| Mesenchymal stem cells(MSCs) are multipotent stem cells that can be easily isolated and cultured in vitro. They can be differentiated into not only mesoderm lineages, such as chondrocytes, osteoceytes and adipocytes, but also ectodermal neural- like cells. The versatility, together with the easier availability, low immunogenicity and lacking ethical controversy make MSCs excellent cell therapy candidates for a wide variety of clinical pathologies. Directed migration of MSCs towards pathologies is essential for cellular transplantation in treatment. It has been reported that MSCs can migrate to the lesion or injured area in the brain, and they participate nervous system reconstruction to improve the function recovery. Transplanted MSCs can differentiate into neural- and glial- like cells, and also can secret various cellular factors which serve to protect neurons and improve tissue repair. Together, MSCs are proposed to be applied in treatment of nervous system diseases, such as peripheral nervous system injury, spinal cord injury, cerebral stroke, and cerebral injury. In recent years, MSCs differentiated into neural like cells in vitro/ in vivo are potential for restorative treatment in Parkinson’s disease. However, only a very limited number of cells successfully reached the damaged areas in vivo, most of the transplanted cells remain at the injection site or dispersed anywhere with the circulation of blood, resulting in the low rate of MSCs replacement. Therefore, ensure enough MSCs migrate to the damage sites is the key for improving the efficacy of treatment.Directed or chemotactic migration of MSCs is regulated by a variety of factors, and cytokines and chemokines play a very important role in this process. It is reported that stromal cell derived factor-1 alpha(SDF-1α) is secreted in damage area of nervous system. SDF-1α binding its receptor CXC R4 specifically activated PI3K/Akt and MAPKs signaling pathways, leading to cytoskeletal recombination, thereby inducing the directional migration of MSCs. This process is extensively involved in various pathological and physiological responses, including angiogenesis, the occurrence of heart and Parkinson’s disease nerve protection, suggesting that SDF-1α is very important in the repair of nervous system injury.It is unlikely that MSC directed migration is regulated by only one factor, chemotactic response and the differentiation of MSCs may also influence their chemotatic migration. It has been reported that MSCs from different tissue sources or in different cell cycle endowed different chemotactic response. MSCs used for implantation are usually at different differentiation states, and cells possess stronger capacity for chemotaxis may be in a special differentiation state. Discovering the characteristics of these cells and the mechanism regulating their higher tendency to chemokine may shed light on optimization of the therapeutic potential of MSCs to be employed for neural diseases.Therefore, different differentiation states of MSCs(undifferentiation, 24-h preinduction, 5-h induction, 18- h and 48-h maintenance) were prepared by basic fibroblast growth factor(b FGF) combination with butyl hydroxy anisol(BHA). Using Boyden chamber and Western blot, we tested the transfilter migration of undifferentiation and diffierentiating MSCs toward SDF-1α. Results are as follows:The migratory capacity of MSCs towards SDF-1α depends on the differentiation states of these cellsTo investgate the chemotactic responses of MSCs to concentration gradients of SDF-1α in relation to their differentiatio n states, we tested the transfilter migration of MSCs toward SDF-1α at concentrations ranging from 0 to 100 ng/ml by microchemotaxis Boyden chamber. Results showed that 25 ng/ml SDF-1α promoted migration of undifferentiated MSCs while higher concentrations inhibited migraton. We then investigated the chemotactic responses of MSCs in different neural differentiation states(24- h preinduction, 5-h induction, 18-h and 48-h maintenance) toward SDF-1α. We found that SDF-1α(5 and 50 ng/ml) did induce migratory response of differentiating MSCs, but the respons ive extently among these cells were different. Otherwise, cells in 24-h preinduction or 5-h induction state underwent chemotaxis when exposed to SDF-1α at 100 ng/ml. Data showed that the level of the chemotactic responses and the migratory capabilities of MSCs toward SDF-1α vary significantly, depending on the neural differentiation states of these cells.MSCs of 24-h preinduction possess the strongest chemotactic migratory capacityTo find a certain differentiated state of MSCs among neural differentiation endowed with stronger chemotactic responsiveness, we examined the response of MSCs under varying differentiation states to the certain concentration of SDF-1α. Since 25 ng/ml was a minimum