Combination of induced pluripotent stem cell-derived motor neuron progenitor cells with irradiated brain-derived neurotrophic factor over-expressing engineered mesenchymal stem cells enhanced restoration of axonal regeneration in a chronic spinal cord injury rat model.

BACKGROUND: Spinal cord injury (SCI) is a disease that causes permanent impairment of motor, sensory, and autonomic nervous system functions. Stem cell transplantation for neuron regeneration is a promising strategic treatment for SCI. However, selecting stem cell sources and cell transplantation based on experimental evidence is required. Therefore, this study aimed to investigate the efficacy of combination cell transplantation using the brain-derived neurotrophic factor (BDNF) over-expressing engineered mesenchymal stem cell (BDNF-eMSC) and induced pluripotent stem cell-derived motor neuron progenitor cell (iMNP) in a chronic SCI rat model. METHOD: A contusive chronic SCI was induced in Sprague-Dawley rats. At 6 weeks post-injury, BDNF-eMSC and iMNP were transplanted into the lesion site via the intralesional route. At 12 weeks post-injury, differentiation and growth factors were evaluated through immunofluorescence staining and western blot analysis. Motor neuron differentiation and neurite outgrowth were evaluated by co-culturing BDNF-eMSC and iMNP in vitro in 2-dimensional and 3-dimensional. RESULTS: Combination cell transplantation in the chronic SCI model improved behavioral recovery more than single-cell transplantation. Additionally, combination cell transplantation enhanced mature motor neuron differentiation and axonal regeneration at the injured spinal cord. Both BDNF-eMSC and iMNP played a critical role in neurite outgrowth and motor neuron maturation via BDNF expression. CONCLUSIONS: Our results suggest that the combined transplantation of BDNF- eMSC and iMNP in chronic SCI results in a significant clinical recovery. The transplanted iMNP cells predominantly differentiated into mature motor neurons. Additionally, BDNF-eMSC exerts a paracrine effect on neuron regeneration through BDNF expression in the injured spinal cord.

Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI. METHODS: A contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro. RESULTS: Stepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation. CONCLUSIONS: Our findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.

Effects of stepwise administration of osteoprotegerin and parathyroid hormone-related peptide DNA vectors on bone formation in ovariectomized rat model.

Osteoporosis is a metabolic bone disease that impairs bone mineral density, microarchitecture, and strength. It requires continuous management, and further research into new treatment options is necessary. Osteoprotegerin (OPG) inhibits bone resorption and osteoclast activity. The objective of this study was to investigate the effects of stepwise administration of OPG-encoded minicircles (mcOPG) and a bone formation regulator, parathyroid hormone-related peptide (PTHrP)-encoded minicircles (mcPTHrP) in osteoporosis. The combined treatment with mcOPG and mcPTHrP significantly increased osteogenic marker expression in osteoblast differentiation compared with the single treatment groups. A model of postmenopausal osteoporosis was established in 12-week-old female rats through ovariectomy (OVX). After 8 weeks of OVX, mcOPG (80 µg/kg) was administered via intravenous injection. After 16 weeks of OVX, mcPTHrP (80 µg/kg) was injected once a week for 3 weeks. The bone microstructure in the femur was evaluated 24 weeks after OVX using micro-CT. In a proof-of-concept study, stepwise treatment with mcOPG and mcPTHrP on an OVX rat model significantly improved bone microstructure compared to treatment with mcOPG or mcPTHrP alone. These results suggest that stepwise treatment with mcOPG and mcPTHrP may be a potential treatment for osteoporosis.

Intranasal administration of induced pluripotent stem cell-derived cortical neural stem cell-secretome as a treatment option for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, resulting in gradual destruction of cognitive abilities. Research on the development of various AD treatments is underway; however, no definitive treatment has been developed yet. Herein, we present induced pluripotent stem cell (iPSC)-derived cortical neural stem cell secretome (CNSC-SE) as a new treatment candidate for AD and explore its efficacy. METHODS: We first assessed the effects of CNSC-SE treatment on neural maturation and electromagnetic signal during cortical nerve cell differentiation. Then to confirm the efficacy in vivo, CNSC-SE was administered to the 5×FAD mouse model through the nasal cavity (5 µg/g, once a week, 4 weeks). The cell-mediated effects on nerve recovery, amyloid beta (Aß) plaque aggregation, microglial and astrocyte detection in the brain, and neuroinflammatory responses were investigated. Metabolomics analysis of iPSC-derived CNSC-SE revealed that it contained components that could exert neuro-protective effects or amplify cognitive restorative effects. RESULTS: Human iPSC-derived CNSC-SE increased neuronal proliferation and dendritic structure formation in vitro. Furthermore, CNSC-SE-treated iPSC-derived cortical neurons acquired electrical network activity and action potential bursts. The 5×FAD mice treated with CNSC-SE showed memory restoration and reduced Aß plaque accumulation. CONCLUSIONS: Our findings suggest that the iPSC-derived CNSC-SE may serve as a potential, non-invasive therapeutic option for AD in reducing amyloid infiltration and restoring memory.

