A novel biodegradable polymer scaffold for in vitro growth of corneal epithelial cells

The shortage of donor corneal tissue worldwide has led to extensive research for alternate corneal equivalents utilizing tissue engineering methods. We conducted experiments using Poly D, L lactic acid polymer along with a copolymer (Eudragit) in varying concentrations to create a biodegradable scaffold suitable for in vitro growth of corneal epithelial stem cells. It was found that stable, spherical, and porous microparticles can be prepared by combining PDLLA and Eudragit RL100 polymers in the ratio of 90:10 and 70:30. The microparticles can then be fused to form scaffold membranes with porous architecture and good water retention capacity at room temperature using methanol, which can withstand handling during transplantation procedures. The scaffolds made using a 70:30 ratio were found to be suitable for the promotion of growth of laboratory corneal epithelial stem cell lines (SIRC cell lines). This innovation can pave way for further developments in corneal stem cell research and growth, thus providing for viable laboratory-derived corneal substitutes.

Updated Molecular Regulation of Hair Growth and Pigmentation by Dermal Papilla Cells / 中国医学科学院学报

There is growing evidence that dermal papilla cells(DPCs)act as the organizing center to induce the cyclic hair regeneration.On one hand,DPCs secrete cytokines or growth factors to regulate the differentiation,proliferation,and migration of epithelial stem cells(EpSCs)and melanocyte stem cells(MeSCs)residing in the bulge region.On the other hand,DPCs manipulate the microenvironment(also termed as niche)for both EpSCs and MeSCs,such as the size of dermal papilla,the distance between dermal papilla and the bulge region,and the lymphatic drainage and sympathetic nerve innervation surrounding the bulge region,thereby orchestrating the cycling hair growth.Recent studies have demonstrated at least four subpopulations existing in dermal papillae,which induce the unilineage transit-amplifying epithelial cells to form the concentric multilayers of hair shafts and sheaths.In addition,emerging study has indicated that sustained psychological stress potentially leads to hyperactivation of the sympathetic nerves that innervate the bulge region.The large amount of norepinephrine released by the nerve endings forces MeSCs to rapidly and abnormally proliferate,resultantly causing the depletion of MeSC pool and the loss of hair pigment.Understanding the molecular regulation of hair growth and pigmentation by DPCs holds substantial promise for the future use of cultured DPCs

Minimal Requirement of Limbal Epithelium for Successful Limbal Cell Transplantation in Rabbit Corneas

PURPOSE: To investigate the minimal requirements of the limbal epithelium for successful limbal stem cell transplantation and the healing process. METHODS: Nine rabbits were divided into 4, 6, and 8 clock-hour transplantation groups. Limbal autografts from the healthy fellow eye were transplanted to the iatrogenic damaged eye. The amniotic membrane served as a stem cell niche. Experimental corneas were evaluated by slit lamp examination and immunohistochemistry. RESULTS: In the over 9 hours transplantation group, the healing process of the epithelium from the limbal stem cell was revealed and cornea-specific keratin k3, transcription factor p63, and connexin 43 were detected by immunohistochemistry. The normal corneal epithelium was regenerated after 60 days postoperatively in the fellow donor eye. CONCLUSIONS: Limbal cell transplantation of over 9 hours seems to be a safe and effective method in the treatment of severe ocular surface disorders. In addition, the donation of limbal epithelium for up to 8 hours did not affect the normal corneal regenerating capability.

Isolation of Putative Corneal Epithelial Stem Cells from Cultured Limbal Tissue

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3x10(4) cell/ml and 8.06x10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21x10(6) cell/ml with a trypsin/EDTA treatment (p<0.05). CFE was 9.67+/-2.13% and 6.63+/-2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61+/-0.42% and 5.21+/-4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67+/-2.24% and 1.17+/-6.13%, respectively (p<0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.

Down-regulation of MHC Expression in Human Stem Cells by Introduction of hCMV US Genes / 대한이식학회지

PURPOSE: Stem cells are considered promising candidates for cell replacement therapy in many devastating diseases. However, it is assumed that stem cells may be rejected on transplantation. Therefore, we introduced human cytomegalovirus (hCMV) US genes, which are known to be able to reduce MHC class I expression on the cell surface after infection, into two known stem cell lines in order to test the feasibility of modifying these cells to reduced MHC class I antigens by the introduction of hCMV US genes. METHODS: The MHC class I expressions of mock-transfected or hCMV US gene-transfected human embryonic neural stem cell line (HB1.F3) and human breast epithelial stem cell line (M13SV1) were examined by FACS. RESULTS: MHC class I expressions in HB1.F3 and M13SV1 cells were dramatically induced by IFN-gamma treatment. In FACS analysis, cells transfected with the hCMV US2, 3, 6 or 11 genes exhibited a dramatic reduction (40~60%) of MHC class I expression compared with mock-transfected cells. CONCLUSION: Our results suggest that human stem cells express high levels of MHC class I antigens, and thus may be rejected on transplantation unless they are odified. In addition introduction of hCMV US genes can be exploited for stemcell transplantation.

The expression and efffects of RSpo1 and ?-catenin in intestinal epithelium with intestinal ischemia-reperfusion injury in mouse / 中国普通外科杂志

Objective To explore the expression and effects of RSp1 and ?-catenin in the intestinal epithelium with intestinal ischemia-reperfusion injury(IIRI)in of mouse.Methods Fifty healthy male kunming mice were randomly divided into control group(n=10)and experimental group(n=40).All mice in control group were only subjected to laparotomy,while the other mice underwent 20 minutes of intestinal mesenteric artery occlusion followed by 6 hours(group A),12 hours(group B),24 hours(group C)and 48 hours(group D)of reperfusion.RT-PCR was used to detect RSpo1 and ?-catenin in small intestine in intestinal ischemia-reperfusion groups and in control group.Results The villous heights of intestinal in experimental groups were significantly lower than that in control group(P