concentration that induced significant difference among migration of cells in all differentiation states, we used this concentration to analyze the migratory response of the differentiating cells. The strongest chemotactic migration appeared in cells of 24- h preinduction showing the most powerful chemotactic response to 25 ng/ml SDF-1α, with 1.53- fold induction compared with unstimulated cells, demonstrating that MSCs of 24-h preinduction display stronger chemotaxis toward 25 ng/ml SDF-1α than cells in other differentiation states.The responses of PI3K/Akt and MAPKs were different among MSCs at different differentiated states after exposed to SDF-1αOur previous stud ies showed that migratory response of MSCs to SDF-1α were closely related to their differentiation states, thus whether PI3K/Akt and MAPKs signaling pathways are involved in cell migration? We then evaluated the effect of SDF-1α on the phosphorylation of PI3K/Akt and MAPKs in MSCs under varying differentiation states, including Akt, ERK1/2, SAPK/JNK, and p38 MAPK. Treatment of undifferentiated MSCs with SDF-1α(0-100 ng/ml) for 30 min led to a concentration-dependent phosphorylation of PI3K/Akt and MAPKs: the phosphorylation of Akt, ERK1/2, and p38 MAPK in undifferentiated MSCs slightly increased after treatment with SDF-1α at 5 ng/ml, and reached a peak by addition of SDF-1α at 25 ng/ml. Then we explore the phosphorylation of PI3K/Akt, ERK1/2, SAPK/JNK, and p38 MAPK of MSCs varying differentiation states. Date showed that MSCs under varying neural differentiation states expressed basal level of phosphorylated PI3K/Akt, ERK1/2, SAPK/JNK, and p38 MAPK. Compared with undifferentiated MSCs, a relatively higher phosphorylation of Akt and ERK1/2 was observed in cells of 5-h induction and 48-h maintenance, with p38 MAPK phosphorylation increased in cells of both 18-h and 48- h maintenance states. The level of SAPK/JNK phosphorylation was significantly elevated in cells of 18-h maintenance and slightly decreased in 24-h preinduction and 48-h maintenance.Activated timepoint and duration of PI3K/Akt and MAPKs signaling moleculars were different among MSCs at different differentiation states after exposed to SDF-1αFollowing SDF-1α treatment, the phosphorylation of Akt was increased at 5 min and sustained for up to 60 min in undifferentiated MSCs and returned to basal levels within 15 min in cells of 18-h maintenance, while no changes were observed in cells of 24- h preinduction and decreased in cells of 5-h induction and 48-h maintenance. Stimulation with SDF-1α led to a slight elevation of ERK1/2 in undifferentiation, 5-h induction, and 18-h maintenance cells at 5 min with different maximal activation and sustained time, while no significant changes were detected in 24-h preinduction and 48-h maintenance. In cells of 24-h preinduction, the SDF-1α-induced SAPK/JNK phosphorylation was significantly elevated at 5 min and prolonged to 60 min, while no significant difference was found in MSCs of undifferentiation and 5-h induction. The level of SAPK/JNK phosphorylation decreased at 5 min and lasted to 60 min in cells of maintenance. Upon SDF-1α treatment, p38 MAPK phosphorylation levels remained unchanged in MSCs of 24-h preinduction, 5-h induction, and 48- h maintenance, but transiently increased at 5 min and 60 min in undifferentiated cells, and markedly elevated at 15 min, virtually returned to basal levels within 30 min, then descended at 60 min in cells of 18-h maintenance.PI3K/Akt and MAPKs signaling pathways are involved in SDF-1α-stimulated migration of differentiation MSCsTo further evaluate the roles of PI3K/Akt and MAPKs in SDF-1α-stimulated migration, MSCs in varying differentiation states were pretreated with 30 μM LY294002(PI3K/Akt inhibitor), 50 μM PD98059(ERK1/2 inhibitor), 30 μM SB203580(p38MAPK inhibitor), or 10 μM SP600125(SAPK/JNK inhibitor) for 30 min prior to the exposure of the gradient of SDF-1α added to the lower well of a microchemotaxis Boyden chamber at a concentration of 25 ng/ml. As shown in results, interference with PI3K/Akt signaling significantly decreased the number of migrated cells of undifferentiation, 18-h and 48- h maintenance states, but not those of 24- h preinduction and 5-h induction. While abolishment of SAPK/JNK signaling suppressed SDF-1α-induced migration of undifferentiated and differentiating MSCs except cells of 5-h induction, decreased migration was only observed in cells of 5-h induction and of 48-h maintenance by ERK1/2 signaling inhibition, and in cells of 18-h and 48-h maintenance by p38 MAPK signaling inhibition. In summary, PI3K/Akt and MAPKs signaling pathways regulate SDF-1α- induced migration of differentiating MSCs, the extent of which depends on the differentiation status.In summary, results presented here raise the possib ility that MSCs at certain