The neuro-protective role of telomerase via TERT/TERF-2 in the acute phase of spinal cord injury.

PURPOSE: To investigate the interaction of telomerase activity and telomere length on neuro-protection or neuro-degeneration effects after spinal cord injury (SCI). METHODS: A contusive SCI model was developed using 56 Sprague-Dawley rats. Seven rats were allocated into acute injury phase groups (1, 3, 8, 24, and 48 h), and sub-acute and chronic injury phase groups (1, 2, and 4 weeks). Telomerase activity was assessed by telomerase reverse transcriptase (TERT) and telomeric repeat binding factor-2 (TERF-2). Differentiation of activated neural stem cells was investigated by co-expression of neuronal/glial cell markers. Apoptosis expression was also investigated by caspase-3, 8, and 9 using terminal deoxynucleotidyl transferase dUTP nick end labelling staining. Immunofluorescence staining and western blotting were performed for quantitative analyses. RESULTS: Expression of TERT increased gradually until 24 h post-injury, and was decreased following SCI (P < 0.05). TERF-2 also was increased following SCI until 24 h post-injury and then decreased with time (P < 0.05). Co-localization of TERT and TERF-2 was higher at 24 h post-injury. High expression of TERT was seen in neurons (Neu N Ab), however, expression of TERT was relatively lower in astrocytes and oligodendrocytes. Apoptosis analysis showed persistent high expression of caspases-3, -9, and -8 during the observation period. CONCLUSIONS: Increased TERT and TERF-2 activity were noted 24 h post-injury in the acute phase of SCI with TERF-2 maintaining telomeric-repeat length. Our results suggest that increased activity of telomere maintenance may be related to neuro-protective mechanisms against subsequent apoptosis resulting from DNA damage after acute SCI.

Mitigating Effect of Estrogen in Alzheimer's Disease-Mimicking Cerebral Organoid.

Alzheimer's disease (AD) is the most common condition in patients with dementia and affects a large population worldwide. The incidence of AD is expected to increase in future owing to the rapid expansion of the aged population globally. Researchers have shown that women are twice more likely to be affected by AD than men. This phenomenon has been attributed to the postmenopausal state, during which the level of estrogen declines significantly. Estrogen is known to alleviate neurotoxicity in the brain and protect neurons. While the effects of estrogen have been investigated in AD models, to our knowledge, they have not been investigated in a stem cell-based three-dimensional in vitro system. Here, we designed a new model for AD using induced pluripotent stem cells (iPSCs) in a three-dimensional, in vitro culture system. We used 5xFAD mice to confirm the potential of estrogen in alleviating the effects of AD pathogenesis. Next, we confirmed a similar trend in an AD model developed using iPSC-derived cerebral organoids, in which the key characteristics of AD were recapitulated. The findings emphasized the potential of estrogen as a treatment agent for AD and also showed the suitability of AD-recapitulating cerebral organoids as a reliable platform for disease modeling and drug screening.

Increased Potential of Bone Formation with the Intravenous Injection of a Parathyroid Hormone-Related Protein Minicircle DNA Vector.

Osteoporosis is commonly treated via the long-term usage of anti-osteoporotic agents; however, poor drug compliance and undesirable side effects limit their treatment efficacy. The parathyroid hormone-related protein (PTHrP) is essential for normal bone formation and remodeling; thus, may be used as an anti-osteoporotic agent. Here, we developed a platform for the delivery of a single peptide composed of two regions of the PTHrP protein (1-34 and 107-139); mcPTHrP 1-34+107-139 using a minicircle vector. We also transfected mcPTHrP 1-34+107-139 into human mesenchymal stem cells (MSCs) and generated Thru 1-34+107-139-producing engineered MSCs (eMSCs) as an alternative delivery system. Osteoporosis was induced in 12-week-old C57BL/6 female mice via ovariectomy. The ovariectomized (OVX) mice were then treated with the two systems; (1) mcPTHrP 1-34+107-139 was intravenously administered three times (once per week); (2) eMSCs were intraperitoneally administered twice (on weeks four and six). Compared with the control OVX mice, the mcPTHrP 1-34+107-139-treated group showed better trabecular bone structure quality, increased bone formation, and decreased bone resorption. Similar results were observed in the eMSCs-treated OVX mice. Altogether, these results provide experimental evidence to support the potential of delivering PTHrP 1-34+107-139 using the minicircle technology for the treatment of osteoporosis.

