Spinal cord injury (SCI) can cause a severe traumatic injury to the central nervous system (CNS).... more Spinal cord injury (SCI) can cause a severe traumatic injury to the central nervous system (CNS). Current therapeutic effects achieved for SCI in clinical medicine show that there is still a long way to go to reach the desirable goal of full or meaningful functional recovery. In basic medical research, however, cell transplantation, gene therapy, application of cytokines and biomaterial scaffolds have been widely used and investigated in treatments for SCI. All these strategies when used separately would help rebuild to some extent the neural circuits in the lesion area of spinal cord. In light of this, it is generally accepted that a combined treatment may be a more effective strategy. This review focuses primarily on our recent series of work on cell transplantation of Schwann cells and adult stem cells, and transplantation of stem cell-derived neural network scaffold with functional synapses. Arising from this, an artificial neural network (an exogenous neuronal relay) has been d...
Journal of biomedical materials research. Part A, Jan 17, 2016
Extracellular matrix (ECM) expression is temporally and spatially regulated during the developmen... more Extracellular matrix (ECM) expression is temporally and spatially regulated during the development of stem cells. We reported previously that fibronectin (FN) secreted by bone marrow mesenchymal stem cells (MSCs) was deposited on the surface of gelatin sponge (GS) soon after culture. In this study, we aimed to assess the function of accumulated FN on neuronal differentiating MSCs as induced by Schwann cells (SCs) in three dimensional transwell co-culture system. The expression pattern and amount of FN of differentiating MSCs was examined by immunofluorescence, Western blot and immunoelectron microscopy. The results showed that FN accumulated inside GS scaffold, although its mRNA expression in MSCs was progressively decreased during neural induction. MSC-derived neuron-like cells showed spindle-shaped cell body and long extending processes on FN-decorated scaffold surface. However, after blocking of FN function by application of monoclonal antibodies, neuron-like cells showed flatten...
Edema in the central nervous system can rapidly result in life-threatening complications. Vasogen... more Edema in the central nervous system can rapidly result in life-threatening complications. Vasogenic edema is clinically manageable, but there is no established medical treatment for cytotoxic edema, which affects astrocytes and is a primary trigger of acute post-traumatic neuronal death. To test the hypothesis that adrenergic receptor agonists, including the stress stimulus epinephrine protects neural parenchyma from damage, we characterized its effects on hypotonicity-induced cellular edema in cortical astrocytes by in vivo and in vitro imaging. After epinephrine administration, hypotonicity-induced swelling of astrocytes was markedly reduced and cytosolic 3'-5'-cyclic adenosine monophosphate (cAMP) was increased, as shown by a fluorescence resonance energy transfer nanosensor. Although, the kinetics of epinephrine-induced cAMP signaling was slowed in primary cortical astrocytes exposed to hypotonicity, the swelling reduction by epinephrine was associated with an attenuated...
There are currently no satisfactory treatments or experimental models showing autonomic dysfuncti... more There are currently no satisfactory treatments or experimental models showing autonomic dysfunction for intramedullary spinal cord gliomas (ISCG). To develop a rat model of ISCG and investigate whether genetically engineered human neural stem cells (F3.hNSCs) could be developed into effective therapies for ISCG. Immunodeficient/Rowett Nude rats received C6 implantation of G55 human glioblastoma cells (10K/each). F3.hNSCs engineered to express either cytosine deaminase gene only (ie, F3.CD) or dual genes of CD and thymidine kinase (ie, F3.CD-TK) converted benign 5-fluorocytosine and ganciclovir into oncolytic 5-fluorouracil and ganciclovir-triphosphate, respectively. ISCG rats received injection of F3.CD-TK, F3.CD, or F3.CD-TK debris near the tumor epicenter 7 days after G55 seeding, followed with 5-FC (500 mg/kg/5 mL) and ganciclovir administrations (25 mg/kg/1 mL/day × 5/each repeat, intraperitoneal injection). Per humane standards for animals, loss of weight-bearing stepping in th...
Severe spinal cord injury (SCI) often causes temporary or permanent damages in strength, sensatio... more Severe spinal cord injury (SCI) often causes temporary or permanent damages in strength, sensation or autonomic functions below the site of the injury. So far there is still no effective treatment for SCI. Mesenchymal stem cells (MSCs) have been used to repair injured spinal cord as an effective strategy. However, the low neural differentiation frequency of MSCs has limited its application. The present study attempted to explore whether the grafted MSC-derived neural-like cells in a gelatin sponge (GS) scaffold could maintain neural features or transdifferentiate into myelin-forming cells in the spinal cord transected. We constructed an engineered tissue by co-seeding of MSCs with genetically enhanced expression of neurotrophin-3 (NT-3) and its high affinity receptor tropomyosin receptor kinase C (TrkC), respectively, into a 3-dimensional GS scaffold to promote the MSCs differentiating into neural-like cells, and transplanted it into the gap of rat spinal cord transected completely....
