Comparison of the proteome patterns of adipose-derived stem cells with those treated with selegiline using a two dimensional gel electrophoresis analysis.

Adipose derived stem cells (ADSCs) are multipotent and can transdifferentiate into neural stem cells. We investigated the transdifferentiation of ADSCs to neural phenotype (NP) cells using selegiline and two-dimensional electrophoresis (2-DE). The perinephric and inguinal fat of rats was collected and used to isolate ADSCs that were characterized by immunophenotyping using flow cytometry. The ADSCs were differentiated into osteogenic and lipogenic cells. The NP cells were generated using 10-9 mM selegiline and characterized by immunocytochemical staining of nestin and neurofilament 68 (NF-68), and by qRT-PCR of nestin, neurod1 and NF68. Total protein of ADSCs and NP cells was extracted and their proteome patterns were examined using 2-DE. ADSCs carried CD73, CD44 and CD90 cell markers, but not CD34. ADSCs were differentiated into osteocyte and adipocyte lineages. The differentiated NP cells expressed nestin, neuro d1 and NF-68. The proteome pattern of ADSCs was compared with that of NP cells and eight spots showed more than a two fold increase in protein expression. The molecular weights and isoelectric points of these highly expressed proteins were estimated using Melanie software. We compared these results with those of the mouse proteomic database using the protein isoelectric point database, and the functions of the eight proteins in differentiation of NP cells were predicted using the UniProt database. The probable identities of the proteins that showed higher expression in NP cells included cholinesterase, GFRa2, protein kinase C (PKC-eta) and RING finger protein 121. The sequences of the proteins identified from mouse database were aligned by comparing them with similar proteins in rat database using the Basic Local Alignment Search Tool (BLAST). The E values of all aligned proteins were zero, which indicates consistency of the matched protein. These proteins participate in differentiation of the neuron and their overexpression causes ADSCs transdifferentiation into NP cells.

Immunocytochemical analysis of valproic acid induced histone H3 and H4 acetylation during differentiation of rat adipose derived stem cells into neuron-like cells.

Valproic acid (VPA) is an inhibitor of histone deacetylases (HDACs) that can regulate differentiation and proliferation of stem cells by epigenetic mechanisms. We investigated VPA induced histone H3 and H4 acetylation in adipose derived stem cells (ADSCs) transdifferentiated into neuron-like cells (NLCs). Rat ADSCs were transdifferentiated into neural stem cells (NSCs) that had been generated from neurospheres. The NSCs were differentiated into NLCs by induction with different concentrations of VPA at 24, 48 and 72 h. The NLCs were evaluated using anti-H3 and -H4 antibodies, and ADSCs, NSCs and NLCs were evaluated using immunofluorescence. The ADSCs were immunoreactive to CD90 and CD49d, but not to CD45 and CD31. Both the neurospheres and NSCs were immunostained with nestin and neurofilament 68. The neurospheres expressed Musashi1, Sox2 and Neu N genes as determined by RT-PCR. Our dose-response study indicated that the optimal concentration of VPA was 1 mM at 72 h. Histone acetylation levels of H3 and H4 immunostaining intensities in NLCs were significantly greater than for ADSCs and NSCs. VPA alters H4 and H3 acetylation immunoreactivities of ADSCs transdifferentiated into NLCs.

Oligoprogenitor cells derived from spermatogonia stem cells improve remyelination in demyelination model.

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.

In vitro maturation of fresh and frozen-thawed mouse round spermatids.

Both initiation and maintenance of spermatogenesis are hormonally regulated by follicle stimulating hormone (rFSH) and testosterone. Co-culture systems also have important roles in the maintenance of spermatogenic cells. In this study, the effects of FSH and testosterone, co-culture system with Vero cells and co-culture supplemented with the hormones for maturation of frozen-thawed spermatids were determined. Testicular cells were suspended from the testis of National Medical Research Institute (NMRI) male mice and divided into two parts. The first aliquot of suspension was allocated for using as fresh and the rest was quickly cryopreserved. The frozen specimens were thawed and washed using Dulbecco modified Eagle's minimum essential medium (DMEM) medium. The fresh specimens were cultured in four groups: control (cultured on DMEM with 10% FBS), hormone (cultured on a medium supplemented with rFSH and testosterone), co-culture (cultured on Vero cells) and co-culture + hormone (cultured on Vero cells combined with rFSH and testosterone). The frozen-thawed specimens were cultured accordingly. The number of spermatids was recorded daily and the survival rates of each group were evaluated using Trypan blue test. The results showed that the number of the elongating spermatids was increased during the first day of the culture of fresh hormone, co-culture and co-culture + hormone groups. Viability rates of all kinds of the spermatid reduced during the 96 h of culturing. Our findings showed that the addition of hormone could support cell viability better than the co-culture. They also confirmed that the fresh round spermatid cells can progress into elongating and elongated spermatid only within the first 2 days of the culture in hormone, co-culture and co-culture + hormone groups. In the frozen-thawed specimens no extra significant increase in the number of cells was observed.

