extended as far into the caudal cord as 5 mm, in contrast to nil in vehicle-infused controls. Fascicular organizations of axon−Schwann cell units within the regenerated tissue cable were better-preserved in enzyme-treated cords than in vehicle-infused controls. We conclude that CS glycoforms deposited during gliosis at the distal graft−host interface could be cleared by the in vivo action of chondroitinase ABC to improve prospects of axonal regeneration into the host spinal cord. Key Words: chondroitin sulfates • regeneration • transplantation • Schwann cells ridging the injured spinal cord with a cellular graft is a promising repair strategy to increase the propensity of regenerating axons across the lesion site. Schwann cells (SCs) (1-3), olfactory ensheathing cells (4, 5), peripheral nerve grafts (6, 7) and fetal tissue B

Respiratory Medicine, 2004

Persistent airway inflammation is an important pathogenetic factor in bronchiectasis, and interle... more Persistent airway inflammation is an important pathogenetic factor in bronchiectasis, and interleukin (IL)-6 is among the mediators implicated in regulation of inflammation in bronchiectatic airways. We postulated that airway secretion with its constituents of cytokines and enzymes would provide an environment for perpetuation of inflammation in vivo. We aimed to determine the action of sputum from patients with bronchiectasis on IL-6 production from cultured normal human bronchial epithelial (NHBE) cells and its modulation by antiinflammatory drugs in vitro. Cultures of NHBE cells were tested with (i) sputum of bronchiectatic patients, (ii) anti-tumor necrosis factor-alpha (TNF-a) pre-treated sputum, or (iii) recombinant human (rh)-TNF-a. Alternatively, NHBE cells were incubated with one of the antiinflammatory drugs before treatment with sputum or rh-TNF-a. IL-6 produced into the medium was assayed by ELISA. Sputum in bronchiectasis stimulated IL-6 production from NHBE cells by 1.9 times. This was largely attributable to TNF-a as pre-incubation of sputum sol with anti-TNFa almost neutralized the sputum effect. Apart from dexamethasone, the other drugs exerted inhibitory effects on IL-6 production. Ibuprofen suppressed sputumstimulated IL-6 production to levels above control and effect levelled off at 50-100 mg/ml, contrasting the dose-dependent suppression to control level with MK-663 (0.1-10 mg/ml) and to sub-control levels with triptolide (20-1000 ng/ml). Our results support that sputum in bronchiectasis can stimulate IL-6 production from NHBE cells, and TNF-a is an important cytokine mediating the process. The suppressive effects observed with ibuprofen, triptolide and MK-663 warrant further study.

Respiratory Medicine, 2008

The airway epithelium participates in chronic airway inflammation by expressing adhesion molecule... more The airway epithelium participates in chronic airway inflammation by expressing adhesion molecules that mediate the transmigration of neutrophils into the inflamed airways. We hypothesize that, in patients with bronchiectasis, cytokines in their bronchial secretions enhance the expression of intercellular cell adhesion molecule (ICAM-1) in the bronchial epithelium and thus contribute to sustained recruitment of neutrophils into the inflamed airways. In the present study, we investigated the effect of bronchial secretions on the regulation of ICAM-1 in bronchial epithelial cells, and its modulation by pharmacologic agents. The expression of ICAM-1 mRNA and protein in human bronchial epithelial cells upon exposure to sputum sol from subjects with bronchiectasis were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and ELISA, respectively. Modulating effects of dexamethasone, ibuprofen, MK-663 or triptolide on ICAM-1 regulation were investigated in vitro. We demonstrated that changes in ICAM-1 expression correlated with levels of TNF-a in the sputum sol, and treatment of sol samples with TNF-a antibodies attenuated both the increase in ICAM-1 mRNA and protein. The role of TNF-a was further demonstrated when TNF-a elicited dose dependent increase in ICAM-1 expression. The sputum effect could also be suppressed dose-dependently by pre-incubation of bronchial epithelial cells with dexamethasone, ibuprofen, MK-663 or triptolide. Evidence is thus provided for the upregulation of bronchial epithelial ICAM-1 expression by airway

Neuroscience Research, 2010

e343 results suggest that the cleavage of p35 to p25 by calpain is controlled by the amount of ca... more e343 results suggest that the cleavage of p35 to p25 by calpain is controlled by the amount of calpastatin in neurons. Interestingly, p35 was degraded without the cleavage to p25 in the Tg mouse brain extract when the extracts was incubated in the presence of Ca 2+. This degradation was suppressed by proteasome inhibitors but not other protease inhibitors. Incubation of the brain extract with Ca 2+ did not increase the proteasome activity but stimulated ubiquitination of p35. Together, these results suggest that Cdk5 activity is regulated by Ca 2+-induced and proteasome-dependent degradation of p35 in neurons.

