3D spheroids of human placenta-derived mesenchymal stem cells attenuate spinal cord injury in mice

Mesenchymal stem cell (MSC) is an absorbing candidate for cell therapy in treating spinal cord injury (SCI) due to its great potential for multiple cell differentiation, mighty paracrine secretion as well as vigorous immunomodulatory effect, of which are beneficial to the improvement of functional recovery post SCI. However, the therapeutic effects of MSC on SCI have been limited because of the gradual loss of MSC stemness in the process of expanding culture. Therefore, in this study, we aimed to maintain those beneficial properties of MSC via three-dimensional spheroid cell culture and then compared them with conventionally-cultured MSCs in the treatment of SCI both in vitro and in vivo with the aid of two-photon microscope. We found that 3D human placenta-derived MSCs (3D-HPMSCs) demonstrated a significant increase in secretion of anti-inflammatory factors and trophic factors like VEGF, PDGF, FGF via QPCR and Bio-Plex assays, and showed great potentials on angiogenesis and neurite morphogenesis when co-cultured with HUVECs or DRGs in vitro. After transplantation into the injured spinal cord, 3D-HPMSCs managed to survive for the entire experiment and retained their advantageous properties in secretion, and exhibited remarkable effects on neuroprotection by minimizing the lesion cavity, inhibiting the inflammation and astrogliosis, and promoting angiogenesis. Further investigation of axonal dieback via two-photon microscope indicated that 3D-HPMSCs could effectively alleviate axonal dieback post injury. Further, mice only treated with 3D-HPMSCs obtained substantial improvement of functional recovery on electrophysiology, BMS score, and Catwalk analysis. RNA sequencing suggested that the 3D-HPMSCs structure organization-related gene was significantly changed, which was likely to potentiate the angiogenesis and inflammation regulation after SCI. These results suggest that 3D-HPMSCs may hold great potential for the treatment of SCI.Subject terms: Spinal cord injury, Mesenchymal stem cells     

Allotransplantation of adult spinal cord tissues after complete transected spinal cord injury: Long-term survival and functional recovery in canines

Science China Life Sciences - Spinal cord injury (SCI), especially complete transected SCI, leads to loss of cells and extracellular matrix and functional impairments. In a previous study, we...     

An allogenic therapeutic strategy for canine spinal cord injury using mesenchymal stem cells

This study was conducted to characterize canine bone marrow-derived mesenchymal stem cells (BMSCs); in vivo tracking in mice, and therapeutic evaluation in canine clinical paraplegia cases. Canine BMSCs were isolated, cultured, and characterized in vitro as per International Society for Cellular Therapy criteria, and successfully differentiated to chondrogenic, osteogenic, and adipogenic lineages. To demonstrate the homing property, the pGL4.51 vector that contained luciferase reporter gene was used to transfect BMSCs. Successfully transfected cells were injected around the skin wound in mice and in vivo imaging was done at 6, 12 and 24 hr post MSCs delivery. In vivo imaging revealed that transfected BMSCs migrated and concentrated predominantly toward the center of the wound. BMSCs were further evaluated for allogenic therapeutic potential in 44 clinical cases of spinal cord injuries (SCI) and compared with conventional therapy (control). Therapeutic potential as evaluated by different body reflexes and recovery score depicted significantly better results in stem cell-treated group compared to control group. In conclusion, allogenic canine BMSCs can serve as potent therapeutic candidate in cell-based therapies, especially for diseases like SCI, where the conventional medication is not so promising.     

Human hepatocyte growth factor promotes functional recovery in primates after spinal cord injury

Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI.     

Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation

Spinal cord injury (SCI) is a severe neurological disease; however, few drugs have been proved to treat SCI effectively. Neuroinflammation is the major pathogenesis of SCI secondary injury and considered to be the therapeutic target of SCI. Salidroside (Sal) has been reported to exert anti‐inflammatory effects in airway, adipose and myocardial tissue; however, the role of Sal in SCI therapeutics has not been clarified. In this study, we showed that Sal could improve the functional recovery of spinal cord in rats as revealed by increased BBB locomotor rating scale, angle of incline, and decreased cavity of spinal cord injury and apoptosis of neurons in vivo. Immunofluorescence double staining of microglia marker and M1/M2 marker demonstrated that Sal could suppress M1 microglia polarization and activate M2 microglia polarization in vivo. To verify how Sal exerts its effects on microglia polarization and neuron protection, we performed the mechanism study in vitro in microglia cell line BV‐2 and neuron cell line PC12. The results showed that Sal prevents apoptosis of PC12 cells in coculture with LPS‐induced M1 BV‐2 microglia, also the inflammatory secretion phenotype of M1 BV‐2 microglia was suppressed by Sal, and further studies demonstrated that autophagic flux regulation through AMPK/mTOR pathway was involved in Sal regulated microglia polarization after SCI. Overall, our study illustrated that Sal could promote spinal cord injury functional recovery in rats, and the mechanism may relate to its microglia polarization modulation through AMPK‐/mTOR‐mediated autophagic flux stimulation.     

Acute leptin treatment enhances functional recovery after spinal cord injury

Background

Spinal cord injury is a major cause of long-term disability and has no current clinically accepted treatment. Leptin, an adipocyte-derived hormone, is best known as a regulator of food intake and energy expenditure. Interestingly, several studies have demonstrated that leptin has significant effects on proliferation and cell survival in different neuropathologies. Here, we sought to evaluate the role of leptin after spinal cord injury.

Findings

Based on its proposed neuroprotective role, we have evaluated the effects of a single, acute intraparenchymal injection of leptin in a clinically relevant animal model of spinal cord injury. As determined by quantitative Real Time-PCR, endogenous leptin and the long isoform of the leptin receptor genes show time-dependent variations in their expression in the healthy and injured adult spinal cord. Immunohistochemical analysis of post-injury tissue showed the long isoform of the leptin receptor expression in oligodendrocytes and, to a lesser extent, in astrocytes, microglia/macrophages and neurons. Moreover, leptin administered after spinal cord injury increased the expression of neuroprotective genes, reduced caspase-3 activity and decreased the expression of pro-inflammatory molecules. In addition, histological analysis performed at the completion of the study showed that leptin treatment reduced microglial reactivity and increased caudal myelin preservation, but it did not modulate astroglial reactivity. Consequently, leptin improved the recovery of sensory and locomotor functioning.

Conclusions

Our data suggest that leptin has a prominent neuroprotective and anti-inflammatory role in spinal cord damage and highlights leptin as a promising therapeutic agent.     

cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury

Central neurons regenerate axons if a permissive environment is provided; after spinal cord injury, however, inhibitory molecules are present that make the local environment nonpermissive. A promising new strategy for inducing neurons to overcome inhibitory signals is to activate cAMP signaling. Here we show that cAMP levels fall in the rostral spinal cord, sensorimotor cortex and brainstem after spinal cord contusion. Inhibition of cAMP hydrolysis by the phosphodiesterase IV inhibitor rolipram prevents this decrease and when combined with Schwann cell grafts promotes significant supraspinal and proprioceptive axon sparing and myelination. Furthermore, combining rolipram with an injection of db-cAMP near the graft not only prevents the drop in cAMP levels but increases them above those in uninjured controls. This further enhances axonal sparing and myelination, promotes growth of serotonergic fibers into and beyond grafts, and significantly improves locomotion. These findings show that cAMP levels are key for protection, growth and myelination of injured CNS axons in vivo and recovery of function.     