Rapid loss of stemness capacity in purified prototype neural stem cells (NSCs) remains a serious ... more Rapid loss of stemness capacity in purified prototype neural stem cells (NSCs) remains a serious challenge to basic and clinical studies aiming to repair the central nervous system. Based on the essential role of mesodermal guidance in the process of neurulation, we hypothesized that co-culture of human NSCs (hNSCs) with human bone marrow-derived mesenchymal stromal stem cells (hMSCs) could enhance the stemness of hNSCs through Notch-1 signaling. We have now tested the hypothesis by assessing behaviors of hNSCs and hMSCs under systematically designed co-culture conditions relative to monocultures, with or without Notch-1 manipulation in vitro. Our data demonstrates that expression levels of Notch-1 and Hes-1 as determined by immunocytochemistry are significantly higher in hNSCs co-cultured with hMSCs than those of controls. Furthermore, co-culturing significantly increases immunoreactivity of CD15, a neural stemness marker but decreases CD24, a marker of neural/neuronal commitment i...
The mixed culture of neurons and glial cells has been widely used as a mechanical insult model fo... more The mixed culture of neurons and glial cells has been widely used as a mechanical insult model for the study of neuron injury in vitro. However, a better model is desirable to eliminate the interference of glial cells during the study. Here we report a new model with exclusive cerebellar granule neurons (CGNs), which can be used for the study of in vitro neuron injury without involvement of glial cells. We found that after scratching insult, there was a decrease in both the survival rate and vitality of injured CGNs. Meanwhile, pathological changes were observed by electron microscopy. With this new model, we also tested the effects of neurotrophin-3 (NT-3) on neuroprotection. The result showed that the vitality of injured CGNs was enhanced by the administration of NT-3. These findings demonstrate that this new model is useful for investigation of the precise effect of mechanical damage on neurons excluding other factors, and to detect the neuroprotective effect of certain factors on mechanically injured neurons.
Rapid progress in the field of nerve tissue engineering has opened up the way for new therapeutic... more Rapid progress in the field of nerve tissue engineering has opened up the way for new therapeutic strategies for spinal cord injury (SCI). Bone marrow-derived mesenchymal stem cells (MSCs) could be differentiated into neural lineages, which can be used as a potential cell source for nerve repair. Schwann cells (SCs) have been reported to support structural and functional recovery of SCI. In this study, we co-cultured neurotrophin-3 (NT-3) gene-modified SCs and NT-3 receptor tyrosine protein kinase C (TrkC) gene-modified MSCs in a three-dimensional porous poly(lactic-acid-co-glycolic acid) (PLGA) conduit with multiple channels in vitro for 14 days. Our results showed that more than 50% of the grafted MSCs were MAP2- and β-III-tubulin-positive cells, and the MSCs expressed a high level of β-III-tubulin detected by Western blotting, indicating a high rate of neuronal differentiation. Furthermore, immunostaining of PSD95 revealed the formation of a synapse-like structure, which was confirmed under electron microscopy. In conclusion, co-culture of NT-3 gene-modified SCs and TrkC gene-modified MSCs in the PLGA multiple-channeled conduit can promote MSCs' differentiation into neuron-like cells with synaptogenesis potential. Our study provides a biological basis for future application of this artificial MSCs/SCs/PLGA complex in the SCI treatment.
Three-dimensional (3D) gelatin sponge (GS) scaffolds were constructed by ensheathing GS with a th... more Three-dimensional (3D) gelatin sponge (GS) scaffolds were constructed by ensheathing GS with a thin film of poly-(lactide-co-glycolide) (PLGA). Rat bone marrow-derived mesenchymal stem cells (MSCs) were isolated, cultured, and then seeded to the scaffolds. Distribution of cells and cell growth, survival, and proliferation within the scaffolds were then determined. Immunofluorescence and Western blot analysis were employed to detect the deposition of fibronectin to the scaffolds on day 3 and day 7 of culture. Scaffolds with or without MSCs were then transplanted into the transected rat spinal cord. One or 8 weeks following transplantation, cavity areas, activated macrophages/microglia, expression of TNF-α and IL-1β, and neovascularization within the grafts were examined and quantified. Deposition of fibronectin (FN) and expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) as potential inducing factors for angiogenesis were also examined. Results showed that 3D GS scaffolds allowed MSCs to adhere, survive, and proliferate and also FN to deposit. In vivo transplantation experiments demonstrated that these scaffolds were biocompatible, and MSCs seeded to the scaffolds played an important role in attenuating inflammation, promoting angiogenesis, and reducing cavity formation. Therefore, the GS scaffolds with MSCs may serve as promising supporting transplants for repairing spinal cord injury.
