Over-Expression of hNGF in Adult Human Olfactory Bulb Neural Stem Cells Promotes Cell Growth and Oligodendrocytic Differentiation

The adult human olfactory bulb neural stem/progenitor cells (OBNC/PC) are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, however effective delivery of NGF into the CNS parenchyma is still challenging due mainly to its limited ability to cross the blood–brain barrier, and intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS is the genetic modification of NSC to overexpress NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation nature, and thus might enhance their therapeutic potential. In this study, we genetically modified adult human OBNS/PC to overexpress human NGF (hNGF) and green fluorescent protein (GFP) genes to provide insight about the effects of hNGF and GFP genes overexpression in adult human OBNS/PC on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB) protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PC-GFP-hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significant faster growth rate and up modulated the expression of oligodendroglia precursor cells markers (PDGFRα, NG2 and CNPase) respect to OBNS/PC-GFP counterparts. These findings suggest an enhanced proliferation and oligodendrocytic differentiation potential for OBNS/PC-GFP-hNGF as compared to OBNS/PC-GFP.

成人嗅球神经干细胞/前体细胞（olfactory bulb neural stem/progenitor cells，OBNC/PC）是治疗创伤性与神经退行性损伤的极具潜力的细胞治疗候选细胞。有研究提出，外源性给予神经生长因子（nerve growth factor，NGF）是治疗创伤性与神经退行性疾病的潜在有效策略，但由于NGF难以穿过血脑屏障（blood-brain barrier），且若经脑室给药会引发难以耐受的副作用，将其有效递送至中枢神经系统（central nervous system，CNS）实质仍面临诸多挑战。确保NGF递送至CNS实质的有效手段，是对神经干细胞（neural stem cell，NSC）进行基因修饰以过表达NGF基因。在成人嗅球神经干细胞/前体细胞中过表达NGF，有望改变其增殖与分化特性，进而提升其治疗潜力。本研究中，我们对成人嗅球神经干细胞/前体细胞（OBNS/PC）进行基因修饰，使其过表达人神经生长因子（human NGF，hNGF）与绿色荧光蛋白（green fluorescent protein，GFP）基因，并采用DNA微阵列（DNA microarray）、免疫表型分析（immunophenotyping）及蛋白质印迹（Western blot，WB）技术，探究hNGF与GFP基因过表达对成人OBNS/PC体外多向分化潜能的影响。分析结果显示，OBNS/PC-GFP与OBNS/PC-GFP-hNGF的分化是一个多维度的过程，涉及主要生物学过程的改变，具体表现为细胞周期与存活标志物、干性标志物及分化标志物等关键标志物的基因表达水平发生变化。通过基因本体（Gene Ontology，GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）分析可知，两类细胞的分化还伴随关键信号通路的调控：OBNS/PC-GFP涉及丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路、ErbB信号通路及神经活性配体-受体相互作用通路；OBNS/PC-GFP-hNGF则涉及轴突导向、电压依赖性钙通道γ亚基7相关通路。与OBNS/PC-GFP对照组相比，分化后的OBNS/PC-GFP-hNGF细胞展现出广泛分支的胞质突起、显著更快的生长速率，且上调了少突胶质前体细胞标志物（PDGFRα、NG2及CNPase）的表达。上述结果表明，相较于OBNS/PC-GFP细胞，OBNS/PC-GFP-hNGF细胞的增殖与少突胶质细胞分化潜能得到了显著增强。