Neuroscience Research, 2009

Chondroitin sulfate (CS) proteoglycans in the mammalian CNS have been implicated in cell prolifer... more Chondroitin sulfate (CS) proteoglycans in the mammalian CNS have been implicated in cell proliferation, migration, neurite outgrowth, and axonal pathfinding. We have been studying the role of CS moieties on the migration of motor neurons in the embryonic hindbrains of Sprague Dawley rats. To dissect the sulfated chondroitins expressed by motile cells as early as E11.5, we followed the emigration of cells from explant cultures of the hindbrain (r3–r5) under time-lapse video microscopy. The cell pattern was then processed for Isl-1 immunostaining and ISH for mRNAs of the chondroitin sulfotransferases. Most of the migrating cells (>90%) were Isl-1-positive and remained connected with each other in chain-like formations via filopodial extensions/retractions. With riboprobes generated for dermatan 4-O-sulfotransferase (D4ST) and chondroitin 6-O-sulfotransferase (C6ST), we found that 90% of Isl-1-positive cells expressed D4ST and subpopulations expressed C6ST. These results suggest that the balance of 4and 6-O-sulfation affects the motile behavior of Isl-1-positive motor neurons.

Neuroscience, 2006

Neurotrophins are known to promote survival after neural injury. To determine the relative import... more Neurotrophins are known to promote survival after neural injury. To determine the relative importance of tyrosine kinase receptors on the survival of axotomized trigeminal nuclear neurons, we examined the temporal expression profile of tyrosine kinase A, tyrosine kinase B and tyrosine kinase C receptors in the mesencephalic trigeminal nucleus and the motor trigeminal nucleus following transection of the masseteric nerve in rats. Axotomized neurons in these nuclei were retrogradely identified with FluoroGold. We found increase in tyrosine kinase A-immunoreactive mesencephalic trigeminal nucleus neurons in the second week after axotomy but no change in the number of tyrosine kinase A-immunoreactive motor trigeminal nucleus neurons. There was no change in the number of tyrosine kinase B-immunoreactive mesencephalic trigeminal nucleus neurons but the significant increase of tyrosine kinase B-immunoreactive motor trigeminal nucleus neurons throughout the period of observation (3 weeks) peaked at ϳ1 week after axotomy. There was no alteration in the number of tyrosine kinase C-immunoreactive mesencephalic trigeminal nucleus neurons but significant increase in tyrosine kinase C-immunoreactive motor trigeminal nucleus neurons observable by 4 days post-axotomy was followed by decline to levels lower than the control in 2 weeks. Temporal changes in the expression of individual tyrosine kinase receptors in mesencephalic trigeminal nucleus and motor trigeminal nucleus neurons following transection of the masseteric nerve suggest differential contribution of tyrosine kinase-specific neurotrophins to the survival of these neurons after axotomy.

Neuroscience, 1999

In decerebrate cats after acute hemilabyrinthectomy, the response sensitivity of extracellularly ... more In decerebrate cats after acute hemilabyrinthectomy, the response sensitivity of extracellularly recorded vestibular nuclear neurons on the lesioned and labyrinth-intact sides were examined quantitatively during constant velocity off-vertical axis rotations with an aim to elucidate the functional contribution of otolithic inputs to the ipsilateral and contralateral vestibular nuclei. The bidirectional response sensitivity, d, was determined as the ratio of the gain during clockwise to that during counterclockwise rotations. A continuum of response sensitivity was identified: one-dimensional neurons showed symmetrically bidirectional response patterns, while two-dimensional neurons showed asymmetrically bidirectional patterns that in some cases approached unidirectional patterns with change in velocity. The proportion of two-dimensional neurons was significantly increased after acute hemilabyrinthectomy. Two-dimensional neurons that responded only to one direction of rotation in at least one of the velocities tested were described as unidirectional neurons. This unidirectional response pattern was observed in one-third of the entire neuronal population studied, but not in cats with both labyrinths intact, thus suggesting that such prominent broadly tuned responses are normally masked by converging otolithic inputs from the contralateral side. These neurons were found in higher proportion on the lesioned side than on the labyrinth-intact side. Among the 70% of unidirectional neurons that exhibited bidirectional response at some velocities and unidirectional response at others, prominent shifts in d values (i.e. between 0/∞ and finite values) with velocity can be computed for each neuron. The shifts in d values correlated with large shifts in the response dynamics and spatial orientation as the response pattern changed with velocity. The response orientations of the unidirectional neurons pointed in all directions on the horizontal plane. When all the two-dimensional neurons (i.e. both the unidirectionally and bidirectionally responsive ones) were pooled, imbalances in the distribution of the response orientations and in response gain were found between the ipsilateral-sidedown/head-down half-circle and the contralateral-side-down/head-up half-circle on the labyrinth-intact side, but not on the lesioned side. These results, derived from spatiotemporal processing of gravitational signals, reveal a novel dimension of imbalance between neuronal populations in the two vestibular nuclear complexes after acute lesion of one labyrinth. This feature would provide, on the one hand, deranged cues of spatial orientation and direction during slow head excursions and, on the other, a framework for the dynamic behavioral deficits associated with hemilabyrinthectomy.