Reports of neuronal differentiation of bone marrow derived mesenchymal stem cells (MSCs) suggeste... more Reports of neuronal differentiation of bone marrow derived mesenchymal stem cells (MSCs) suggested the possibility that these cells could serve as a source of treatment for spinal cord injury. However, the percentages of neuron-like cells differentiated from the MSCs were relatively low both in vitro and in vivo. Here, we investigated whether co-culture of human neurotrophin-3 (NT-3) gene modified Schwann cells (SCs) and human NT-3 receptor tyrosine protein kinase C (TrkC) gene modified MSCs could increase differentiation of neuron-like cells from MSCs. It was shown that MSCs were significantly promoted to differentiate into neuron-like cells, as evidenced immunocytochemically by the expression of neuronal markers, including nestin, beta-III-tubulin, MAP2 and PSD95, 7 days after co-culture. However, the expression of glial fibrillary acidic protein (GFAP)--an astrocyte marker in these cells--was not so obvious. These results demonstrate that the binding of overexpressed NT-3 in SCs and its receptor TrkC in MSCs can be considered to stimulate the increased rate of neuronal differentiation.
Intervertebral disk (IVD) degeneration is a common, chronic, and complex degeneration process tha... more Intervertebral disk (IVD) degeneration is a common, chronic, and complex degeneration process that frequently leads to back pain and disability, resulting in a major public health issue. In this review we describe biological therapies under preclinical or clinical development with an emphasis on stem cell-based multimodal approaches that target prevention and treatment of IVD degeneration. Systematical review of the basic science and clinical literature was performed to summarize the current status of devising biological approaches to treating IVD degeneration. Since the exact mechanisms underlying IVD degeneration have not yet been fully elucidated and conservative managements appear to be mostly ineffective, current surgical treatment focuses on removal of the pathological disk tissues combined with spinal fusion. The treatment options, however, often produce insufficient efficacy and even serious complications. Therefore, there have been growing demands and endeavors for developi...
Spinal cord injury (SCI) can cause a severe traumatic injury to the central nervous system (CNS).... more Spinal cord injury (SCI) can cause a severe traumatic injury to the central nervous system (CNS). Current therapeutic effects achieved for SCI in clinical medicine show that there is still a long way to go to reach the desirable goal of full or meaningful functional recovery. In basic medical research, however, cell transplantation, gene therapy, application of cytokines and biomaterial scaffolds have been widely used and investigated in treatments for SCI. All these strategies when used separately would help rebuild to some extent the neural circuits in the lesion area of spinal cord. In light of this, it is generally accepted that a combined treatment may be a more effective strategy. This review focuses primarily on our recent series of work on cell transplantation of Schwann cells and adult stem cells, and transplantation of stem cell-derived neural network scaffold with functional synapses. Arising from this, an artificial neural network (an exogenous neuronal relay) has been d...
Journal of biomedical materials research. Part A, Jan 17, 2016
Extracellular matrix (ECM) expression is temporally and spatially regulated during the developmen... more Extracellular matrix (ECM) expression is temporally and spatially regulated during the development of stem cells. We reported previously that fibronectin (FN) secreted by bone marrow mesenchymal stem cells (MSCs) was deposited on the surface of gelatin sponge (GS) soon after culture. In this study, we aimed to assess the function of accumulated FN on neuronal differentiating MSCs as induced by Schwann cells (SCs) in three dimensional transwell co-culture system. The expression pattern and amount of FN of differentiating MSCs was examined by immunofluorescence, Western blot and immunoelectron microscopy. The results showed that FN accumulated inside GS scaffold, although its mRNA expression in MSCs was progressively decreased during neural induction. MSC-derived neuron-like cells showed spindle-shaped cell body and long extending processes on FN-decorated scaffold surface. However, after blocking of FN function by application of monoclonal antibodies, neuron-like cells showed flatten...