Astrogliosis in different zones of the spinal cord gray matter after sciatic nerve axotomy in the newborn rat: a morphometric and immunohistochemical study.

Astrocytic response following unilateral sciatic nerve axotomy was examined in the spinal gray matter of newborn rats. Using an antiserum to glial fibrillary acidic protein (GFAP), immunoreactive astrocytes were studied in the ventral, dorsal and transitional region between the dorsal and ventral gray matters (TDVG) at intervals of one day, one week, two weeks and one month postaxotomy. The axotomized side showed an obvious increase in the number of immunoreactive astrocytes at one week, two weeks and one month after surgery. The numerical density per area of the glial cells (N(a)) was determined in all groups on both the intact and axotomized sides, and it increased in all groups at the axotomized sides. The percentage of glial cell increase (Pgi) was also determined. At the ventral horn Pgi increased at day one and continued to increase in all groups, while the increase in TDVG and the dorsal horn occurred at later time points. The total motoneuron count in the ventral horn at the axotomized and intact sides was done at all time points, and the percentage of motoneuron reduction (Pmr) was calculated, the highest Pmr being noticed at one month (41%). A nonlinear regression for Pmr and Pgi showed that the rate of Pgi was approximately double that of Pmr. The rate of glial cell increase at each time point (one day, one week, two weeks and one month groups) was calculated, and the highest rate of glial cell increase in the ventral horn occurred one week after axotomy, while the highest rate in the dorsal horn and TDVG occurred at the second week. The conclusion of the study is that there may be an initial post-axotomic proliferative phase of the glial cells, which was followed by a differentiation phase. Also a gradient of an increase in the rate glial cell proliferation was noticed from the ventral horn toward the dorsal horn, maybe due to stimulation by a paracrine factor.

Sciatic nerve transection in neonatal rats induces apoptotic neuronal death in L5 dorsal root ganglion.

Transection of a peripheral nerve in neonatal rats induces death of the axotomized neurons which may be due to either necrosis or apoptosis. In the present investigation, neuronal cell death in L5 dorsal root ganglion was evaluated after unilateral sciatic nerve transection in rats at 1, 3, 5, 7 and 10 days age. After 5 days, right (experimental) and left (control) dorsal root ganglia in all groups were removed, fixed, processed and embedded for either light or electron microscopy. Normal nucleoli were counted in paraffin embedded serial sections, and correction factors for split and multiple nucleoli were applied as well as the physical disector. The number of neurons in the right dorsal root ganglia, as compared with the controls, was significantly lower in all groups, and the percentage of the reduction at 1, 3, 5, 7 and 10 days was 32.4, 27.2, 23.8, 22.8 and 21.8% respectively. On the other hand, the results of neuronal counts using the disector method showed 34.0, 25.7, 20.2, 20.0 and 14.2% reduction in the number of neurons at 1, 3, 5, 7 and 10 days, respectively. The microscopic and ultrastructural results indicated that there were typical morphological changes similar to those of apoptosis, including condensed basophilic nuclei, formation of nuclear caps, cell shrinkage and apoptotic body formation. We concluded that there is an increase in apoptosis in dorsal root ganglia following sciatic nerve axotomy with the greatest neuronal loss on postnatal day 1.

Ultrastructure of apoptotic oligodendrocytes in the spinal cord of adult rat with long-standing axotomised sciatic nerve.

The apoptosis in the oligodendrocytes was evaluated in adult rat which were axotomised and sacrificed three months aftersurgery. The apoptotic cell had electron dense organelles with condensed cytoplasm, the nucleus showed chromatin condensation with formation of band-like structure of clumped chromatin, and subsequently the cell underwent progressive shrinkage. The advanced stage of apoptosis showed electron lucency in the chromatin band, followed by disintegration of the nucleus, while the cytoplasm was replaced with high electron dense granular substance forming round electron dense apoptotic cells. The autophagy was also noticed in the cytoplasm of these cells, consisting of membrane bounded cytoplasmic organelles. Because the autophagy was seen in the apoptotic cell, this indicates that the autophagy is not limited to autophagic type programmed cell death.