NeuroReport, 2003

The response properties of extracellularly recorded Y group neurons on the lesioned side were exa... more The response properties of extracellularly recorded Y group neurons on the lesioned side were examined in decerebrate cats after acute hemilabyrinthectomy, with the use of constant velocity off-vertical axis rotations (OVAR) to stimulate the remaining intact otolith receptors. During rotation in the clockwise or counterclockwise direction, Y group neurons displayed a spectrum of position-dependent bidirectional response sensitivities, ranging from one- to two-dimensional. Some two-dimensional neurons even exhibited unidirectional responses with change in OVAR velocity. These findings indicate that Y group neurons have the capacity to code spatiotemporal signals arising from the contralateral otolith. The best response orientations of one-dimensional and two-dimensional neurons were found predominantly along the antero-posterior direction, thus providing a spatial framework for the otolithic reflexes.

Neural Development, 2012

Background Establishing correct neuronal circuitry is crucial to proper function of the vertebrat... more Background Establishing correct neuronal circuitry is crucial to proper function of the vertebrate nervous system. The abundance of chondroitin sulfate (CS) proteoglycans in embryonic neural environments suggests that matrix proteoglycans regulate axonal projections when fiber tracts have not yet formed. Among the early-born neurons, the vestibular nucleus (VN) neurons initiate commissural projections soon after generation at E12.5 and reach the contralateral target by E15.5 in the rat hindbrain. We therefore exploited 24-hour cultures (1 day in vitro (DIV)) of the rat embryos and chondroitinase ABC treatment of the hindbrain matrix to reveal the role of CS moieties in axonal initiation and projection in the early hindbrain. Results DiI tracing from the VN at E12.5(+1 DIV) showed contralaterally projecting fibers assuming fascicles that hardly reached the midline in the controls. In the enzyme-treated embryos, the majority of fibers were unfasciculated as they crossed the midline at...

The Journal of Comparative Neurology, 2010

We examined the functional maturation of canal-related brainstem neurons in Sprague-Dawley rats a... more We examined the functional maturation of canal-related brainstem neurons in Sprague-Dawley rats at postnatal day (P)1 to adult. Conscious animals were subjected to cycles of angular acceleration and deceleration so as to selectively activate hair cells of the horizontal semicircular canals. Brainstem neurons were monitored for c-fos expression by immuno-hybridization histochemistry as an indicator of neuronal activation. Fos-immunoreactive canal-related neurons were identifiable from P4 onwards in the vestibular nucleus and downstream vestibular relay stations, prepositus hypoglossal nucleus, and inferior olive. In the vestibular nucleus and prepositus hypoglossal nucleus, the number of canal-related neurons increased progressively with age, reaching the adult level by P21. Those in the inferior olive increased in number from P4 to P14 but decreased significantly afterwards until adulthood. The topography was not clear in the vestibular nucleus and prepositus hypoglossal nucleus. Canal-related neurons in P4-7 rats were spread throughout the rostrocaudal length of each subnucleus but clusters of canal-related neurons tended to form within specific subnuclei by P21. These were concentrated in the caudal halves of medial and spinal vestibular nuclei and the rostral parts of superior vestibular nucleus and prepositus hypoglossal nucleus. In the inferior olive, the topography was evident early in the course of development. Canal-related neurons were exclusively located in four subnuclei: dorsal medial cell column, dorsal cap, subnucleus A, and subnucleus C, but not in other subnuclei. Taken together, our data revealed the developmental profile of neuronal subpopulations within the horizontal canal system, thus providing an internal neural representation for postnatal coding of horizontal head rotations in spatial perception.