Edema in the central nervous system can rapidly result in life-threatening complications. Vasogen... more Edema in the central nervous system can rapidly result in life-threatening complications. Vasogenic edema is clinically manageable, but there is no established medical treatment for cytotoxic edema, which affects astrocytes and is a primary trigger of acute post-traumatic neuronal death. To test the hypothesis that adrenergic receptor agonists, including the stress stimulus epinephrine protects neural parenchyma from damage, we characterized its effects on hypotonicity-induced cellular edema in cortical astrocytes by in vivo and in vitro imaging. After epinephrine administration, hypotonicity-induced swelling of astrocytes was markedly reduced and cytosolic 3'-5'-cyclic adenosine monophosphate (cAMP) was increased, as shown by a fluorescence resonance energy transfer nanosensor. Although, the kinetics of epinephrine-induced cAMP signaling was slowed in primary cortical astrocytes exposed to hypotonicity, the swelling reduction by epinephrine was associated with an attenuated...
There are currently no satisfactory treatments or experimental models showing autonomic dysfuncti... more There are currently no satisfactory treatments or experimental models showing autonomic dysfunction for intramedullary spinal cord gliomas (ISCG). To develop a rat model of ISCG and investigate whether genetically engineered human neural stem cells (F3.hNSCs) could be developed into effective therapies for ISCG. Immunodeficient/Rowett Nude rats received C6 implantation of G55 human glioblastoma cells (10K/each). F3.hNSCs engineered to express either cytosine deaminase gene only (ie, F3.CD) or dual genes of CD and thymidine kinase (ie, F3.CD-TK) converted benign 5-fluorocytosine and ganciclovir into oncolytic 5-fluorouracil and ganciclovir-triphosphate, respectively. ISCG rats received injection of F3.CD-TK, F3.CD, or F3.CD-TK debris near the tumor epicenter 7 days after G55 seeding, followed with 5-FC (500 mg/kg/5 mL) and ganciclovir administrations (25 mg/kg/1 mL/day × 5/each repeat, intraperitoneal injection). Per humane standards for animals, loss of weight-bearing stepping in th...
Severe spinal cord injury (SCI) often causes temporary or permanent damages in strength, sensatio... more Severe spinal cord injury (SCI) often causes temporary or permanent damages in strength, sensation or autonomic functions below the site of the injury. So far there is still no effective treatment for SCI. Mesenchymal stem cells (MSCs) have been used to repair injured spinal cord as an effective strategy. However, the low neural differentiation frequency of MSCs has limited its application. The present study attempted to explore whether the grafted MSC-derived neural-like cells in a gelatin sponge (GS) scaffold could maintain neural features or transdifferentiate into myelin-forming cells in the spinal cord transected. We constructed an engineered tissue by co-seeding of MSCs with genetically enhanced expression of neurotrophin-3 (NT-3) and its high affinity receptor tropomyosin receptor kinase C (TrkC), respectively, into a 3-dimensional GS scaffold to promote the MSCs differentiating into neural-like cells, and transplanted it into the gap of rat spinal cord transected completely....
Rapid loss of stemness capacity in purified prototype neural stem cells (NSCs) remains a serious ... more Rapid loss of stemness capacity in purified prototype neural stem cells (NSCs) remains a serious challenge to basic and clinical studies aiming to repair the central nervous system. Based on the essential role of mesodermal guidance in the process of neurulation, we hypothesized that co-culture of human NSCs (hNSCs) with human bone marrow-derived mesenchymal stromal stem cells (hMSCs) could enhance the stemness of hNSCs through Notch-1 signaling. We have now tested the hypothesis by assessing behaviors of hNSCs and hMSCs under systematically designed co-culture conditions relative to monocultures, with or without Notch-1 manipulation in vitro. Our data demonstrates that expression levels of Notch-1 and Hes-1 as determined by immunocytochemistry are significantly higher in hNSCs co-cultured with hMSCs than those of controls. Furthermore, co-culturing significantly increases immunoreactivity of CD15, a neural stemness marker but decreases CD24, a marker of neural/neuronal commitment i...
The mixed culture of neurons and glial cells has been widely used as a mechanical insult model fo... more The mixed culture of neurons and glial cells has been widely used as a mechanical insult model for the study of neuron injury in vitro. However, a better model is desirable to eliminate the interference of glial cells during the study. Here we report a new model with exclusive cerebellar granule neurons (CGNs), which can be used for the study of in vitro neuron injury without involvement of glial cells. We found that after scratching insult, there was a decrease in both the survival rate and vitality of injured CGNs. Meanwhile, pathological changes were observed by electron microscopy. With this new model, we also tested the effects of neurotrophin-3 (NT-3) on neuroprotection. The result showed that the vitality of injured CGNs was enhanced by the administration of NT-3. These findings demonstrate that this new model is useful for investigation of the precise effect of mechanical damage on neurons excluding other factors, and to detect the neuroprotective effect of certain factors on mechanically injured neurons.
