Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells.

Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.

[Prokaryotic expression of Nanog gene and preparation of anti-Nanog antibody].

AIM: To express Nanog fusion protein in Escherichia coli ( E.coli), and to prepare rabbit anti-mouse polyclonal antibodies to the Nanog fusion protein. METHODS: Mouse Nanog gene was amplified from the pNA992 recombinant plasmid and inserted into pET-32a vector to construct a recombinant expression vector pET-32a-Nanog. The recombinant vector was transfected into E.coli BL21 and induced by IPTG to express in them. The acquired Nanog fusion protein was purified with HisTrap affinity column and injected as an antigen into rabbits for preparing polyclonal antibodies. At last, the titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA, Western blotting and immunocytochemical staining, respectively. RESULTS: The recombinant expression vector pET-32a-Nanog was successfully prepared, transfected and induced to obtain the high expression of the Nanog fusion protein in a form of inclusion bodies in E.coli. After purification, its purity was up to 97%. The titer of anti-Nanog antibodies was 1:32 000 in the immunized rabbit serum, and exhibited a high specificity to Nanog protein. CONCLUSION: The rabbit anti-mouse polyclonal antibodies have been prepared successfully with a high titer and specificity to the Nanog fusion protein.

In vitro production of haploid sperm cells from male germ cells of foetal cattle.

The purpose of this study was to isolate the foetal cattle male germ cells (mGCs) and then induce them into sperm cells. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the vasa and the c-kit positive cells were 95.34+/-2.25% and 53.3+/-1.03% by using flow cytometry analysis (FCA), respectively. In feeder-free culture system, the half-suspending cells appeared and formed a 16-cell rosary in medium after the mGCs were cultured for 6-8 days. On immunocytochemical staining during the second passage, some single cells adhering to the plate appeared to be both Oct-4 and alpha6-integrin positive. During the third passage, the mGCs were induced for 48 h by retinol acid (RA) on Sertoli cell-feeder layer, followed by 5-7 days culture in an RA-free medium. Some elongated sperm-like cells appeared in the medium at this stage. We found that the most effective concentration of RA for the inducement was 10(-7)moll(-1) (P<0.01). The haploid cells in suspension were identified by FCA. The elongated sperm-like cells showed proacrosome-like structure and the flagellum with fibre construct under electron microscopy. The mRNA of outer dense fibre-3 (ODF-3) and transcription protein-1 (TP-1) could be detected in the suspended cells by using reverse transcription polymerase chain reaction (RT-PCR). About 23.1% bovine oocytes could be activated to perform cleavage by intracytoplasmic injection with the sperm-like cells, but embryos did not further develop. Our investigation further demonstrated that foetal cattle mGCs could be induced in vitro into haploid sperm in the short term.

[Establishment of the isolation and culture system for monocloning human pancreatic stem cell].

The present study aimed to establish an isolation and culture system for the monoclonal human pancreatic stem cells and monoclonal human pancreatic stem cell line. Some factors which would influence the proliferation of the monoclonal human pancreatic stem cells were assessed. Pancreatic tissues, taken from abortive fetuses by sterile procedures, were dissected into 1 mm3 segments and digested with 0.1% type IV collagenase. The isolated cells were grown in media containing low glucose Dulbecco's Modified Eagles's Medium supplemented with 10% fetal bovine serum (FBS), 3.7 g/L sodium pyruvate, 0.08 g/L penicillin and 0.1 g/L streptomycin. These cells were further digested with 0.25 g/L trypsin and 0.4 g/L EDTA for propagation. The monoclonal human pancreatic stem cells were selected by clone-ring, and further proliferated after addition of 10 ng/mL epidermal growth factor (EGF) in culture media. The cell chromosome set was determined by karyotype analysis. The growth curve was made by the 3-(4, 5)-dimethylthiahiazo (-z-yl)-3, 5-di-phenytetrazoliumromide (MTT) method. The results showed that pancreatic tissues were digested to many single cells and cell clusters with collagenase. Adherently cultured, primary epidermal-like pancreatic stem cells grew clonally. After several times of dissociation and propagation, pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the monoclonal human pancreatic stem cells were obtained. Continuously propagated, a monoclonal human pancreatic stem cell line which was derived from a male abortive fetus of 4 month-old had been passed through 50 generations. Karyotype analysis demonstrated that the chromosome set of the monoclonal human pancreatic stem cell line was normal diploid. Growth curve revealed that monoclonal human pancreatic stem cells grew slowly in initial 1-4 days. Then, they entered the logarithmic growth period in next 5-6 days. Their proliferation was speeded up by supplementation with 15% FBS in culture media, which was even more quickly after addition of the 15 ng/mL EGF or 10 ng/mL insulin growth factor II (IGF-II). The result identified that the monoclonal human pancreatic stem cell line could be obtained by the cell isolation and culture system.

[Research on differentiation features of the monoclonal human pancreatic stem cell].