Figure S4. Flow cytometry analysis of rat BMSCs. Rat BMSCs (A) between passage number 5 and 8 wer... more Figure S4. Flow cytometry analysis of rat BMSCs. Rat BMSCs (A) between passage number 5 and 8 were analyzed by flow cytometry. In all panels, isotype controls are represented by red lines, while immunopositivity for respective cell surface markers are represented by blue lines. Percentages of positive cells as shown within individual panels are representative of one sample. Immunopositivity for CD90, STRO-1, and CD73 in isolated rat BMSCs were 95.70 ± 1.21 %, 95.91 ± 1.86 %, and 92.80 ± 1.46 %, respectively (B–D). Immunopositivity for nestin was 11.31 ± 1.25 % (E). Immunopositivity towards the hematopoietic progenitor marker CD45 was negligible (F). Mean ± SD, n = 4. (PDF 288 kb)

Figure S2. Isotype controls for immunocytochemistry. (A, B) Mouse and rabbit isotype control anti... more Figure S2. Isotype controls for immunocytochemistry. (A, B) Mouse and rabbit isotype control antibody-stained neurospheres derived following normoxic and hypoxic treatment, respectively. (C, D) Mouse and rabbit isotype control antibody-stained SCLCs derived from normoxia- and hypoxia-treated BMSCs, respectively. (E, F) Mouse and rabbit isotype control antibody-stained fate-committed Schwann cells generated from normoxia- and hypoxia-treated BMSCs, respectively. (PDF 211 kb)

Figure S1. Work flow for the derivation of fate-committed Schwann cells from BMSCs. BMSCs isolate... more Figure S1. Work flow for the derivation of fate-committed Schwann cells from BMSCs. BMSCs isolated by means of adherence to tissue culture plastic were dissociated and maintained for 12 days in the presence of EGF/bFGF on low-attachment culture plates. Thereafter, resultant neurospheres were plated onto PDL/laminin-coated culture plates and differentiated into Schwann cell-like cells (SCLCs) in glial differentiation medium containing β-heregulin, bFGF, and PDGF for 7 days. In order to direct SCLCs to fate commitment, they were co-cultured with purified and partially dissociated dorsal root ganglia (DRG) neurons for a further 15 days. (PDF 159 kb)

Sheng li xue bao : [Acta physiologica Sinica], 2014

The capability of the central vestibular system in utilizing cues arising from the inner ear dete... more The capability of the central vestibular system in utilizing cues arising from the inner ear determines the ability of animals to acquire the sense of head orientations in the three-dimensional space and to shape postural movements. During development, neurons in the vestibular nucleus (VN) show significant changes in their electrophysiological properties. An age-dependent enhancement of membrane excitability is accompanied by a progressive increase in firing rate and discharge regularity. The coding of horizontal and vertical linear motions also exhibits developmental refinement in VN neurons. Further, modification of cell surface receptors, such as glutamate receptors, of developing VN neurons are well-orchestrated in the course of maturation, thereby regulating synaptic efficacy and spatial coding capacity of these neurons in local circuits. Taken together, these characteristic features of VN neurons contribute to developmental establishment of space-centered coordinates within t...

Sheng li xue bao : [Acta physiologica Sinica], 2014

The availability of human stem cells heralds a new era for in vitro cell-based modeling of neurod... more The availability of human stem cells heralds a new era for in vitro cell-based modeling of neurodevelopmental and neurodegenerative diseases. Adding to the excitement is the discovery that somatic cells of patients can be reprogrammed to a pluripotent state from which neural lineage cells that carry the disease genotype can be derived. These in vitro cell-based models of neurological diseases hold promise for monitoring of disease initiation and progression, and for testing of new drug treatments on the patient-derived cells. In this review, we focus on the prospective applications of different stem cell types for disease modeling and drug screening. We also highlight how the availability of patient-specific induced pluripotent stem cells (iPS cells) offers a unique opportunity for studying and modeling human neurodevelopmental and neurodegenerative diseases in vitro and for testing small molecules or other potential therapies for these disorders. Finally, the limitations of this te...

Development, 2000

The effects of removing chondroitin sulfate from chondroitin sulfate proteoglycan molecules on gu... more The effects of removing chondroitin sulfate from chondroitin sulfate proteoglycan molecules on guidance of retinal ganglion cell axons at the optic chiasm were investigated in a brain slice preparation of mouse embryos of embryonic day 13 to 15. Slices were grown for 5 hours and growth of dye-labeled axons was traced through the chiasm. After continuous enzymatic digestion of the chondroitin sulfate proteoglycans with chondroitinase ABC, which removes the glycosaminoglycan chains, navigation of retinal axons was disrupted. At embryonic day 13, before the uncrossed projection forms in normal development, many axons deviated from their normal course, crossing the midline at aberrant positions and invading the ventral diencephalon. In slices from embryonic day 14 embryos, axons that would normally form the uncrossed projection at this stage failed to turn into the ipsilateral optic tract. In embryonic day 15 slices, enzyme treatment caused a reduction of the uncrossed projection that d...

