Co-transplantation Of HbFGF-expressing Amniotic Epithelial Cells And Neural Stem Cells Promote Functional Recovery In Spinal Cord-Injured Rats

World wide, an estimated 2.5 million people live with spinal cord injury (SCI), with more than 130.000 new injuries reported each year. There are no fully restorative therapies for SCI. SCI has a significant impact on quality of life. Spinal cord repair strategies including:①bridging of the lesion site by a regeneration-facilitating tissue;②neuroprotective therapies: delivery of neurotrophins(NTFs) or antibodies against MAIFs;③prevention of scar formation. Many strategies, including surgical, pharmacological, neurophysiological, and technological approaches, have been used in attempts to develop new therapies that will allow patients to recovery.Recently, evidence of axonal regeneration and functional recovery has been reported in animal spinal cord injury models. Developmental biology of neural stem cells (NSCs) contributes to the understanding of the neural regenerations. NSCs isolated and expanded from multiple regions of the CNS showed multiple differentiation phenotypes after transplantation. NSCs could enhance neural repair after SCI either by replacing host cells that have died or by promoting host axonal regeneration. However, survival and neural differentiation following transplantation to the adult CNS have been poor, likely due to a lack of neurotrophic factors. It has been suggested that amniotic epithelial cells(AECs) be known to produce a"cocktail"of trophic factors, such as brain-derived neurotrophic factor(BDNF),neurotrophin-3(NT-3),nerve growth factor(NGF),IL-1,IL-4,IL-6 and so on. It has been reported that bFGF up-regulates the TrkB receptor as a preceding condition for BDNF activity and BDNF, in turn, together with NT-3, may up-regulate the TrkC receptor in anticipation of subsequent NT-3 activity. Thus, cotransplant of NSCs and AECs modified with bFGF gene may be a useful clinical strategy.1. Expression of specific proteins of neural cell in rat amniotic epithelial cellsTo detect specific proteins of neural cells in AECs, AECs were dissociated and purified from the amnion of pregnancy 12 -14d rats. The expression of specific markers of neural stem cells (Nestin, Musashi) and differentiated cells (MAP-2, GFAP) in the AECs were detected by immunocytochemistry. The cultured AECs displayed positive immunoreactivity to MAP-2, GFAP, Nestin and Musashi. In addition, RT-PCR analysis was performed to confirm the expression of BDNF (757bp) and NT3 (796bp) mRNA. The presence of rat BDNF and NT3 protein in CM of RAECs or transwell media were determined by Western bloting. The major bands were observed at the molecular weight 27 kDa (BDNF) or 29kDa (NT3). Cultured RAECs were also found to exhibit immunocytochemically detectable levels of BDNF or NT3 protein. Results indicate that RAEC can secrete NT-3 and BDNF, but not NGF, RAECs are similar with neural cells and it may be useful as a sustained source to improve outcome of neural stem cells transplantation.2. NSCs were co-cultured with amniotic epithelial cells (AECs)Dissociated NSCs from the brain of E12-14 rats by serum-free medium technique. After 1 weeks of expansion, neurospheres were found to be immunoreactive for Bromodeoxyuridine (Brdu),Musashi and Nestin. The cells when plated in the differentiation medium, they rapidly adhered to the tissue culture treated plates and these cells showed positive immunoreactivity to the NSE and GFAP. The present results suggested that these cells have the capable of differentiation into neural cells and glial cells.In the present study, NSCs cultures failed to grow and thrive following withdrawl of bFGF. In contrast, AECs provided a supportive environment that was capable of maitaining NSCs cultures even in the basic media. The study confirmed that AECs could provide a supportive environment for the differentiation of NSCs, appear to promote differentiation toward a neuronal phenotype and prolong the length of neurite of MAP-2-positive cells. The differentiation of NSCs and the length of neurite were enhanced even more in the DC group, which may indicate that the physical limitations of the distance supportive agents can diffuse, or cell-cell contact provided additional and independent support. It also confirmed that the presence of bFGF enhanced the function of AECs. The results showed that AECs may regard as a critical component of NSCs niche and the presence of bFGF enhanced the function of AECs.Such information would circumvent the need for AECs-NSCs coculture and potentially facilitate the production of neurons for future clinical applications.3. Transduction of AECs with retroviral vectors①Cloning human bFGF gene by reverse transcriptase-polymerase chain reaction (RT-PCR);②Ligation the PCR product to TA vector to construct the pMD-hbFGF vector and identification the right clone by digestion of restriction enzyme and sequencing;③Digestion cDNA fragment of bFGF by Bgl-Ⅱand Sal-Ⅰ;④Construction the retroviral vectors pLEGFP-hbFGF by introducing bFGF-2 cDNA into the cloning sites of plEGFP-C1;⑤Transfection PA317 packaging cells with plEGFP-C1, pLEGFP-hbFGF using the lipofection protocol. Transduction of AECs with the supernatant;⑥Detecting the protein expression of bFGF gene in AECs by immunofluorescence.We successfully recombinated pLEGFP-hbFGF and the RAECs transducted by pLEGFP-hbFGF were expressed bFGF protein.4. Co-transplantation of NSCs and AECs modified with hbFGF geneAdult female Wistar rats (300–350 g) were received a 20g contusion SCI at the 9th-to-11th thoracic (T9-T11) spinal vertebrae using an impactor. Forty-six rats were divided into a graft NSCs group (n = 10), a graft NSCs and AECs/ pLEGFP-hbFGF co-transplantation group (n = 10), a graft NSCs and AECs/ plEGFP-C1 co-transplantation group (n = 10), a physiologic saline group (NaCl group, n = 10). The pseduoperation group (n = 6) was served as control group. NSCs labeled with Hoechst 33342.Hindlimb motor function was assessed by using the open-field BBB scoring system. Comparing with the NaCl groups, AECs/ pLEGFP-hbFGF can promote the regeneration and improve the hindlimb motor function. The electrophysiological results showed that the MEP and SEP were easily detected with an amplitude (0.24±0.002; 0.29±0.007) and latency (2.89±0.03; 2.86±0.03) in NSCs and AECs/ pLEGFP-hbFGF co-transplanted rats and were closed to uninjured rats (0.34±0.01; 2.76±0.06 and 0.35±0.03; 2.66±0.12).The histological results showed when transplanted into the injured spinal cord, NSCs or AECs were survived well for a lone time (4 weeks) and integrated well with the host tissue. Immunohistological results showed AECs/ pLEGFP-hbFGF promoted surviving (vs NaCl group: 194±9.17 vs 103.6±13.05 )and neural differention(vs NaCl group: 14.24±1.11 vs 7±0.63 ) of co-transplanted NSCs. They also rescued the atrophy of synapse.Together, these results suggest that AECs were similar to neural cells and could integrate well with host tissue. Both of transplants (AECs and NSCs) completely filled the lesion in the host spinal cord. AECs/ pLEGFP-hbFGF were serving as a NSCs differentiated niche and be useful as a source of sustained trophic supported to improve NSCs differentiation towards neuron in vivo. These findings suggest that AECs/ pLEGFP-hbFGF could have potential benefits for SCI. However, this study represents an initial step toward defining potential applications; additional animal studies are necessary both to establish the mechanisms of recovery and to evaluate the potential of these cells for possible therapeutic use.