Injectable collagen scaffold promotes swine myocardial infarction recovery by long-term local retention of transplanted human umbilical cord mesenchymal stem cells

Stem cell therapy is an attractive approach for recovery from myocardial infarction(MI) but faces the challenges of rapid diffusion and poor survival after transplantation. Here we developed an injectable collagen scaffold to promote the long-term retention of transplanted cells in chronic MI. Forty-five minipigs underwent left anterior descending artery(LAD) ligation and were equally divided into three groups 2 months later(collagen scaffold loading with human umbilical mesenchymal stem cell(hUMSC) group, hUMSC group, and placebo group(only phosphate-buffered saline(PBS) injection)). Immunofluorescence staining indicated that the retention of transplanted cells was promoted by the collagen scaffold. Echocardiography and cardiac magnetic resonance imaging(CMR) showed much higher left ventricular ejection fraction(LVEF) and lower infarct size percentage in the collagen/hUMSC group than in the hUMSC and placebo groups at 12 months after treatment. There were also higher densities of vWf-, α-sma-, and cTnT-positive cells in the infarct border zone in the collagen/cell group, as revealed by immunohistochemical analysis, suggesting better angiogenesis and more cardiomyocyte survival after MI. Thus, the injectable collagen scaffold was safe and effective on a large animal myocardial model, which is beneficial for constructing a favorable microenvironment for applying stem cells in clinical MI.     

P2X7 receptor inhibition improves recovery after spinal cord injury

Secondary injury exacerbates the extent of spinal cord insults, yet the mechanistic basis of this phenomenon has largely been unexplored. Here we report that broad regions of the peritraumatic zone are characterized by a sustained process of pathologic, high ATP release. Spinal cord neurons expressed P2X7 purine receptors (P2X7R), and exposure to ATP led to high-frequency spiking, irreversible increases in cytosolic calcium and cell death. To assess the potential effect of P2X7R blockade in ameliorating acute spinal cord injury (SCI), we delivered P2X7R antagonists OxATP or PPADS to rats after acute impact injury. We found that both OxATP and PPADS significantly improved functional recovery and diminished cell death in the peritraumatic zone. These observations demonstrate that SCI is associated with prolonged purinergic receptor activation, which results in excitotoxicity-based neuronal degeneration. P2X7R antagonists inhibit this process, reducing both the histological extent and functional sequelae of acute SCI.     

Mesenchymal stem cell graft improves recovery after spinal cord injury in adult rats through neurotrophic and pro-angiogenic actions

Numerous strategies have been managed to improve functional recovery after spinal cord injury (SCI) but an optimal strategy doesn't exist yet. Actually, it is the complexity of the injured spinal cord pathophysiology that begets the multifactorial approaches assessed to favour tissue protection, axonal regrowth and functional recovery. In this context, it appears that mesenchymal stem cells (MSCs) could take an interesting part. The aim of this study is to graft MSCs after a spinal cord compression injury in adult rat to assess their effect on functional recovery and to highlight their mechanisms of action. We found that in intravenously grafted animals, MSCs induce, as early as 1 week after the graft, an improvement of their open field and grid navigation scores compared to control animals. At the histological analysis of their dissected spinal cord, no MSCs were found within the host despite their BrdU labelling performed before the graft, whatever the delay observed: 7, 14 or 21 days. However, a cytokine array performed on spinal cord extracts 3 days after MSC graft reveals a significant increase of NGF expression in the injured tissue. Also, a significant tissue sparing effect of MSC graft was observed. Finally, we also show that MSCs promote vascularisation, as the density of blood vessels within the lesioned area was higher in grafted rats. In conclusion, we bring here some new evidences that MSCs most likely act throughout their secretions and not via their own integration/differentiation within the host tissue.     

Gramine promotes functional recovery after spinal cord injury via ameliorating microglia activation

In recent years, a large number of studies have reported that neuroinflammation aggravates the occurrence of secondary injury after spinal cord injury. Gramine (GM), a natural indole alkaloid, possesses various pharmacological properties; however, the anti-inflammation property remains unclear. In our study, Gramine was investigated in vitro and in vivo to explore the neuroprotection effects. In vitro experiment, our results suggest that Gramine treatment can inhibit release of pro-inflammatory mediators. Moreover, Gramine prevented apoptosis of PC12 cells which was caused by activated HAPI microglia, and the inflammatory secretion ability of microglia was inhibited by Gramine through NF-κB pathway. The in vivo experiment is that 80 mg/kg Gramine was injected orthotopically to rats after spinal cord injury (SCI). Behavioural and histological analyses demonstrated that Gramine treatment may alleviate microglia activation and then boost recovery of motor function after SCI. Overall, our research has demonstrated that Gramine exerts suppressed microglia activation and promotes motor functional recovery after SCI through NF-κB pathway, which may put forward the prospect of clinical treatment of inflammation-related central nervous diseases.     