Neuroembryology and Aging, 2009

The expression patterns of the two subunits of the metabotropic GABAB receptor, GABABR1 and GABAB... more The expression patterns of the two subunits of the metabotropic GABAB receptor, GABABR1 and GABABR2, vary among various neural regions and their relative expression in the rat central vestibular system remains unknown. To identify the expression patterns of these two subunits of the metabotropic GABAB receptor in the vestibular nucleus of adult rats, double immunofluorescence experiments were performed. GABABR1 or GABABR2 immunoreactivity was observed in neurons and neuropilar elements of the vestibular nuclear complex and its subgroups x and y. These neurons were oval, multipolar, fusiform or triangular in shape. In contrast to other brain regions, individual neurons or neuropil within the various vestibular subnuclei and subgroups displayed comparable levels of GABABR1 and GABABR2 immunoreactivity. Double immunofluorescence experiments further demonstrated the coexpression of GABABR1 and GABABR2 receptor subunits in individual central vestibular neurons. The present findings provi...

Stem Cell Research & Therapy, 2016

Background Bone marrow stromal cells (BMSCs) are attractive as a source of neural progenitors for... more Background Bone marrow stromal cells (BMSCs) are attractive as a source of neural progenitors for ex vivo generation of neurons and glia. Limited numbers of this subpopulation, however, hinder translation into autologous cell-based therapy. Here, we demonstrate rapid and efficient conditioning with hypoxia to enrich for these neural progenitor cells prior to further expansion in neurosphere culture. Method Adherent cultures of BMSCs (rat/human) were subjected to 1 % oxygen for 24 h and then subcultured as neurospheres with epidermal growth factor (EGF) and basic fibroblast growth factor supplementation. Neurospheres and cell progeny were monitored immunocytochemically for marker expression. To generate Schwann cell-like cells, neurospheres were plated out and exposed to gliogenic medium. The resulting cells were co-cultured with purified dorsal root ganglia (rat) neurons and then tested for commitment to the Schwann cell fate. Fate-committed Schwann cells were subjected to in vitro ...

Urological Research, 1987

The inhibitory activity of urine on calcium oxalate crystallization was measured as an inhibitory... more The inhibitory activity of urine on calcium oxalate crystallization was measured as an inhibitory index using a gel model modified from Schneider et al. [8]. Urine sampies from 36 recurrent stone formers and 21 controls in 3 separate periods (morning, afternoon and evening) were tested. Mean inhibitory indices of > 0 were observed among normal controls in the 3 sampled periods and among stone formers in the morning and afternoon samples. A significantly lower (negative) value was observed in the evening samples of stone formers, indicating a higher tendency towards crystallization than normal. The gel method may be applied to identify individuals at risk of stone formation if sufficient numbers of period-defined urine samples from the individual are tested for a statistically significant mean inhibitory index but whether this is practical in a clinical laboratory will need further evaluations.

Tissue Engineering Part A, 2011

Mesenchymal stem cell (MSC)-based engineering is promising for cartilage repair. However, the com... more Mesenchymal stem cell (MSC)-based engineering is promising for cartilage repair. However, the compositional mechanical relationship of the engineered structures has not been extensively studied, given the importance of such relationship in native cartilage tissues. In this study, a novel human MSC-collagen microsphere system was used to study the compositional mechanical relationship during in vitro chondrogenic differentiation using histological and biochemical methods and a microplate compression assay. The mechanical property was found positively correlating with newly deposited cartilage-relevant matrices, glycosaminoglycan, and type II collagen, and with the collagen crosslinker density, in agreement with the presence of thick collagen bundles upon structural characterization. On the other hand, the mechanical property negatively correlates with type I collagen and total collagen, suggesting that the initial collagen matrix scaffold of the microsphere system was being remodeled by the differentiating human MSCs. This study also demonstrated the application of a simple, sensitive, and nondestructive tool for monitoring the progression of chondrogenic differentiation of MSCs in tissue-engineered constructs and therefore contributes to future development of novel cartilage repair strategies.