Integrity of the Untorn Articular-Sided Tendon in Bursal-Sided Partial-Thickness Rotator Cuff Tear: A Comparative Study of Apoptotic Activity in Torn and Untorn Layers.

BACKGROUND: Conversion to full-thickness tear in partial-thickness rotator cuff tears (PTRCTs) is based on the quality and thickness of the normal-looking untorn rotator cuff layer. However, whether the untorn tendon is a healthy tissue remains to be elucidated. PURPOSE: To compare the apoptotic gene expression of the untorn articular layer with the torn bursal layer in PTRCTs. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon tissues were harvested from 20 patients undergoing arthroscopic surgery for partial-thickness rotator cuff repair. As a control group, the tissues were harvested during intramedullary nail fixation in 10 proximal humeral fractures. In the experimental group, the samples were harvested from 2 sites: the torn bursal-sided tendon and the untorn articular-sided tendon. Hematoxylin and eosin (H&E) staining was conducted for basic histological evaluation, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to detect apoptosis of tissue cells. The expression of caspase 3, 8, and 9 was confirmed immunohistochemically. Western blot analysis was used to assay the caspase activities. RESULTS: In H&E staining, the direction of collagen bundles in untorn tendon was disoriented when compared with those of control tendon. However, the shape of the nuclei was not different, although the nuclei of the untorn tendon showed apoptosis in the TUNEL staining similar to those of the torn tendon. The immunohistochemical staining of caspase 3, 8, and 9 was increased concomitantly in untorn and torn tendons. All of the caspase activities in the untorn articular layer and torn bursal layer were significantly higher than in controls ( P < .05). However, no significant differences were found between the two layers ( P > .05). CONCLUSION: The study demonstrates that apoptotic gene expression is increased not only in the torn bursal layer but also in the untorn articular layer of PTRCTs. CLINICAL RELEVANCE: The untorn articular layer of PTRCTs is abnormal, which triggers postoperative pain and further rotator cuff tears. Therefore, treatment of the abnormal untorn articular layer is essential in bursal-sided PTRCTs.

Induction of Endogenous Neural Stem Cells By Extracorporeal Shock Waves After Spinal Cord Injury.

STUDY DESIGN: Animal experimental study OBJECTIVES.: The purpose of this study is to investigate the effects of extracorporeal shock waves (ESWs) on endogenous neural stem cells (NSCs) proliferation after spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: Exogenous stem cell transplantation for SCI still has many limitations to be addressed such as ideal cell sources, timing of transplantation, and fate of the transplanted cells. Moreover, the efficacy is another issue owing to a peculiar pathologic condition in the chronic phase of SCI. METHODS: Contusive SCI was made using 24 Sprague-Dawley rats, and ESWs were applied at post-injury 4 weeks in rats. Proliferation and differentiation of endogenous NSCs (DCX, Sox-2) and axonal sprouting (GAP-43 and MAP-2) were observed at 6 weeks after application of ESWs. Differentiation of the activated neural stem cells was also investigated by coexpression of neuronal/glial cell markers (GFAP, Neu N, and CC-1). Immunofluorescence staining and western blotting were performed for quantitative analysis, and these results were compared with those in the control group. For clinical assessment, the BBB locomotor rating scale was performed. RESULTS: More proliferation of endogenous neural stem cells was noted in the experimental groups, and these activated cells were mainly founded in the ependymal layer of the central canal and the injured posterior horn. Differentiation into neuronal and glial cells was also noted in a limited number of cells. With respect to axonal regeneration, GAP-43 and MAP-2 expressions in the experimental groups were also significantly higher than those in the control group. During 6 weeks' clinical observation following ESWs application, functional improvement of the hindlimb was observed without clinical deterioration by trials. CONCLUSION: Collectively, these findings indicate that ESWs on the chronic phase of SCI induce activation of endogenous NSCs and consequent functional improvement. LEVEL OF EVIDENCE: N/A.

Transplantation of Mesenchymal Stem Cells for Acute Spinal Cord Injury in Rats: Comparative Study between Intralesional Injection and Scaffold Based Transplantation.