The in vitro and in vivo differentiation features of the monoclonal human pancreatic stem cell (mhPSC) line derived from the pancreatic tissues of a male abortive fetus at 4 month-old were studied. The mhPSCs were plated in culture dishes that had been coated with 0.1% gelatin in phosphate-buffered saline without calcium and magnesium. After proliferated for 3 days, the mhPSCs were induced in modified high-glucose Dulbecco's Modified Eagles's Medium for 25 days. The changes of the cell morphology were observed by phasecontrast microscope during inducement course. The results of the mhPSCs in vitro induced to differentiate into functional pancreatic islets were identified using dithizone staining, RT-PCR and stimulation-glucose secreting insulin and C-peptide radioimmunoassay. The mhPSCs suspension was separately injected under the groin hypoderm of male nude mice. On the 30th day, the grafts were taken off. The immunochemistry reactions were performed by the SP method. The in vivo differentiation ability of the mhPSCs in nude mice was assessed. In vitro proliferation culture, the mhPSCs adhesively grew and showed polygon epithelioid morphology. After proliferation a layer, the mhPSCs showed the gravelstone-like. During in vitro directional inducement, the mhPSCs gradually turned from polygon to round, suspended to grow and assembled pancreatic islets-like clusters. On the 15th inducement day, only a few cells of pancreatic islet-like clusters were induced into the beta cells that became crimson with dithizone staining. However, till the 25th inducement day, most cells of pancreatic islet-like clusters had differentiated into the beta cells, as identified by dithizone staining, which expressed transcription factor of insulin. Respectively stimulated with different concentration glucose, the induced pancreatic islets not only secreted insulin and C-peptide, but also the secretion volumes of the insulin and C-peptide were markedly increased after the stimulation with higher concentration glucose (0.01

[Activation of Stra 8 gene during the differentiation of spermatogonial stem cells].

Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra 8 (Stimulated by Retinoic Acid) was one of the gene in this group. In mouse, Stra 8 is restrictively expressed in male germ line cells, and its function is related to the development of sperm. In order to investigate the feature of Stra 8 gene expression,the 1.4 kb (-1407 - +7) promoter region of Stra 8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4 kb promoter region, and the reporter gene of EGFP that was regulated by 1.4kb Stra 8 promoter. To investigate the specificity of Stra 8 promoter,the vector pStro-EGFP was transfected into undifferentiated mouse stem cells such as ES-129, bone marrow mesenchymal stem cell (mMSC) and spermatogonial stem cell (mSSC). The results showed that the expression of GFP was only observed in the mSSC cells,which indicated that Stra 8 gene was specially regulated in testis tissue. As the gene marker,vector pStra8-EGFP was then transfected to undifferentiated mMSC cells. After being selected by G418 for 2 weeks,the mMSC cells were induced by retinoic acid. After 12 hours induction, some induced cells started to express GFP protein, which was observed under the fluorescence microscope. At the same time, several stem cell specificity biomarkers such as Oct4, and spermatogonial stem cell biomarkers such as CyclinA2 and Stra 8 were detected in the induced cells by RT-PCR method. These results showed that the mMSCs would differentiated to spermatognial stem cells after induced by Retinoic Acid.

[Study on pluripotency and cultivation of ES-like cells derived from male germ stem cells of bovine fetuses].

Male germ stem cells (mGSCs), which is in testis after sex differentiation, derive from primordial germ cells. In this study, bovine mGSCs were isolated from testis of 20 weeks fetuses. Number of CD9 positive cells of the cells through two-steps adhering plates velocity different was 95.8% by flow cytometer. The carina-type cells clones and the plane-type cells clones appeared in co-cultured system. One cells lines had been successively maintained for 4 passages, and the cells clusters showed AKP positive staining. The cells clusters showed nest-shape in third passage showed SSEA1 and Oct-4 positive staining. These cells can also spontaneously differentiate into c-kit positive staining germ cells, and the cells were directional induced to formaactin positive staining cardiac-like cells cluster and NF positive staining neuron-like cells. The conclusion showed that male germ stem cells from 20 weeks bovine fetuses could be in vitro formed like embryonic stem cells.

[Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.

[The origin and nature of embryonic stem cells].

The inner cell mass (ICM), blastomeres, epiblasts and primordial germ cells (PGCs) are usually used as primary materials for the establishment of embryonic stem cell (ESC) lines. ES-like cells have even been isolated from neonatal mouse testis. ESC are traditionally regarded as ICM cells, though some scholars believe they more closely resemble cells from the epiblast. However, recent evidence of ESC molecular markers indicate that the characteristics of ESC resemble those of early germ cells. The unknown origin and nature of ESC may limit the successful establishment of ESC lines from many different species. Here we review the progress of research regarding embryonic pluripotent cells, early germ cells and ESC. We find ESC can be derived from many cell types. Future study should elucidate the origin of ESC by comparing different ESC lines so as to determine the nature of ESC and improve the efficiency of ESC derivation.

[Nuclear transfer and therapeutic cloning].

Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.