英文假说

Regulation of NPC Differentiation from Bone Marrow StromalCells by Construction of Novel Inducing SystemAbstract:Neural precursor cells (NPC) exploited as “seed cells” in tissue engineering are the perfect cell source for the treatment of degenerative diseases and trauma of central nervous system. However, these cells are somewhat limited in their utility due to source-related ethics or legislations, and the diffculties in acquiring from autologous tissue, suggesting discovery and development of novel approach that could obtain sufficient NPCs are needed. In recent years, the utility of bone marrow derived mesenchymal stem cells (BMSCs) to differentiate into neural cells have become a potential method for NPCs acquisition. Increasing research trend to utilize growth factors (GFs) as an inducer to evoke BMSCs for neural differentiation, however the related differetiation mechanisms induced by GFs are still largely unclear. Recently, many evidences have proved that PI3K/AKT signaling pathway was involved in neural differentiation of BMSCs. In addition, it has also been demonstrated that GFs play an important role in the activation of PI3K/AKT signaling pathway. Therefore we propose a hypothesis that mechanisms involved in the differetiation of MSCs into neuron induced by GFs is meditated by its subsequent induction of PI3K/AKT signaling pathway. Basis on the mechanism, we could attempt to utilize neuroblastoma cells (NBCs) as a potential GFs pool for the constant secrection of many GFs, which may jointly activte this signaling pathway and efficiently promote the neural differentiation of MSCs. On the other hand, bFGF, one type of GFs, is demonstrated to be able to prolong the transformation of NPCs into mature neural cells. To better use these NBCs and bFGF, we could utilize the technique of multilayered alginate microcapsules to make the coalition of them for the NPC differentiation of BMSCs. Overall, we hypothesis that NBCs microcapsulated in multilayeredalginate microcapsules with bFGF supplement could efficiently induce the differentiation of MSCs into NPCs through PI3K/AKT signaling pathway activated by the GFs. If possible, this hypothesis might open an attractive approach for clinical NPC transfortation to boost neuron regeneration and synapse reconstruction, and provide the relevant theoretical basis for treatment of central nervous system diseases.IntruductionNeuro-degenerative diseases and central nervous system trauma have been reported as one of the leading causes of disability or mortality. The limited potentials of central nervous system in nerve regeneration and function reconstruction is currently a hard nut to crack for the treatment of these diseases. Transplantion of stem cells, particularly neural precursor cells(NPCs)/ neural stem cells(NSCs) have been proposed as a promising therapeutic strategy for neuron repairation [1]. Plenty of studies identifies that NPCs could facilitate CNS regeneration due to their differentiation plasticity, which might lead to the substitution of dead cells and functional integration [2-3]. It is also proved that NPCs possess the capacity to release neuroprotective factors which are capable of stimulating endogenous regeneration[4-5]. However, the reliable source of NPCs is one of the most challenging technical issues. In recent years, the differentiation of BMSCs into neural cells has become a focus of current tissue engineering researches [6-8]. BMSCs that are capable of self-renewal [9-10] have been widely demonstrated to possess the capacity of neural differentiation in the presence of GFs [11-12]. In addition, some literatures confirmed that MSCs can be converted into NSCs/NPCs in vitro under the multi-cell factors [13-14]. However, themechanisms involved in the differetiation of MSC into neuron induced by GFs is still unclear. On the other hand, although MSCs have the ability to differentiate into nerve cells, the differentiation rates were relatively low in most experiments, especially, the directed differentiation rate of NPC. Therefore this paper could provide some correlative hypothesis to discuss these issues.1. The effect of growth factors on MSCs differetiation is associated withactivation of PI3k/AKT signaling pathwayRecently, various data suggested that several intracellular signal transduction pathways including PI3K/AKT, cAMP-PKA and JAK-STAT3 signaling pathway could trigger and control the neuronal differentiation of MSCs [15-17]. Particularly, PI3K/AKT signaling has been revealed to possess the addition action of regulating neurite development and neuroprotection [18-20]. Therefore, we focused on the relationship between PI3K/AKT signaling pathway and neuronal differetiation of MSC.PI3K( phosphoinositol-3-kinases), a family of kinases, could generate PIP2 into PIP3 (phosphoinositide-3, 4,5 -triphosphate), which is a second messenger that activates many target molecules including AKT/PKB protein. PI3K/AKT signaling pathway has been shown to contribute to the regulation of BMSCs differentiation through up-regulation of proteins that mediate cell survival such as mammalian target of rapamycin (mTOR) [21]. In addition, it is reported that the activity of PI3K/AKT signalling pathway could be evoked by various GFs such as platelet-derived growth factor (PDGF), Epidermal Growth Factor (EGF), basicfibroblast growth factor (bFGF), and VEGF [19-22]. These findings, together with the observations from Hermann et al [13] and Hu Wei et al[14] that testified the indispensable role of multi-GFs in NPCs/NSCs differentiation from MSCs. Hence, we hypothesis that the mechanisms involved in the differentiation of MSCs into neuron induced by GFs is mediated by its subsequent induction of PI3K/AKT signaling pathway. Base on the mechanisms, maybe we could utilize the superiority of immortal cell lines such as NBCs to continuously secrete many GFs in Micro-environment，the alliance of GFs jointly activte this signaling pathway and contribute to the growth differentiation of BMSCs into neural cells.2. The alliance of GFs secreted by NBCs are invoved in regulation of MSCs differentiationNeuroblastoma is a kind of neuroendocrine tumor, which derived from the original pluripotent sympathetic neurocyte in the neural crest. It has been shown that various growth factors are expressed in NBCs. These factors such as VEGF-B, VEGF-C, bFGF, PDGF play an important role in cell growth, progression