Experimental stem cell therapy for spinal cord injury (SCI) has been extensively investigated. The selection of effective cell transplantation route is also an important issue. Although various types of scaffold have been widely tried as a carrier of stem cells to the injured spinal cord, there was little comparative study to investigate the efficacy of transplantation comparing with conventional transplantation route. A total of 48 Sprague-Dawley rats were subjected to standardized SCI, followed by transplantation of allogeneic mesenchymal stem cells (MSCs), either via intralesional injection (IL group), or via the poly (lactic-co-glycolic acid) (PLGA) scaffold (IP group) or chitosan scaffold (IC group). Engraftment and differentiation of the transplanted cells, expression of neurotrophic factors in the injured spinal cord, and functional recovery were compared with those of the control group. The mean numbers of engrafted MSCs in the IL, IP, and IC groups were 20.6 ± 0.7, 25.6 ± 1.7 and 26.7 ± 1.8 cells/high power filed (HPF), respectively. Results showed higher success rate of MSCs engraftment in the scaffold groups compared to the IL group. Expression of neuroprotective growth factors in the SCI lesions showed no significant differences between the IL, IP, and IC groups. The mean Basso, Beattie and Bresnahan locomotor scales at 6 weeks post-transplantation in the IL, IP, IC, and control groups were 7.9 ± 1.1, 7.9 ± 2.1, 8.7 ± 2.1, and 2.9 ± 1.0, respectively. The functional improvement was most excellent in the IC group. The scaffold based MSC transplantation for acute SCI presented the better cell engraftment and neuroprotective effect compared to the intralesional injection transplantation.

Theoretical Investigation of the Radical-Radical Reaction of O((3)P) + C2H3 and Comparison with Gas-Phase Crossed-Beam Experiments.

Herein, we present an ab initio study of the prototypal radical-radical reactions of ground-state atomic oxygen [O((3)P)] with the vinyl (C2H3) radical using density functional theory and a complete basis set model. Two distinctive pathways on the lowest doublet potential energy surfaces (PESs) were predicted to be in competition: addition and abstraction. The barrierless addition of O((3)P) to the hydrocarbon radicals leads to energy-rich intermediate formation followed by subsequent isomerization and decomposition to yield various products: CH2CO (ketene) + H, CO + CH3, C2HOH (acetylenol) + H, (3,1)CCHOH + H, H2O + C2H, (3,1)CH2 + HCO, H2CO (formaldehyde) + CH, C2H2 (acetylene) + OH, and (3,1)CCH2 + OH. The competing but minor H-atom abstraction mechanisms produce C2H2 + OH and (1,3)CCH2 + OH. The optimized structures of the reactants, products, intermediates, and transition states and the reaction mechanisms were obtained on the lowest doublet PESs. The major pathway was predicted to be the formation of CH2CO + H through the low-barrier, single-step cleavages of the addition intermediates. The Levine-Bernstein prior method, statistical surprisal approach, and microcanonical Rice-Ramsperger-Kassel-Marcus theory were applied to deduce the energy distributions of H atoms and OH products and quantitative rate constants. On the basis of the statistical theory and the population analysis, the predicted energy distributions were compared to the kinetic energy release of H and the preferential population of the Π(A') component of OH products reported in recent gas-phase crossed-beam investigations (Park, M. J.; Jang, S. C.; Choi, J. H. J. Chem. Phys. 2012, 137, 204311), and their kinetic and dynamic characteristics were discussed.

Effects of Therapeutic Hypothermia on Apoptosis and Autophagy After Spinal Cord Injury in Rats.

STUDY DESIGN: Animal study. OBJECTIVE: To further investigate the effects of therapeutic hypothermia (TH), the present study compared autophagy and apoptosis after treatment with either therapeutic moderate systemic hypothermia or methylprednisolone sodium succinate (MP) in a rat model of acute spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: The neuroprotective effects of TH have recently become an important topic in the field of SCI research. METHODS: All rats were subjected to a 25-g/cm spinal cord contusion over the ninth thoracic vertebrae. After the induction of SCI, the control group did not receive any further treatment, TH group immediately received moderate systemic hypothermia for 4 hours, and MP group was administered high-dose MP. The rats were killed either 2 or 7 days after SCI, and the injured spinal cord tissues were obtained. Apoptosis and autophagy were assessed by immunohistochemical analyses and Western blot analyses. In addition, the microarchitecture of the autophagosomes was evaluated using transmission electron microscopy, and the motor activity of the rats was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale. RESULTS: Compared with controls, there was a significant reduction in the expression levels of cleaved caspase-8, -9, and -3 in the TH- and MP-treated groups 2 days after SCI. Moreover, compared with the control group, the expression of LC3II and Beclin-1 exhibited a significant decrease on day 2 after treatment with TH. The numbers of transferase dUTP nicked-end labeling and LC3-positive cells were significantly lower on days 2 and 7. The Basso-Beattie-Bresnahan ratings were significantly higher 6 weeks after SCI in both the TH- and MP-treated groups than in the control group. CONCLUSION: Both TH and MP have neuroprotective effects on injured spinal cord tissues via the inhibition of apoptosis and autophagy. Thus, the application of moderate systemic hypothermia may be a useful treatment modality after acute SCI. LEVEL OF EVIDENCE: N/A.

