Transformation of human amniotic epithelial cells into neuron-like cells in the microenvironment of traumatic brain injury in vivo and in vitro

Survival and differentiation of transplanted cells is closely related to the local microenvironment.The present study cultured human amniotic epithelial cells（HAECs） in a simulated microenvironment in vitro comprising RPMI 1640 culture medium and the solution extracted from injured brain tissues.Some HAECs were round,triangular in form or irregularly shaped,with extended neuron-like processes;some of the processes were interconnected,representing neuron-like morphology and some HAECs were microtubule-associated protein 2-positive.HAECs survived for at least 4 weeks following transplantation into the center and edges of the trauma focus with traumatic brain injury,and were microtubule-associated protein 2-positive.Moreover,the motor function of rat hind limbs was significantly improved.

Effect of human autologous serum and fetal bovine serum on human corneal epithelial cell viability,migration and proliferation in vitro

AIM： To analyze the concentration-dependent effects of autologous serum （AS） and fetal bovine serum （FBS） on human corneal epithelial cell （HCEC） viability, migration and proliferation. METHODS： AS was prepared from 13 patients with non- healing epithelial defects Dulbecco＇s modified eagle medium/ Ham＇s F12 （DMEM/F12） with 5% FBS, 0.5% dimethyl sulphoxide （DMSO）, 10 ng/mL human epidermal growth factor, 1% insulin-transferrin-selenium, then were incubated in serum media： DMEM/F12 supplemented by 5%, 10%, 15% or 30% AS or FBS. HCEC viability was analyzed using cell proliferation kit XTI＇, migration using a wound healing assay, proliferation by the cell proliferation enzyme-linked immunosorbent assay （ELISA） BrdU kit. Statistical analysis was performed using the generalized linear model, the values at 30% AS or 30% FBS were used as the baselines. RESULTS： HCEC viability was the highest at 30% AS or 15% FBS and the lowest at 10% AS or 30% FBS application. HCEC migration was the quickest through 30% AS or 30% FBS and the slowest through 5% AS or 5% FBS concentrations. Proliferation was the most increased through 15% AS or 5% FBS and the least increased through 30% AS or 30% FBS concentrations. HCEC viability at 10% and 15% AS was significantly worse （P=0.001, P=0.023） compared to baseline and significantly better at 15% FBS （P=0.003） concentrations. HCEC migration was significantly worse （P〈0.007） and HCEC proliferation significantly better （P〈0.001） in all concentration groups compared to baseline. CONCLUSION： For the best viability of HCEC 30% AS or 15% FBS, for HCEC migration 30% AS or 30% FBS, for proliferation 15% AS or 5% FBS should be used. Therefore, we suggest the use of 30% AS in clinical practice.

Role of soluble factors and three-dimensional culture in in vitro differentiation of intestinal macrophages

AIM: To examine the factor(s) involved in differentiation of intestinal macrophages (IMACs) using a recently established in vitro model. METHODS: To test whether soluble or membrane bound factors induce IMAC-differentiation, freshly elutriated monocytes (MO) were incubated with conditioned media or cell membranes of intestinal epithelial cells (IEC) or cultured with IEC in transwell systems. To determine the importance of an active migration of MO, three- dimensional aggregates from a 1:1-mixture of MO and IEC were examined by immunohistochemistry and flow cytometry. Apoptosis was examined by caspase-3 Western blots. Extracellular matrix production in differentiation models was compared by immunohistochemistry. RESULTS: IMAC differentiation was observed in a complex three-dimensional co-culture model (multicellular spheroid, MCS) with IEC after migration of MO into the spheroids. By co-culture of MO with conditioned media or membrane preparations of IEC no IMAC differentiation was induced. Co-culture of MO with IEC in transwell- cultures, with the two cell populations separated by a membrane also did not result in intestinal-like differentiation of MO. In contrast to IEC-spheroids with immigrating MO in mixed MCS of IEC and MO only a small subpopulation of MO was able to survive the seven day culture period. CONCLUSION: Intestinal-like differentiation of MO in vitro is only induced in the complex three-dimensional MCS model after immigration of MO indicating a roleof cell-matrix and/or cell-cell interactions during the differentiation of IMACs.

Effect of Aβ protein on inhibiting proliferation and promoting apoptosis of retinal pigment epithelial cells

AIM： To identify the effect and regulatory mechanism of amyloid β （Aβ） protein on retinal pigment epithelial （RPE） cells in cell proliferation and apoptosis, and clarify Aβ role in the pathogenesis of age-related macular degeneration （AMD）. METHODS： The model of Aβ25-35 protein cytotoxicity in RPE cell was successfully established to investigate the effect of Aβ protein on RPE cells in vitro. Based on Aβ protein, the specific inhibitors （HY-50682 or BAY11-7082） or activating agent （lipopolysaccharide） was used to analyze the regulatory mechanism of Aβ protein to RPE cells on cell proliferation and apoptosis by flow cytometry, real-time polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay and dual-luciferase reporter gene assay. RESULTS： The number of RPE cells, treated with Aβ25-35 from 0.3 to 60 μmol/L, significantly reduce （P〈0.01）, and had the dose-dependent effect. Aβ protein 60 μmol/L inhibits the G1/S phase transition （P〈0.01） and down-regulated cyclin E mRNA level （P〈0.01）. Similarly, Aβ25-35 induced a significant increase of cell apoptosis, accompanied by the significantly higher level of activated caspase 3 protein. Furthermore, nuclear factor-kappaB （NF-κB） activity and hosphorylated Iκ-Ba level would significantly lower in treated RPE cells. Using specific inhibitors or activating agent based on the Aβ, the cell numbers, NF-κB activity, phosphorylated Iκ-Ba level, receptor for advanced glycation endproducts （RAGE） gene expression levels, cyclin E mRNA level and activated caspase 3 level had accordingly changed by different methods, confirming that RAGE/NF-κB signaling pathway involved in the regulation of Aβ protein on RPE cell apoptosis and proliferation. CONCLUSION： Aβ protein inhibits cell proliferation and activates apoptosis via inactivation of the RAGE/NF-κB signaling pathway in RPE cell.