| IntroductionSpinal cord injury (SCI) is one of the most devastating forms of trauma experienced by humans, often resulting in permanent disability. As reported by the Foundation for Spinal Cord Injury Prevention, Care, and Cure, there are approximately450,000people living with SCI, and an additional11,000new SCI cases occur every year in the US. The repair of SCI is still a major therapeutic challenge at present, because endogenous repair following SCI in adult mammals is restricted. Damage caused by inflammatory cells, glia scar formation and decreased intrinsic growth drive of adult neurons, as well as the inhibitory effect of myelin in the central nervous system, may all play a part.Exogenous intervention strategies are necessary to enhance recovery. As many studies performed, cell transplantation therapy has emerged as a powerful and promising repair strategy for enhancing restitution of the lost function. The goals of cell transplantation therapy were vary widely include replacing damaged neurons, filling the cystic cavity, enhancing axonal regeneration by creating a regenerative environment, and supporting or inducing remyelination. Different types of cells have been evaluated as therapeutic strategies for post-SCI cell transplantation including embryonic stem cells, neural or glial precursor cells, genetically modified fibroblasts, mesenchymal stem cells, olfactory ensheathing cells, and Schwann cells. Although recent dramatic progress in cellular transplantation has heightened the optimism about future cures for such injuries, development of powerful strategies to treat SCI is still a major clinical challenge.Previous studies report that stem cells isolated from neural crest-derived tissue have multidifferentiation ability, such as in dental tissue. Haekwon Kim et al isolated neural crest-like stem cells from human eyelid adipose tissue. These human eyelid adipose-derived stem cells (hEASCs) are of the bipolar neuronal shape, whereas other Mesenchymal stem cells (MSCs) are spindle shaped. hEASCs and MSC are not only different in morphology, but cells characteristics are also different. Undifferentiated hEASCs spontaneously express many neural cell-related mRNAs and proteins, most of which are observed in human neural crest cells. In contrast, MSCs from the trunk adipose tissues do not show most of these neural cell-like characters unless be induced. Therefore hEASCs have unique characteristics that favor their use in transplantation strategies for SCI repair. To the best of our knowledge, no previous study used this cell for treatment SCI. Thus, in this study we conducted to assess the effect of hEASCs transplantation in a rat SCI model for SCI repair. However, previous studies also show that the low survival rate of graft stem cells alone after transplantation into lesion tissue is a major obstacle for successful stem cell therapy.Accordingly, several experimental studies have shown17-β-estradiol (E2) has neuroprotective properties and produces therapeutic effects in various models of central nervous diseases. E2has a protective effect against oxidant, inflammatory and apoptotic, able to attenuate cell death in vitro and reduce secondary damage in vivo investigations. Recent research suggests that E2decreases lesion volume and attenuates apoptotic cell death following SCI.Previous many studies have reported that estrogen alone plays the role of SCI repair. However, the protective potential for combination E2and stem cell has not yet been investigated in SCI. Therefore, we put forward a hypothesis that combination pre-administration E2with hEASCs transplantation after SCI in a rat model will promote functional recovery of paralyzed rats, beneficial for SCI repair. A broader understanding of the histopathology and functional outcomes of thoracic SCI could hasten the identification of appropriate therapeutic targets for this injury and support the translation of potential therapeutics to the sizable thoracic SCI population.Stage1Isolation and characterization of hEASCsAim:hEASCs were isolated from human eyelid adipose after eyelid reshaping surgery, cultured and characterized.Methods and results:Cell culture and fluorescence-activated cell sorting (FACS) analysis.The multi-differentiation potential, gene expression profile and proliferation assay, neural differentiation capacity and neural specific genes and protein markers of hEASCs were investigated in vitro. The results showed that hEASCs exhibited some parallel characteristics typical of MSCs (CD105, CD29, CD166and CD44), and null expression of hemopoietic stem cell marker(CD34)and bone marrow stromal cell maker (CD18). hEASCs have the capbiltiy of osteogenic, adipogenic and chondrogenic differentiation, and spontaneous expressed many neural cell-related mRNAs and proteins which would be enhanced after induced.Conclusion:This study thus demonstrated that the use of hEASCs as a source for human transplant populations not only possess the inherently broad capacity of expansion and differentiation, but also offers advantages over other cell types, and do not violate ethical. Stage2Transplantation hEASCs into rat spinal cord model in biodegradable scaffold with E2for spinal cord repairAim:The aim of this study was to investigate their therapeutic potentials for SCI repair, and whether the combination of HEACs and E2is a potential therapy method for SCI.Methods and results:We first set up the animal model of SCI at10th thoracic vertebras (T10) by hemisection at the right side. A lateral slit in the dura was generated, and then right hemisection was created at T10. The SCI rats were randomly divided into three groups,17animals for each group. The first group (sham control) underwent sterile phosphate buffer saline (PBS) injection. The second group was injected with hEASCs after SCI7days. The third group was treated with the combination of E2subcutaneous injection and hEASCs transplantation. In the third group E2was also administered at a dose of100μg/kg15min after SCI, daily for the next14days. After6weeks, hEASCs in vivo continue to express motor neuron marker microtubule associated protein (MAP2), and oligodendrocyte marker galactosidase (GALAC), do not express astrocyte markers glial fibrillary acidic portein (GFAP). These results suggested the implanted hEASCs maybe differentiate to neurons and oligodendrocytes, however not contribute to astrocyte, which is beneficial to the SCI repair. Furthermore, the hEASCs groups obviously reduced cavity formation compared SCI vehicle groups. The xenograft cell expressing growth factors (igf-1, ngf, hgf) in vivo might have the effect of improve local environment, reduce hollow, and promote axonal regeneration in transplantation treatment of SCI. These results suggested this cells hEASCs can be used as a promising seed cell for the treatment of SCI. Compared the pure cells group, the E2-hEASCs group have obvious improvement. We detected the human (3-actin expression after transplanted cell7days. The combination significantly promotes the survival of hEASCs at acute phase of SCI when plenty of cell death happens. Such survival should conduce to the consequence of increased remyelination, and subsequently significantly improvement in hind limb motor function as determined by Basso, Beattie, and Bresnahan locomotor open field behavioral rating test (BBB) and Grid-walking test. From Toluidine Blue staining, E2increased peripheral mylinated axons in grafts. Furthermore, the E2-hEASCs group effectively reduced the apoptotic cell death and caspase-3activity, compared the only cells group after SCI.Conclusion:This study demonstrated that unique properties of hEASCs combined with E2might cooperatively work, significantly increase hEASCs survival after transplantation, promote axonal regeneration and improves histological outcomes that correlate with improved recovery. This finding highlights that the combination of E2and hEASCs transplantation may be a highly efficient therapeutic approach for SCI.
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