BMP inhibition enhances axonal growth and functional recovery after spinal cord injury

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor-β superfamily. BMPs regulate several crucial aspects of embryonic development and organogenesis. The reemergence of BMPs in the injured adult CNS suggests their involvement in the pathogenesis of the lesion. Here, we demonstrate that BMPs are potent inhibitors of axonal regeneration in the adult spinal cord. The expression of BMP-2/4 is elevated in oligodendrocytes and astrocytes around the injury site following spinal cord contusion. Intrathecal administration of noggin – a soluble BMP antagonist—leads to enhanced locomotor activity and reveals significant regrowth of the corticospinal tract after spinal cord contusion. Thus, BMPs play a role in inhibiting axonal regeneration and limiting functional recovery following injury to the CNS.     

Transplantation of specific human astrocytes promotes functional recovery after spinal cord injury

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system.     

Clinical analysis of the treatment of spinal cord injury with umbilical cord mesenchymal stem cells

Background aimsThe purpose of this study was to observe the clinical effect and safety of umbilical cord mesenchymal stem cells (UC-MSCs) in treating spinal cord injury (SCI) by intrathecal injection.MethodsFrom January 2008 to October 2010, we treated 22 patients with SCI with UC-MSCs by intrathecal injection; dosage was 1 × 10⁶ cells/kg body weight once a week given four times as a course. Four patients received two courses, one patient received three courses and all other patients received one course. American Spinal Injury Association scoring system and International Association of Neurorestoratology Spinal Cord Injury Functional Rating Scale were used to evaluate neural function and ability to perform activities of daily living.ResultsTreatment was effective in 13 of 22 patients; nine patients had no response. Among patients with incomplete SCI, the response to treatment was 81.25%; there was no response to treatment among six patients with complete SCI. Five patients with a response to treatment received two to three courses of therapy, and effects in these patients were further enhanced. In most patients in whom treatment was effective, motor or sensory functions, or both, were improved, and bowel and bladder control ability was improved. In 22 patients 1 month after therapy, algesia, tactile sensation, motion and activity of daily living scale were significantly improved (P < 0.01). During therapy, common adverse effects were headache (one case) and low back pain (one cases); these disappeared within 1–3 days. No treatment-related adverse events occurred during a follow-up period ranging from 3 months to 3 years.ConclusionsUC-MSC therapy by intrathecal injection is safe and can improve neurologic function and quality of life in most patients with incomplete SCI.     

骨髓间充质干细胞移植对急性脊髓损伤脱髓鞘病变的保护作用

骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)已被广泛应用于治疗脊髓损伤,但目前对其治疗机制了解甚少。BMSCs被移植至脊髓钳夹损伤模型大鼠,以研究其保护作用。通过LFB(Luxol fast blue)染色、锇酸染色、TUNEL(Td T-mediated d UTP nick-end labeling)染色和透射电镜对白质有髓神经纤维进行观察。免疫印迹检测BMSCs移植对脑源性神经营养因子(Brain derived neurotrophic factor,BDNF)和caspase 3蛋白表达的影响。通过脊髓损伤后1、7、14 d三个时间点移植BMSCs并进行后肢运动评分(Basso,beattie and bresnahan;BBB评分)和CNPase(2′,3′-cyclic-nucleotide 3′-phosphodiesterase)、髓鞘碱性蛋白(Myelin basic protein,MBP)、caspase 3蛋白水平的检测。免疫荧光观察BMSCs移植到受损脊髓后分化情况及CNPase-caspase 3~+共表达情况。骨髓间充质干细胞移植7 d后,部分移植的BMSCs可表达神经元和少突胶质细胞标记物,大鼠后肢运动能力和髓鞘超微结构特征均明显改善。骨髓间充质干细胞移植后BDNF蛋白表达水平增加,caspase 3蛋白表达水平则降低。相对于脊髓损伤后1 d和14 d,7 d移植BMSCs后MBP和CNPase蛋白表达水平最高;caspase 3蛋白表达水平则最低。骨髓间充质干细胞移植后CNPase-caspase 3~+细胞散在分布于脊髓白质。结果表明,急性脊髓损伤后,BMSCs移植到受损脊髓有分化为神经元和少突胶质细胞的倾向,并促进BDNF的分泌介导抗少突胶质细胞凋亡而对神经脱髓鞘病变有保护作用,且最佳移植时间为脊髓损伤后7 d。     