differentiation state might possess stronger capacity of chemotaxis, highlighting a pivotal role of the differentiation states in the chemotactic responses of MSCs. We can take advantage of MSCs at certain differentiation state possessing stronger migration to cell transplantation, might promote the structural and functional repair of the damaged brain more efficiently which is of great importance in the context of repair after brain injury.However, loss of neurons and the resulted inhibitory micro environment at the lesion site becomes a physical barrier for axon extension. Glial scar is easy to form after injury, which impede neuronal axons regeneration and extention, and resulted in difficulty for establishing new synaptic connections and the reconstruction of the central nervous system function. Therefore, MSCs arrived at the site of injury should differentiate into neural- like cells, and establish synaptic contacts, then replace the damaged nerve tissue to improve functional reconstruction of the nervous system.For above question, suitable biomimetic materials, which simulate extracellular matrix perplexing protein network structure, can provide suitable supports for MSCs growth and guide the orientation and migration towards damaged area to enhance neural repair.Bombyx mori silk fibroin(SF) has become a potential substitute of medical tissue engineering, as it is easy to be processed into various shapes and slowly to degrade in vivo. Now, simulate natural extracellular matrix is the inevitable trend of tissue engineering in injury repair. The second part of this paper will be to investigate the regulating roles of SF nanofibrous scaffolds with different diameters and orientations on supporting growth and migration of MSCs wit h differentiation states.For this purpose, we induced neural differentiation of MSCs in vitro and engineered mechanically stable 3D SF scaffolds similar to the natural extracellular matrix fibers with different diameters(100, 200 and 400 nm) and orientations(random and aligned) by electrospinning to mimic the structure of the ECM and retain its integral mechanical properties, to investigate the regulating roles of SF nanofibrous scaffolds with different diameters and orientations on supporting growth and migration of MSCs with differentiation states.100 nm and 200 nm SF fiber promote the adhesion and growth of MSCs, and SF also support the neural differentiation of MSCs.Basically, the scaffolds used in tissue are designed to support cell growth as well as to maintain cell morphology and physiological functions. In our experiments, cell number at different time point was assessed to investigate viability of MSCs grown on SF scaffolds. It was observed that few dead cells were detected on SF fibers with different diameter and PLL after culturing for 3, 6, 9 days, suggesting that SF fibers display a good biocompatibility with MSCs and support the survival and growth of MSCs. O n day 3, significantly more cells were observed on 100 nm and 200 nm SF fibers than PLL, while there is no statistically significant difference was observed between 400 nm SF scaffold and PLL. As time goes on, 100 nm and 200 nm SF fiber suppprted the proliferation of MSCs on day 9, s ignificantly higher than control group. These results suggest that 100 nm and 200 nm SF fibers promote the growth and propagation of MSCs in early stage, and the reason might be that surface area increased as fiber diameter decreased, and smaller diameter fibers with a high specific surface area could adsorb adhesion proteins from the culture medium and thereby offer more focal adhesion points for cell to attach and grow. Additionally, we assess the effect of SF fibers-diameter on the growth of MSCs under varying differentiation states, the results showed that SF fibers support the neural differentiation of MSCs as well as PLL.SF fibe rs enhance migration speed of MSCs at some certain differentiation states, while the diameter of SF fibers and the differentiation states of MSCs affect the migration speed.Directed migration of MSCs is critically important for restoration after injury because deficient motility of MSCs is tightly related to treatment effectiveness. If much effort be devoted to understand of MSC migratory behavior after combining with tissue engineered materials, the therapeutic application of MSCs by effective transplantation protocols would bring the hope. In this study, MSCs at passage 3 through 10 were seeded on different diameter SF fibers(100, 200 and 400 nm) and different orientations(rando m or aligned), then time- lapse video microscopy was used to monitor MSC behavior during a 4 h period, migration speed and migration efficiency were further analyzed and calculated. Q uantitative analysis showed that MSCs of undifferentiation, 24-h preinduction and 5- h induction culturing on 100 nm and 200 nm SF fibers migrated faster than PLL, while 400 nm SF fibers only enhanced the migration speed of cells in undifferentiation and 24-h preinduction. Furthermore, MSCs of 24- h preinduction possess stronger migration speed than cells in other differentiation states regardless of fiber diameter.200 nm and 400 nm aligned SF fibers improve migration efficiency of MSCs under varying differentiation states.Migration behavior of MSCs contains two valuable paramenters, one is migration speed, and another is migration efficiency which is determined by calculating the forward migration index(FMI); that is, the ratio of the most direct the cell progressed to its total path length. 