Expression of Tubulin Beta II in Neural Stem/Progenitor Cells and Radial Fibers During Human Fetal Brain Development

Abstract

SUMMARY:Recent studies revealed that the "radial glia" in fetal rodent brains are dividing neuronal precursor cells. However, in fetal primate brains, this issue remains unclear, with previous reports indicating that radial glia are a specialized form of astroglia. To investigate the relationship between radial fibers (RFs) and neural stem/progenitor cells in the fetal human brain, we generated polyclonal antibodies to human nestin protein and developed a new mAb, KNY-379, by screening for antibodies that immunostained RFs on paraffin-embedded human fetal brain specimens (12 gestational weeks). The immunostaining for KNY-379 antigen and nestin was seen over the RFs in brains at 8 gestational weeks. Furthermore, KNY-379 antigen and nestin were also detected in human neural stem/progenitor cells in neurosphere cultures. At 12 to 15 gestational weeks, the KNY-379 immunostaining of RFs remained in the periventricular zone and the deep part of the intermediate zone, but it also appeared in outgrowing axons in the cortical plate, in the superficial portion of the intermediate zone, and in apical dendrites in the molecular layer. In the later stages of fetal development (18 -40 gestational weeks), this antigen remained in the outgrowing axons and dendrites, but was no longer associated with RFs. Expression cloning and immunoblot analysis demonstrated the antigen to be tubulin beta II, which would thus be a good marker for studying RFs and neural stem/progenitor cells in the early developing human brain. (Lab Invest 2003, 83:479 -489).

Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells

“…In the microarray data, Retinol binding Protein 1 (RBP1, gene encoding the carrier protein for transport of vitamin A) which is necessary for growth and differentiation of epithelial tissues was 3.9-fold increased in fetal dermal skin cells and confirmation with RT-PCR at 6.45-fold higher expression. Others such as Tubulin beta 2B (up-regulated at 1.58) have been shown to be highly present in fetal brain development (Nakamura et al 2003). Other studies emphasize that wound healing is regulated by many cytokines, growth factors and their receptors (Constant et al, 1997;Peled et al, 2001;Werner and Grose, 2003;Yang et al, 2003) and it is suggested that efficient wound healing in fetal skin at early gestation is a result of the unique cytokine or growth factor profile.…”

Transfection with <i>pax6</i> Gene of Mouse Embryonic Stem Cells and Subsequent Cell Cloning Induced Retinal Neuron Progenitors, Including Retinal Ganglion Cell-Like Cells, in vitro

“…Cells cultured for 4 days in DMEM/F12 medium with N2 supplement and fibronectin (differentiation medium) expressed neural cell-associated mRNA, TUB [31] , neurofilament middle chain (NFM) [32] and ␤ -catenin ( fig. 2 A) [33] .…”

D2-40 antibody immunoreactivity in developing human brain, brain tumors and cultured neural cells

“…The differentiated cells were also fixed in PBS containing 4% PHA for 20 min at room temperature. Frozen sections of neurospheres were incubated with two primary antibodies to nestin (rabbit polyclonal; 1:500) 14 and D2-40 in PBS/0.1% Triton-X/10% normal goat serum at 41C overnight. Nuclei were stained with TO-PRO-3 s (Molecular Probes Inc., Eugene, OR, USA).…”

“…Cell suspensions were grown using the neurosphere method 16 in a defined DMEM/F-12 (1:1)-based growth medium supplemented with human recombinant (hr-) epidermal growth factor (20 ng/ml, Invitrogen), hr-fibroblast growth factor 2 (20 ng/ml, PeproTech Inc, Rocky Hill, NJ, USA), hr-LIF(10ng/ml, Chemicon), heparin (5 mg/ml, Sigma, St Louis, MS, USA), B27 supplement (Invitrogen), 15 mM HEPES as previously reported. 14,16,17 To induce differentiation, neurospheres were plated on poly-ornithine-coated glass coverslips and cultured in the medium with all D2-40 in brain Y Nakamura et al three growth factors withdrawn and supplemented with 1% FBS. Cells were cultured for 14 days before fixation for immunocytochemistry.…”

“…In the search for immunohistochemical markers of the developing human brain, we raised several novel antibodies such as tubulin beta II as a neural stem cell and neuronal cell lineage marker, 14 and KIAA0864 protein as a Purkinje cell and its dendrite/axon marker. 15 Several commercially available antibodies such as nestin, vimentin and GFAP were also evaluated.…”