The FASEB Journal, 2003

Grafting of Schwann cell-seeded channels into hemisected adult rat thoracic spinal cords has been... more Grafting of Schwann cell-seeded channels into hemisected adult rat thoracic spinal cords has been tested as a strategy to bridge the injured cord. Despite success in guiding axonal growth into the graft, regeneration across the distal graft−host interface into the host spinal cord was limited. We hypothesized that chondroitin sulfate (CS) glycoforms deposited at the gliotic front of the interface constitute a molecular barrier to axonal growth into the host cord. Because CS glycoforms deposited by purified astrocytes in vitro were removable by digestion with chondroitinase ABC, we attempted to achieve likewise by infusion of the enzyme to the host side of the interface. By 1 month post-treatment, significant numbers of regenerating axons crossed an interface that was subdued in macrophage/microglia reaction and decreased in CSimmunopositivity. The axons extended as far into the caudal cord as 5 mm, in contrast to nil in vehicle-infused controls. Fascicular organizations of axon−Schwann cell units within the regenerated tissue cable were better-preserved in enzyme-treated cords than in vehicle-infused controls. We conclude that CS glycoforms deposited during gliosis at the distal graft−host interface could be cleared by the in vivo action of chondroitinase ABC to improve prospects of axonal regeneration into the host spinal cord. Key Words: chondroitin sulfates • regeneration • transplantation • Schwann cells ridging the injured spinal cord with a cellular graft is a promising repair strategy to increase the propensity of regenerating axons across the lesion site. Schwann cells (SCs) (1-3), olfactory ensheathing cells (4, 5), peripheral nerve grafts (6, 7) and fetal tissue B

Respiratory Medicine, 2004

Persistent airway inflammation is an important pathogenetic factor in bronchiectasis, and interle... more Persistent airway inflammation is an important pathogenetic factor in bronchiectasis, and interleukin (IL)-6 is among the mediators implicated in regulation of inflammation in bronchiectatic airways. We postulated that airway secretion with its constituents of cytokines and enzymes would provide an environment for perpetuation of inflammation in vivo. We aimed to determine the action of sputum from patients with bronchiectasis on IL-6 production from cultured normal human bronchial epithelial (NHBE) cells and its modulation by antiinflammatory drugs in vitro. Cultures of NHBE cells were tested with (i) sputum of bronchiectatic patients, (ii) anti-tumor necrosis factor-alpha (TNF-a) pre-treated sputum, or (iii) recombinant human (rh)-TNF-a. Alternatively, NHBE cells were incubated with one of the antiinflammatory drugs before treatment with sputum or rh-TNF-a. IL-6 produced into the medium was assayed by ELISA. Sputum in bronchiectasis stimulated IL-6 production from NHBE cells by 1.9 times. This was largely attributable to TNF-a as pre-incubation of sputum sol with anti-TNFa almost neutralized the sputum effect. Apart from dexamethasone, the other drugs exerted inhibitory effects on IL-6 production. Ibuprofen suppressed sputumstimulated IL-6 production to levels above control and effect levelled off at 50-100 mg/ml, contrasting the dose-dependent suppression to control level with MK-663 (0.1-10 mg/ml) and to sub-control levels with triptolide (20-1000 ng/ml). Our results support that sputum in bronchiectasis can stimulate IL-6 production from NHBE cells, and TNF-a is an important cytokine mediating the process. The suppressive effects observed with ibuprofen, triptolide and MK-663 warrant further study.

Respiratory Medicine, 2008

The airway epithelium participates in chronic airway inflammation by expressing adhesion molecule... more The airway epithelium participates in chronic airway inflammation by expressing adhesion molecules that mediate the transmigration of neutrophils into the inflamed airways. We hypothesize that, in patients with bronchiectasis, cytokines in their bronchial secretions enhance the expression of intercellular cell adhesion molecule (ICAM-1) in the bronchial epithelium and thus contribute to sustained recruitment of neutrophils into the inflamed airways. In the present study, we investigated the effect of bronchial secretions on the regulation of ICAM-1 in bronchial epithelial cells, and its modulation by pharmacologic agents. The expression of ICAM-1 mRNA and protein in human bronchial epithelial cells upon exposure to sputum sol from subjects with bronchiectasis were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and ELISA, respectively. Modulating effects of dexamethasone, ibuprofen, MK-663 or triptolide on ICAM-1 regulation were investigated in vitro. We demonstrated that changes in ICAM-1 expression correlated with levels of TNF-a in the sputum sol, and treatment of sol samples with TNF-a antibodies attenuated both the increase in ICAM-1 mRNA and protein. The role of TNF-a was further demonstrated when TNF-a elicited dose dependent increase in ICAM-1 expression. The sputum effect could also be suppressed dose-dependently by pre-incubation of bronchial epithelial cells with dexamethasone, ibuprofen, MK-663 or triptolide. Evidence is thus provided for the upregulation of bronchial epithelial ICAM-1 expression by airway

Neuroscience Research, 2010

e343 results suggest that the cleavage of p35 to p25 by calpain is controlled by the amount of ca... more e343 results suggest that the cleavage of p35 to p25 by calpain is controlled by the amount of calpastatin in neurons. Interestingly, p35 was degraded without the cleavage to p25 in the Tg mouse brain extract when the extracts was incubated in the presence of Ca 2+. This degradation was suppressed by proteasome inhibitors but not other protease inhibitors. Incubation of the brain extract with Ca 2+ did not increase the proteasome activity but stimulated ubiquitination of p35. Together, these results suggest that Cdk5 activity is regulated by Ca 2+-induced and proteasome-dependent degradation of p35 in neurons.