200 and 400 nm aligned SF fibers enha nced migration efficiency of both undifferentiated and differentiating MSCs, while 100 nm SF fibers only support cells of undifferentiated MSCs and cells of 24-h preinduction and 5-h induction.SF fibers stimulated phosphorylation of PI3K/Akt and ERK1/2 i n MSCs under varing differentiation states depend on their diameterIncreasing evidences have been shown that MSCs could migrate to injured, inflamed tissues from blood and exert therapeutic effects. The mechanisms by which MSCs migrate to the target tissues are not yet fully understood, however, extensive studies have shown that directed migration of MSCs is of great importance in the context of repair after tissue injure. In our present study, PI3K/Akt and ERK1/2 signaling are involved in cell migration b y various growth factors activating the downstream signaling molecules. Thus, whether SF enhances the migration speed of MSCs vary differentiated states also through the PI3K/Akt and ERK1/2 signaling? Then we choose 200 nm and 400 nm SF fibers(materials could promote MSC migration efficiency in varying neuronal differentiation states) and three different differentiated states(undifferentiated, 24-h preinduction and 18-h maintenance), which represent control, high migration capability and low high migratio n capability of MSCs in varying neuronal differentiation states. Results show that the phosphorylation of ERK1/2 increased in all the three different differentiated states of MSCs growing on 200 nm SF fibers, while 400 nm SF fibers activated the ERK1/2 signaling of cells in undifferentiation and 24-h preinduction states, but not in 18-h maintenance state. Meanwhile, PI3K/Akt signaling was activated in cells of 24-h preinduction both on 200 and 400 nm SF fibers, however, the phosphorylation of Akt elevated in MSCs of undifferentiation on 200 nm SF fibers and MSCs of 18-h maintenance on 400 nm SF fibers.PI3K/Akt and ERK1/2 signaling pathways are involved in migration of the differentiating MSCs on SF fibersTo further evaluate the roles of PI3K/Akt and ERK1/2 signaling in MSCs of varying differentiation states on 200 nm and 400 nm SF fibers, three different differentiated states of MSCs were pretreated with 30 μM LY294002(PI3K/Akt inhibitor) and 50 μM PD98059(ERK1/2 inhibitor) for 30 min prior to record by time- lapse video microscopy during 4 h. Results showed that inhibition of PI3K/Akt signaling, migration speed of MSCs of undifferentiated and 18-h maintenance states was attenuated on 200 nm SF fibers, while abolishment of ERK1/2 signaling significantly decreased the migration speed of cells in 24-h preinduction and 18-h maintenance states. Meanwhile, on 400 nm SF fibers, blocking of PI3K/Akt or ERK1/2 signaling impaired the migration speed of undifferentiated and 24-h preinduction MSCs.The results of first part illustrate that different differentiation states of MSCs have different migration abilities, and chemotactic response to different concentrations of cytokines is also different, intriguing possibility that MSCs at a certain level of differentiation state endowed with stronger migratory capacity or chemotactic responsiveness than cells in other differentiation states, highlighting a pivotal role of the differentiation state in the chemotactic responses of MSCs. The results of second part find that SF could promote MSC growth and migration of different differentiation states. Aligned SF, diameter between 200 nm and 400 nm represent valuable fibers for efficient migration of neuronal differentiating MSCs, and MSCs of 24 h preinduction exhibit the most effective migration and become the best cell state of neuronal differentiation. Moreover, PI3K/Akt and ERK1/2 signaling are involved in SF enhancing MSC migration.In conclusion, taking advantage of certain differentiation state endow stronger migration efficiency, and the suitable diameter of alignment SF fibers to promote the structural and functional repair of a wide variety of neurologic diseases more efficiently, undoubtedly bring new hope to the nerve tissue regeneration after injury. |
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