Valproic acid attenuates blood-spinal cord barrier disruption by inhibiting matrix metalloprotease-9 activity and improves functional recovery after spinal cord injury

In a retinal ischemic ex vivo model, we have reported protective effects of somatostatin (SRIF) receptor 2 (sst(2) ). As an ischemic condition not only causes cell death but also induces a vascular response, we asked whether vascular endothelial growth factor (VEGF) is altered in this model and whether its expression, release or localization are affected by sst(2) activation. Ex vivo retinas of wild-type (WT) and sst(1) KO mice (which over-express sst(2) ) were incubated in ischemic conditions with SRIF, octreotide (OCT) or a VEGF trap. Ischemia in WT retinas caused increase of VEGF release and decrease of VEGF mRNA. Both effects were counteracted by SRIF or OCT. VEGF immunoreactivity was in retinal neurons and scarcely in vessels. Ischemia caused a significant shift of VEGF immunoreactivity from neurons to vessels. The increase of vascular VEGF was reduced in sst(1) KO retinas and in WT retinas treated with SRIF or OCT. VEGF trap also limited this increase, demonstrating that vascular VEGF was of extracellular origin. Together, the data show a VEGF response to ischemia, in which VEGF released by damaged neurons reaches the retinal capillaries. The activation of sst(2) protects neurons from ischemic damage, thereby limiting VEGF release and the VEGF response.     

Umbilical cord blood mesenchymal stem cells application in hematopoietic stem cells expansion on nanofiber three-dimensional scaffold

Umbilical cord blood (UCB) hematopoietic stem cells (HSCs) transplantation (HSCTs) is considered as a therapeutic strategy for malignant and nonmalignant hematologic disorders. Nevertheless, the low number of HSCs obtained from each unit of UCB can be a major challenge for using these cells in adults. In addition, UCB is a rich source of mesenchymal stem cells (MSCs) creating hopes for nonaggressive and painless treatment in tissue engineering compared with bone marrow MSCs. This study was designed to evaluate the effects of UCB-MSCs application in UCB-HSCs expansion on the nanoscaffold that mimics the cell's natural niche. To achieve this goal, after flow cytometry confirmation of isolated HSCs from UCB, they were expanded on three-dimensional (3D) poly-l -lactic acid (PLLA) scaffolds fabricated by electrospinning and two-dimensional (2D)-culture systems, such as (1) HSCs-MSCs culturing on the scaffold, (2) HSCs culturing on the scaffold, (3) HSCs-MSCs culturing on 2D, and (4) HSCs culturing on 2D. After 7 days, real-time polymerase chain reaction (PCR) was performed to evaluate the CXCR4 gene expression in the mentioned groups. Moreover, for the next validation, the number of total HSCs, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay, scanning electron microscopy imaging, and colony-forming unit assay were evaluated as well. The results of the study indicated that UCB-MSCs interaction with HSCs in 3D-culture systems led to the highest expansion of UCB-HSCs on day 7. Flow cytometry results showed the highest purity of HSCs cocultured with MSCs. Real-time PCR showed a significant increase in gene expression of CXCR4 in the mentioned group. The highest viability and clonogenicity were detected in the mentioned group too. Considered together, our results suggest that UCB-HSCs and MSCs coculturing on PLLA scaffold could provide a proper microenvironment that efficiently promotes UCB-HSCs expansion and UCB-MSCs can also be considered as a promising candidate for UCB-HSCTs.