Does extracorporeal shock wave introduce alteration of microenvironment in cell therapy for chronic spinal cord injury?

STUDY DESIGN: Animal experimental study. OBJECTIVE: To present experimental evidence for cell therapy for spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: In chronic SCI, the efficacy of cell engraftment has been known to be low due to its distinct pathology. Alteration of microenvironment was tried using extracorporeal shock waves (ESW) for chronic SCI, and the efficacy of cell therapy was investigated. METHODS: A chronic contusive SCI model was made in 36 Sprague-Dawley rats. The rats were allocated into (1) control group (SCI only), (2) ESW control group (SCI + ESW), (3) IV group (SCI + intravenous transplantation of mesenchymal stem cells; MSCs), and (4) ESW + IV group (SCI + MSCs IV transplantation after ESW). ESW were applied at the energy determined by our preliminary trials. Engraftment of the cells and expressions of growth factors (brain-derived neurotrophic factor, neuronal growth factor) and cytokines (SDF-1, CXCR4, VEGF) at the epicenter were assessed. The Basso, Beattie, and Bresnahan locomotor scale was used for the clinical assessment. RESULTS: The mean numbers of engrafted cells were higher in the ESW+ IV than that in the IV with a statistical significance. The expression of SDF-1 was higher in the ESW groups than that in the control or IV group. CXCR4 was highly expressed in the transplanted groups. The expressions of growth factors in the treated group were higher in the treated group than those in the control group. However, various statistical significances were noted. The improvement of locomotor was higher in the transplanted groups than that in the control and ESW only group. CONCLUSION: At a given energy level, ESW presented more engraftment of the transplanted MSCs without any clinical deterioration in a chronic SCI. Based on this promising result and possible explanations, ESW may cause an alteration of the microenvironment for the cell therapy in chronic SCI. LEVEL OF EVIDENCE: N/A.

Bone marrow-derived mesenchymal stem cell transplantation for chronic spinal cord injury in rats: comparative study between intralesional and intravenous transplantation.

STUDY DESIGN: Animal experimental study. OBJECTIVE: To present experimental evidence for mesenchymal cell therapy for spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: Prior to clinical application of stem cell therapy for SCI, many critical issues have to be addressed including efficiency, safety, method of transplantation, and differentiation of the transplanted cells. METHODS: Chronic contusive SCI was induced in 36 Sprague-Dawley rats and randomly assigned to the intralesional (IL), intravenous (IV), or control groups. At 6 weeks post injury, allogenic mesenchymal stem cells (MSCs, 1 × 10 cells) were transplanted either intralesionally or intravenously for the intervention groups. Engraftment of the transplanted MSCs was evaluated with PKH 26 staining. Differentiation was evaluated using double stain with neuronal and glial cell markers. Brain-derived neurotrophic factor and nerve growth factor (NGF) were used for neurotrophic factor expression. Basso, Beattie, and Bresnahan locomotor rating scale was used for evaluation of functional recovery. RESULTS: The estimated engraftment percentage of the transplanted cells in the IL group and IV group were 36.5%, and 15.5%, respectively. The engraftment of the transplanted MSCs was higher in the IL group than in the IV group. Most of the transplanted MSCs were colocalized with GFAP in both transplantation groups. Brain-derived neurotrophic factor and NGF expression (Western blot and real-time polymerase chain reaction) in the injured spinal cord was higher in both transplanted groups compared with those in the control group. At 6 weeks post transplantation, the mean Basso, Beattie, and Bresnahan locomotor scales in the IL, IV, and control groups were 5.63 ± 0.89, 5.63 ± 1.03, and 2.88 ± 0.44, respectively. The functional recovery seen in the rats that underwent transplantation was significantly better than that in the control group (P < 0.05). CONCLUSION: Although the number of engrafted cells and expression of neurotrophic factors were lower in the IV group than those in the IL group, both IL and IV transplantation of MSC in the chronic SCI gave a significant clinical improvement. However, there were no differences in differentiation of the transplanted cells between the IL group and IV group. Astrocytic differentiation of the transplanted cells was predominant. LEVEL OF EVIDENCE: N/A.