Rapid progress in the field of nerve tissue engineering has opened up the way for new therapeutic... more Rapid progress in the field of nerve tissue engineering has opened up the way for new therapeutic strategies for spinal cord injury (SCI). Bone marrow-derived mesenchymal stem cells (MSCs) could be differentiated into neural lineages, which can be used as a potential cell source for nerve repair. Schwann cells (SCs) have been reported to support structural and functional recovery of SCI. In this study, we co-cultured neurotrophin-3 (NT-3) gene-modified SCs and NT-3 receptor tyrosine protein kinase C (TrkC) gene-modified MSCs in a three-dimensional porous poly(lactic-acid-co-glycolic acid) (PLGA) conduit with multiple channels in vitro for 14 days. Our results showed that more than 50% of the grafted MSCs were MAP2- and β-III-tubulin-positive cells, and the MSCs expressed a high level of β-III-tubulin detected by Western blotting, indicating a high rate of neuronal differentiation. Furthermore, immunostaining of PSD95 revealed the formation of a synapse-like structure, which was confirmed under electron microscopy. In conclusion, co-culture of NT-3 gene-modified SCs and TrkC gene-modified MSCs in the PLGA multiple-channeled conduit can promote MSCs' differentiation into neuron-like cells with synaptogenesis potential. Our study provides a biological basis for future application of this artificial MSCs/SCs/PLGA complex in the SCI treatment.
Three-dimensional (3D) gelatin sponge (GS) scaffolds were constructed by ensheathing GS with a th... more Three-dimensional (3D) gelatin sponge (GS) scaffolds were constructed by ensheathing GS with a thin film of poly-(lactide-co-glycolide) (PLGA). Rat bone marrow-derived mesenchymal stem cells (MSCs) were isolated, cultured, and then seeded to the scaffolds. Distribution of cells and cell growth, survival, and proliferation within the scaffolds were then determined. Immunofluorescence and Western blot analysis were employed to detect the deposition of fibronectin to the scaffolds on day 3 and day 7 of culture. Scaffolds with or without MSCs were then transplanted into the transected rat spinal cord. One or 8 weeks following transplantation, cavity areas, activated macrophages/microglia, expression of TNF-α and IL-1β, and neovascularization within the grafts were examined and quantified. Deposition of fibronectin (FN) and expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) as potential inducing factors for angiogenesis were also examined. Results showed that 3D GS scaffolds allowed MSCs to adhere, survive, and proliferate and also FN to deposit. In vivo transplantation experiments demonstrated that these scaffolds were biocompatible, and MSCs seeded to the scaffolds played an important role in attenuating inflammation, promoting angiogenesis, and reducing cavity formation. Therefore, the GS scaffolds with MSCs may serve as promising supporting transplants for repairing spinal cord injury.
Reports of neuronal differentiation of bone marrow derived mesenchymal stem cells (MSCs) suggeste... more Reports of neuronal differentiation of bone marrow derived mesenchymal stem cells (MSCs) suggested the possibility that these cells could serve as a source of treatment for spinal cord injury. However, the percentages of neuron-like cells differentiated from the MSCs were relatively low both in vitro and in vivo. Here, we investigated whether co-culture of human neurotrophin-3 (NT-3) gene modified Schwann cells (SCs) and human NT-3 receptor tyrosine protein kinase C (TrkC) gene modified MSCs could increase differentiation of neuron-like cells from MSCs. It was shown that MSCs were significantly promoted to differentiate into neuron-like cells, as evidenced immunocytochemically by the expression of neuronal markers, including nestin, beta-III-tubulin, MAP2 and PSD95, 7 days after co-culture. However, the expression of glial fibrillary acidic protein (GFAP)--an astrocyte marker in these cells--was not so obvious. These results demonstrate that the binding of overexpressed NT-3 in SCs and its receptor TrkC in MSCs can be considered to stimulate the increased rate of neuronal differentiation.
Intervertebral disk (IVD) degeneration is a common, chronic, and complex degeneration process tha... more Intervertebral disk (IVD) degeneration is a common, chronic, and complex degeneration process that frequently leads to back pain and disability, resulting in a major public health issue. In this review we describe biological therapies under preclinical or clinical development with an emphasis on stem cell-based multimodal approaches that target prevention and treatment of IVD degeneration. Systematical review of the basic science and clinical literature was performed to summarize the current status of devising biological approaches to treating IVD degeneration. Since the exact mechanisms underlying IVD degeneration have not yet been fully elucidated and conservative managements appear to be mostly ineffective, current surgical treatment focuses on removal of the pathological disk tissues combined with spinal fusion. The treatment options, however, often produce insufficient efficacy and even serious complications. Therefore, there have been growing demands and endeavors for developi...
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