SPSS software version For multiple group comparisons, analysis of variance with Tukey's post hoc test was applied. The distribution appeared scattered in cell suspension, but with excellent refraction. At day 3 in primary culture, small cell spheres arose in the liquid and grew in a clustered and floating manner.
At day 5 in primary culture, spherical colonies formed by dozens of cells could be observed. With the development of time, the number and the diameter of the spherical colonies markedly increased. Following continuous passaging, at P1 and P2, greater numbers of NSCs were obtained, whose growth characteristics were similar to that of the original generation Fig.
In close approximation of our expectations, the rate of Nestin-positive expression was The GFAP-positive rate was DAPI stained the nuclei with blue florescence middle. White arrows represent the positive cells. Cells from five fields in each well were collected, and each detection in vitro was prepared for 6 plates 6-pore plate of cells. D Representative bar graph for the rate of positive cells. NGF had a positive rate of White arrows represented the positive cells.
Cells from five fields in each well were collected, each detection in vitro was prepared for six plates six-pore plate of cells. Over time, the locomotor function could be partially restored. At day 7 post-injury, the hind-limb locomotor functions had recovered spontaneously to an approximate BBB score of By contrast, the acute group did not show any functional recovery compared with the acute control group Fig. B The chronic group exhibited significantly better functional recovery than the chronic control group at day 16 post-injury.
Arrowheads indicate the transplantation time.
Historical Development of Neural Transplantation
At day 16 post-injury in the chronic group, a large number of surviving NSCs with blue nuclei staining labeled by Hoechst could be found in the spinal cord of the NSC-transplanted TSs near the needle passage , whereas there were no Hoechst-positive cells in the control group; the results indicated that NSCs could survive and migrate in the spinal cord around the injection site Fig.
In order to detect the differentiation of NSCs in the host spinal cord, the immunohistochemical staining of specific markers was performed to recognize neurons and astrocytes, as performed in vitro. The merged images revealed that in the chronic group, certain grafted cells differentiated into NeuN-positive cells A NSCs labeled with Hoechst blue fluorescence, left survived and migrated at day 16 post-injury in vivo. The control exhibited no positive reactivity right. White arrows indicate the positive cells.
The images were captured 1 cm below the lesion.
NSC group. Following transplantation in the chronic phase of hSCI, the BBB score revealed that the locomotor function in the hind-limbs was improved, but that there was no statistical significance in the acute-phase transplantation, when compared with the control group. To the best of our knowledge, these findings, for the first time, indicate that the transplantation of NSCs from TSs is available for neurological function improvement following hSCI, but only in the acute phase, and that the expression of multiple NTFs linked with the upregulation of NGF is probably involved in the underlying mechanism.
In the experiments assessing the proliferation of NSCs from TSs, it was confirmed that the NSCs possessed the capacity to proliferate in vitro and in vivo. The area of neurosphere formation kept increasing as well throughout the culture, which innovatively illustrated that NSCs from TSs can successfully proliferate in vitro. These results suggested that NSCs from TSs could be considered as an available cell source for the treatment of disease. The majority of NSC experimental models have been focused on rodents and few studies have involved the use of primates or humans due to the associated ethical issues and the lack of availability 33 - Compared with rats, TSs exhibit biological characteristics and gross anatomy that are more similar to those of humans 24 , Furthermore, TSs have a lower economic cost and are a more convenient resource than other animals.
The present study verified that NSCs from TSs could differentiate into neurons and astrocytes in vivo and in vitro , which may replace the damaged nerve cells to restore the structure of the injured spinal cord following transplantation. Over the past decades, NSCs have been reported to exhibit multi-directional differentiation to neurons and astrocytes 13 , 21 , 36 , However, the differentiation ability of NSCs in non-human primates, such as TSs, was previously unknown.
To the best of our knowledge, the present study was the first to demonstrate the differentiation characteristics of NSCs from TSs. The present experiments therefore provided crucial evidence that NSCs can differentiate into neurons and glial cells, which assist in reconstructing neural injury.
In the present study, the BBB score exhibited no significant difference in terms of varying observation points in the acute phase group compared with those in the acute control group; this indicated that NSC implantation in the chronic phase could contribute to the recovery of nervous function in TSs, but not in the acute phase. A number of studies found that the degenerative degree of the spinal cord tissue near the spinal cord transection or contusion was reduced significantly following NSC transplantation in the chronic phase 40 , 41 , but associated mechanisms involved in the NTFs were not mentioned, let alone the non-human primate tree shrew model.
