[Isolation, culture, and identification of human spermatogonial stem cells].

OBJECTIVE: To isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application. METHODS: We detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro. RESULTS: The isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity. CONCLUSION: CD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.

Fibroblast growth factor 4 is required but not sufficient for the astrocyte dedifferentiation.

Our recent studies demonstrated that mature astrocytes from spinal cord can be reprogrammed in vitro and in vivo to generate neural stem/progenitor cells (NSPCs) following treatment with conditioned medium collected from mechanically injured astrocytes. However, little is known regarding the molecular mechanisms underlying the reprogramming of astrocytes. Here, we show that fibroblast growth factor 4 (FGF4) exerts a critical role in synergistically converting astrocytes into NSPCs that can express multiple neural stem cell markers (nestin and CD133) and are capable of both self-renewal and differentiation into neurons and glia. Lack of FGF4 signals fails to elicit the dedifferentiation of astrocytes towards NSPCs, displaying a substantially lower efficiency in the reprogramming of astrocytes and a slower transition through fate-determined state. These astrocyte-derived NSPCs displayed relatively poor self-renewal and multipotency. More importantly, further investigation suggested that FGF4 is a key molecule necessary for activating PI3K/Akt/p21 signaling cascades, as well as their downstream effectors responsible for directing cell reprogramming towards NSPCs. Collectively, these findings provide a molecular basis for astrocyte dedifferentiation into NSPCs after central nervous system (CNS) injury and imply that FGF4 may be a clinically applicable molecule for in situ neural repair in the CNS disorders.

Involvement of α7 nAChR signaling cascade in epigallocatechin gallate suppression of ß-amyloid-induced apoptotic cortical neuronal insults.

Excessive generation and accumulation of the ß-amyloid (Aß) peptide in selectively vulnerable brain regions is a key pathogenic event in the Alzheimer's disease (AD), while epigallocatechin gallate (EGCG) is a very promising chemical to suppress a variety of Aß-induced neurodegenerative disorders. However, the precise molecular mechanism of EGCG responsible for protection against neurotoxicity still remains elusive. To validate and further investigate the possible mechanism involved, we explored whether EGCG neuroprotection against neurotoxicity of Aß is mediated through the α7 nicotinic acetylcholine receptor (α7 nAChR) signaling cascade. It was shown in rat primary cortical neurons that short-term treatment with EGCG significantly attenuated the neurotoxicity of Aß1-42, as demonstrated by increased cell viability, reduced number of apoptotic cells, decreased reactive oxygen species (ROS) generation, and downregulated caspase-3 levels after treatment with 25-µM Aß1-42. In addition, EGCG markedly strengthened activation of α7nAChR as well as its downstream pathway signaling molecules phosphatidylinositol 3-kinase (PI3K) and Akt, subsequently leading to suppression of Bcl-2 downregulation in Aß-treated neurons. Conversely, administration of α7nAChR antagonist methyllycaconitine (MLA; 20 µM) to neuronal cultures significantly attenuated the neuroprotection of EGCG against Aß-induced neurototoxicity, thus presenting new evidence that the α7nAChR activity together with PI3K/Akt transduction signaling may contribute to the molecular mechanism underlying the neuroprotective effects of EGCG against Aß-induced cell death.