Neuroscience Research, 2009

Chondroitin sulfate (CS) proteoglycans in the mammalian CNS have been implicated in cell prolifer... more Chondroitin sulfate (CS) proteoglycans in the mammalian CNS have been implicated in cell proliferation, migration, neurite outgrowth, and axonal pathfinding. We have been studying the role of CS moieties on the migration of motor neurons in the embryonic hindbrains of Sprague Dawley rats. To dissect the sulfated chondroitins expressed by motile cells as early as E11.5, we followed the emigration of cells from explant cultures of the hindbrain (r3–r5) under time-lapse video microscopy. The cell pattern was then processed for Isl-1 immunostaining and ISH for mRNAs of the chondroitin sulfotransferases. Most of the migrating cells (>90%) were Isl-1-positive and remained connected with each other in chain-like formations via filopodial extensions/retractions. With riboprobes generated for dermatan 4-O-sulfotransferase (D4ST) and chondroitin 6-O-sulfotransferase (C6ST), we found that 90% of Isl-1-positive cells expressed D4ST and subpopulations expressed C6ST. These results suggest that the balance of 4and 6-O-sulfation affects the motile behavior of Isl-1-positive motor neurons.

Neuroscience, 2006

Neurotrophins are known to promote survival after neural injury. To determine the relative import... more Neurotrophins are known to promote survival after neural injury. To determine the relative importance of tyrosine kinase receptors on the survival of axotomized trigeminal nuclear neurons, we examined the temporal expression profile of tyrosine kinase A, tyrosine kinase B and tyrosine kinase C receptors in the mesencephalic trigeminal nucleus and the motor trigeminal nucleus following transection of the masseteric nerve in rats. Axotomized neurons in these nuclei were retrogradely identified with FluoroGold. We found increase in tyrosine kinase A-immunoreactive mesencephalic trigeminal nucleus neurons in the second week after axotomy but no change in the number of tyrosine kinase A-immunoreactive motor trigeminal nucleus neurons. There was no change in the number of tyrosine kinase B-immunoreactive mesencephalic trigeminal nucleus neurons but the significant increase of tyrosine kinase B-immunoreactive motor trigeminal nucleus neurons throughout the period of observation (3 weeks) peaked at ϳ1 week after axotomy. There was no alteration in the number of tyrosine kinase C-immunoreactive mesencephalic trigeminal nucleus neurons but significant increase in tyrosine kinase C-immunoreactive motor trigeminal nucleus neurons observable by 4 days post-axotomy was followed by decline to levels lower than the control in 2 weeks. Temporal changes in the expression of individual tyrosine kinase receptors in mesencephalic trigeminal nucleus and motor trigeminal nucleus neurons following transection of the masseteric nerve suggest differential contribution of tyrosine kinase-specific neurotrophins to the survival of these neurons after axotomy.

Neuroscience, 1999

In decerebrate cats after acute hemilabyrinthectomy, the response sensitivity of extracellularly ... more In decerebrate cats after acute hemilabyrinthectomy, the response sensitivity of extracellularly recorded vestibular nuclear neurons on the lesioned and labyrinth-intact sides were examined quantitatively during constant velocity off-vertical axis rotations with an aim to elucidate the functional contribution of otolithic inputs to the ipsilateral and contralateral vestibular nuclei. The bidirectional response sensitivity, d, was determined as the ratio of the gain during clockwise to that during counterclockwise rotations. A continuum of response sensitivity was identified: one-dimensional neurons showed symmetrically bidirectional response patterns, while two-dimensional neurons showed asymmetrically bidirectional patterns that in some cases approached unidirectional patterns with change in velocity. The proportion of two-dimensional neurons was significantly increased after acute hemilabyrinthectomy. Two-dimensional neurons that responded only to one direction of rotation in at least one of the velocities tested were described as unidirectional neurons. This unidirectional response pattern was observed in one-third of the entire neuronal population studied, but not in cats with both labyrinths intact, thus suggesting that such prominent broadly tuned responses are normally masked by converging otolithic inputs from the contralateral side. These neurons were found in higher proportion on the lesioned side than on the labyrinth-intact side. Among the 70% of unidirectional neurons that exhibited bidirectional response at some velocities and unidirectional response at others, prominent shifts in d values (i.e. between 0/∞ and finite values) with velocity can be computed for each neuron. The shifts in d values correlated with large shifts in the response dynamics and spatial orientation as the response pattern changed with velocity. The response orientations of the unidirectional neurons pointed in all directions on the horizontal plane. When all the two-dimensional neurons (i.e. both the unidirectionally and bidirectionally responsive ones) were pooled, imbalances in the distribution of the response orientations and in response gain were found between the ipsilateral-sidedown/head-down half-circle and the contralateral-side-down/head-up half-circle on the labyrinth-intact side, but not on the lesioned side. These results, derived from spatiotemporal processing of gravitational signals, reveal a novel dimension of imbalance between neuronal populations in the two vestibular nuclear complexes after acute lesion of one labyrinth. This feature would provide, on the one hand, deranged cues of spatial orientation and direction during slow head excursions and, on the other, a framework for the dynamic behavioral deficits associated with hemilabyrinthectomy.