Therefore, the present study obtained novel findings that NSC transplantation into TSs in the chronic phase could effectively improve nervous function, which may be linked to the secretion of NTFs in the chronic group. According to previous studies, there are two main hypotheses for the effectively promotion of neural functional recovery by NSCs: The alternative theory and the nutrition theory.
Previous studies have demonstrated that NSC transplantation can differentiate into neurons and glial cells to repair the neuronal necrosis, but little evidence shows that grafted NSCs can integrate into the neural networks of the host. Therefore, whether grafted NSCs can integrate into host neural network has become important for research, as this may help to further interpret the role of NSCs transplantation in the restoration of nerve function 14 - 17 , 42 - These results will aid in understanding the molecular mechanisms for stem cell therapy in diseases of non-human primates, which may ultimately become available to future patients in the clinic.
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Wang D and Zhang J: Effects of hypothermia combined with neural stem cell transplantation on recovery of neurological function in rats with spinal cord injury. Mol Med Rep. Chin J Tissue Eng Res. Iran J Basic Med Sci. Nat Commun. Virol J. Grytz R and Siegwart JT Jr: Changing material properties of the tree shrew sclera during minus lens compensation and recovery. Invest Ophthalmol Vis Sci. Dongwuxue Yanjiu. Neurosci Lett. Lab Anim Res. Reynolds BA and Weiss S: Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.
Vroemen M, Aigner L, Winkler J and Weidner N: Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways.
Eur J Neurosci. Sci Rep. J Neurosci Res. Mol Brain. June Volume 41 Issue 6. Sign up for eToc alerts. Data Availability: All relevant data are within the paper and its Supporting Information files.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Proper brain function requires a strict balance between neuronal excitation and inhibition [ 1 — 2 ]. Reduced inhibition e. Cell-based therapy to replace lost or malfunctioning inhibitory interneurons has been hailed as a potential biologic therapeutic for these disorders [ 6 — 10 ]. Previous studies have demonstrated that neural stem and progenitor cells from animal embryos and fetuses possess the capacity to differentiate into GABAergic interneurons that form functional synaptic connections and integrate into the host brain circuitry when transplanted into animals [ 11 — 12 ].
Transplanted human embryonic and fetal stem cells in both younger and adult animals can develop into regionally appropriate neuron types including interneurons [ 13 — 20 ]. Previous studies have revealed that transplanted animal and human embryonic stem cell-derived GABAergic neuron precursors can attenuate behavioral deficits in rodent models of human disorders [ 2 , 5 , 7 , 17 , 23 , 30 — 32 ].
Clinical benefit has been reported in some patients with human stem cell transplantation, such as Huntington's disease [ 33 ], amyotrophic lateral sclerosis [ 34 ] and Pelizaeus-Merzbacher Disease [ 35 ]. The major goal of human stem cell transplantation for neurodegenerative disorders is to elucidate its role in disease treatment. To achieve this goal it is essential to investigate both the specific phenotypes of transplanted stem cells and the ability of these cells to influence the behavior of the host neural circuitry in animal studies.
Transplanted animal stem and progenitor cells that can generate different types of neurons have been studied intensively.
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However, human stem cell transplantation has not been investigated to the same degree. This study investigated the electrophysiological and histological properties of different types of neurons derived from transplanted human neural precursor cells hNPCs. GABAergic interneurons can be distinguished by their electrophysiology and expression of specific molecular markers [ 37 ]. GABAergic interneurons expressing the calcium-binding proteins, parvalbumin PV or calretinin CR , or the neuropeptide, somatostatin SS , comprise three separate families of interneurons, which account for the majority of neocortical GABAergic interneurons [ 37 — 38 ].
Postnatal day 2 P2 mice received transplantation of hNPCs into the neocortex. Offspring were weaned on P Male or female offspring were used for electrophysiological and histological experiments at 8 weeks 8 W, P56—P61 after transplantation. All procedures were performed in accordance with guidelines approved by the National Institutes of Health and the Institutional Animal Care and Use Committee at the University of Florida. Human NPCs were derived from the telencephalon of a single fetus after routine legal abortion at ten weeks of age, as previously published [ 39 — 41 ].
Fresh medium was added the next day. Cells were serially passaged using the non-adherent culturing technique called the Neurosphere Assay [ 43 — 45 ]. The culture was passaged after every 10 days by first collecting and pelleting the neurospheres.