and metastasis of NB [23-24], due to their effect of regulating cell proliferation and differentiation.The role of conditioned medium (CM) from NBCs in the modification of cell differentiation become an emerging focus in resent years. Fuetal. et al have demonstrated that the differetiation of oligodendrocyteprecursor cells could be available by inducing neural stem cells in the presence of conditioned medium(CM) obtained from B104 neuroblastoma cells (B104-CM), bFGF and PDGF-AA in B104CM are 2 key factors that stimulate oligodendrocyte precursor cell (OPC)proliferation and differentiation [25]. Moreover, DEBORAL.et al have shown that conditioned medium(CM) of B104 neuroblastoma cells was capable of inducing differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) to a neuronal phenotype[26]. Thus, above studies some extent confirmed that the combined effects of multiple GFs secreted by NBCs could trigger and promote the neural differentiation.3. The tissue engineering technology of cells microcapsulationBased on the potential of conditioned medium (CM) from NBCs to induce neural differentiation, we suggest an approach to obtain sufficient and constant GFs from conditioned medium (CM) of NBCs is needed. Cells microencapsulation as a promising strategy have achieved important progress for clinical treatment. Cell encapsulation technology, as the name implies, is an approach that immobilizate cells inside a semipermeable membrane. It provides the unique cultivation of 3D environment and promotes the passage of oxygen, therapeutic proteins, nutrients, growth factors due to its thin membrane and relatively high ratio of surface-to-volume [27]. Cell encapsulation can also protect the cell from the host’s immune system and mechanical stress [28]. In addition the technology could achieve continuous deliver of GFs by the encapsulated cells over time [29-30]. Indeed, the characteristics of microencapsulated tumor cells (TCs) have acquired the most interest in rescent years. Qiuyan Wangal et al. detected that the culture of hepatocarcinoma cells in alginate-poly-lysine-alginate (APA) microcapsules in vitro could proliferate TCs with high viability, formingmulticellular spheroid just as the cytoarchitecture of TCs in vivo [31]. Shaoling Wu et al. showed that the implants of microencapsulated rat pheochromocytoma (PC12) cells could secrete catecholamines especially dopamine to decrease the coldallodynia behavior of rats that had the neuropathic pain [32]. They also found that the cell-loaded group did not occur neoplasia in the rats’ spinal cords [33]. Therefore, we hypothesis that the superiority of cell microencapsulation technique can not only enhance NBCs proliferation and boost the effect on the neural differentiaon of MSCs, but also segregate NBCs from normal cells, thus reducing their harmful effect of carcinogenicity.4. Strategy of microencapsulated NBCs together with bFGF supplement for NPC/NSC differetion derivered from MSCs (sig.1)Several experiments have found that the expression of nestin (a marker of neural stem cells) gradually decrease in the process of the neuronal differetiation of MSCs, indicating that NPC/NSC derivered from MSC differentiated into mature neural cells (MNCs) with time. The bFGF, one type of GFs, has been reported to be able to prolong the transformation of NPCs into mature neural cells and lead to neuronal cells dedifferentiate and appear similar to neural stem cells [34], suggesting the utilize of bFGF could stimulate the differetiation of NPC/NSC rather than mature neural cells. Recently, bFGF has also been used as the the major constituent of neural stem cell medium. Based on these data, we hypothesis the special characteristic of bFGF may be applied to the issue of NPC/NSC differetiation from BMSCs. Moreover, according to the technique of contemporary tissue engineering, we can projecte a procedure to synthesize multilayered alginatemicrocapsules called APA as a induction system. In this system, The alginate core was coated with a permselective poly-L-ornithine (PLO) layer which could facilitate the permeability of solutes into the microcapsules [35], the inner layer can be used for NB encapsulation, while the outer layer can be utilized for bFGF encapsulation. It is reported that the addition of the outer layer could keep the bFGF sustained release for more than 18 days without altering the permselectivity of the microcapsule coat [36]. Meanwhile the bFGF also could promote the capacity of proliferation of NBCs, together with the ability of NBCs to delivery the bFGF, and keep the high concentration of bFGF. we suppose the mutual promotion between NBCs and bFGF in the induction system may play a prominent part in NPC/NSC differetiation derivered from MSCs.Conclusion:Recently, the tissue engineering technology of neural differetiation from BMSCs appears bright developing prospects in treatment for central nervous system diseases. In the process of neuronal differentiation of MSCs, PI3K/AKT signaling pathway that could be activated by GFs have been proved to play an essential role in this transformation, suggesting a hypothesis that GFs could activate its subsequent induction of PI3K/AKT signaling pathway and promote differetiation of MSCs into neuron. Base on this mechanism, we exploit to co-culture the microencapsulation of NBC and bFGF with MSCs for promotion of its neural differentiation. The novel induction system may greatly stimulate the directed differetiation rate of NPC/NSC derivered from MSCs. However, subsequent experimental studies are required to further validate the potentialbenefits of the novel induction system.Conflict of interestNone.Figure 1References:[1]. Martino G, Franklin RJ, Van Evercooren AB, Kerr DA. Stem cell transplant-ation in multiple sclerosis: current status and future prospects. Nat RevNeurol 2010; 6:247–255.[2]. Poss KD. Advances in understanding tissue regenerative capacity andMechanisms in animals. Nat Rev Genet 2010; 11:710–722.[3]. Ingber DE, Levin M. What lies at the interface of regenerative medicine anddevelopmental biology? Development 2007; 134:2541–2547.[4]. Pera MF, Tam PP. Extrinsic regulation of pluripotent stem cells. Nature2010;465:713–720.[5]. Uccelli A, Laroni A, Freedman MS. Mesenchymal stem cells for thetreatment of multiple sclerosis and other neurological diseases. LancetNeurol 2011;10:649–656.[6]. 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