NeuroReport, 2003

The response properties of extracellularly recorded Y group neurons on the lesioned side were exa... more The response properties of extracellularly recorded Y group neurons on the lesioned side were examined in decerebrate cats after acute hemilabyrinthectomy, with the use of constant velocity off-vertical axis rotations (OVAR) to stimulate the remaining intact otolith receptors. During rotation in the clockwise or counterclockwise direction, Y group neurons displayed a spectrum of position-dependent bidirectional response sensitivities, ranging from one- to two-dimensional. Some two-dimensional neurons even exhibited unidirectional responses with change in OVAR velocity. These findings indicate that Y group neurons have the capacity to code spatiotemporal signals arising from the contralateral otolith. The best response orientations of one-dimensional and two-dimensional neurons were found predominantly along the antero-posterior direction, thus providing a spatial framework for the otolithic reflexes.

Neural Development, 2012

Background Establishing correct neuronal circuitry is crucial to proper function of the vertebrat... more Background Establishing correct neuronal circuitry is crucial to proper function of the vertebrate nervous system. The abundance of chondroitin sulfate (CS) proteoglycans in embryonic neural environments suggests that matrix proteoglycans regulate axonal projections when fiber tracts have not yet formed. Among the early-born neurons, the vestibular nucleus (VN) neurons initiate commissural projections soon after generation at E12.5 and reach the contralateral target by E15.5 in the rat hindbrain. We therefore exploited 24-hour cultures (1 day in vitro (DIV)) of the rat embryos and chondroitinase ABC treatment of the hindbrain matrix to reveal the role of CS moieties in axonal initiation and projection in the early hindbrain. Results DiI tracing from the VN at E12.5(+1 DIV) showed contralaterally projecting fibers assuming fascicles that hardly reached the midline in the controls. In the enzyme-treated embryos, the majority of fibers were unfasciculated as they crossed the midline at...

The Journal of Comparative Neurology, 2010

We examined the functional maturation of canal-related brainstem neurons in Sprague-Dawley rats a... more We examined the functional maturation of canal-related brainstem neurons in Sprague-Dawley rats at postnatal day (P)1 to adult. Conscious animals were subjected to cycles of angular acceleration and deceleration so as to selectively activate hair cells of the horizontal semicircular canals. Brainstem neurons were monitored for c-fos expression by immuno-hybridization histochemistry as an indicator of neuronal activation. Fos-immunoreactive canal-related neurons were identifiable from P4 onwards in the vestibular nucleus and downstream vestibular relay stations, prepositus hypoglossal nucleus, and inferior olive. In the vestibular nucleus and prepositus hypoglossal nucleus, the number of canal-related neurons increased progressively with age, reaching the adult level by P21. Those in the inferior olive increased in number from P4 to P14 but decreased significantly afterwards until adulthood. The topography was not clear in the vestibular nucleus and prepositus hypoglossal nucleus. Canal-related neurons in P4-7 rats were spread throughout the rostrocaudal length of each subnucleus but clusters of canal-related neurons tended to form within specific subnuclei by P21. These were concentrated in the caudal halves of medial and spinal vestibular nuclei and the rostral parts of superior vestibular nucleus and prepositus hypoglossal nucleus. In the inferior olive, the topography was evident early in the course of development. Canal-related neurons were exclusively located in four subnuclei: dorsal medial cell column, dorsal cap, subnucleus A, and subnucleus C, but not in other subnuclei. Taken together, our data revealed the developmental profile of neuronal subpopulations within the horizontal canal system, thus providing an internal neural representation for postnatal coding of horizontal head rotations in spatial perception.