Fibroblast Feeder Layers Research Articles

Long-term culture and transplantation of spermatogonial stem cells from BALB/c mice

Objective To establish a long-term culture system for mouse spermatogonial stem cells(SSCs). Methods Testis cells from 4-6 days postpartum male transgenic BALB/C mice were collected by a modified two-step enzymatic digestion method.After three differential adherence selections,the enriched germ cells were finally suspended in StemPro-34 SFM medium supplemented with other nutrients factors and plated on mouse embryonic fibroblast(MEF)feeder layer.20 ng/ml Glial cell line-derived neurotrophic factor,10 ng/ml basic fibroblast growth factor and 200 ng/ml GDNF-family receptor al were added to the serum-free medium to promote SSCs proliferation.Aduh male BALB/C mice,4-5 weeks old,underwent intraperitoneal injection of 40 mg/kg busulfan as recipient mice.Cultured SSCs were also injected into the seminiferous tubules of the left recipient testis through micromanipulator and right testis as self-control.Testes of recipient mice were observed by a fluorescence stereomicroscope and HE stains at 2 months after transplantation. Results By improved digestion method,the vitality of isolated testis cells was more than 98%and the stem cells was enriched about 18.5 fold. 1-2 days after transferred to MEF feeder, the round germ cells started to proliferate and had the shape of paired or aligned undifferentiated spermatogonia connected by cytoplasmic bridges. After 3-4 days, SSCs proliferated continuously and formed typical colonies. SSCs from BALB/c mice could be cultured and passaged in a steady state for 3 months. Cryostat section through the transplanted testis showed that most of seminiferous tubules were filled with germ cells expressing EGFP.HE staining further showed clearly that seminiferous tubules contained complete spermatogenesis.Conclusions SSCs from BALB/c mice could be cultured in an improved culture system for 3 months.The culture system could facilitate understanding the regulatory mechanism that governs SSCs and might provide an opportunity for the cure of infertility. Key words: Spermatogonia; Stem cells; Cell culture techniques; Transplantation; Mice

101. ISOLATION OF PUTATIVE EMBRYONIC STEM CELLS FROM CLONED PIG EMBRYOS

The isolation of embryonic stem cells from cloned embryos (NT-ESC) from domestic animals would have a number of biomedical and agricultural applications. Putative ESC lines from in vivo derived and in vitro produced pig embryos were recently established using a new isolation method1. The aim of the current study was to determine whether NT-ESC lines could be isolated from cloned pig embryos using this method. To do this we determined initially whether the treatment of embryos with Trichostatin A (TSA), a histone deacetylase inhibitor, could increase the number of cloned embryos that develop to the blastocyst stage because TSA has been shown to increase blastocyst development and NT-ESC isolation efficiencies in mice2. Cloned embryos were produced as described previously3. Briefly, in vitro matured sow oocytes were enucleated, fused with adult fibroblasts using an electrical pulse and activated about 1.5 hrs later with a second electrical pulse. Reconstructed embryos were then cultured in modified NCSU23 with or without 50nM TSA treatment for the initial 24 hours of culture. Embryo development was assessed on day 6. Treatment with TSA increased the number of cloned embryos that developed to the blastocyst stage (143/471; 30%) compared with control (54/353; 15%; P < 0.0001). Blastocysts were then plated by mechanical depression onto mitotically inactivated mouse embryonic fibroblast feeder layers in a serum-free culture system on day 7. There was no significant difference in the efficiencies of establishment of homogeneous primary outgrowths between TSA treated (17/96; 18%) and control blastocysts (8/43; 19%). Thirteen homogenous outgrowths from the TSA treated group were vitrified at passage 2 or 3. Sublines are currently being characterised to determine their pluripotency.

CGI-58 Is an α/β-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an alpha/beta-hydrolase fold and is also known as the alpha/beta-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca(2+) conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation.

Preliminary Experimental Study of Tissue-Engineered Urethral Reconstruction Using Oral Keratinocytes Seeded on BAMG

Introduction: This study investigates the feasibility of replacing urinary epithelial cells with oral keratinocytes seeded on a bladder acellular matrix graft (BAMG) to reconstruct a tissue-engineered urethra. Materials and Methods: After enzymatic treatment of a small segment of rabbit buccal mucosa (1.0 × 0.4 cm), the epidermis and dermis were separated mechanically. Oral keratinocytes were isolated from the epidermis and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin (i3T3) or on a culture dish without i3T3, respectively. These cells were expanded and applied to sterilized BAMG to obtain a tissue-engineered mucosa. Oral keratinocytes were assessed by growth curve, morphology and immunofluorescence staining. The tissue-engineered mucosa was assessed using morphology, immunohistochemistry staining and scanning electron microscopy. Results: Oral keratinocytes seeded onto a feeder layer of i3T3 had finer morphous and better amplification capability, and could be passaged for 8 generations. Oral keratinocytes that were cultured without i3T3 could only be subcultured 2 generations before ageing. Passage 2 oral keratinocytes cultured with i3T3 were expanded and seeded onto sterilized BAMG to obtain a tissue-engineered mucosa for urethral reconstruction. The compound graft was assessed using HE staining and scanning electron microscopy. Oral keratinocytes had good compatibility with BAMG. Conclusions: Rabbit oral keratinocytes of can be cultured in vitro and attain magnitude in quantity. Oral keratinocytes also had good compatibility with BAMG and the compound graft achieved could become a new choice in urethral reconstruction.

Development of Defined Media for the Serum-Free Expansion of Primary Keratinocytes and Human Embryonic Stem Cells

Primary keratinocyte (Kc) cells and human embryonic stem (hES) cells are routinely propagated on a mouse fibroblast feeder layer in media containing fetal bovine serum or other nondefined factors. One disadvantage of using these nondefined factors is that they may inadvertently contaminate the culture system with infectious agents; thus, there remains a need to develop safe culture conditions free from poorly defined and/or animal products. Our laboratory has discovered that growth factors (GFs) and vitronectin (VN) can bind to each other resulting in synergistic short-term functional effects in several cell types. The aim of the current study was to determine whether primary Kc and hES cells can be established and serially propagated serum-free using medium containing VN, insulin-like growth factor-I, and insulin-like growth factor binding protein-3 (VN:GF). Here we demonstrate that primary Kc cells can be isolated, established, serially propagated, and re-form an epidermal layer using the VN:GF combination. Additionally, cell proliferation studies indicate that the Kcs proliferate using the VN:GF combination at a rate comparable to cells grown using serum. Similarly, we verified that this VN:GF combination could be employed for the serial propagation of hES cells. Importantly, both the Kc and hES cells retain their undifferentiated phenotype when cultured using the VN:GF combinations as a serum-free medium for up to 4 passages for Kc and at least 10 passages for hES cells as indicated by the expression of a range of cell surface markers. This study demonstrates that the novel, fully defined VN:GF medium is a viable alternative to media containing serum and highlights the potential of this technology for generating therapeutically viable cells and tissues.

Serum free culture and characterization of human embryonic germ cells

Many proteins and growth factors in serum play important roles in the propagation of embryonic stem (ES) cells. But there are some disadvantages in using the serum to culture ES cells. We examined the effects of a chemically-defined culture system knock-out serum replacement (KSR) in the absence of FBS on the growth and differentiation of human primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs collected from gonadal ridges and mesenteries of 413 week fetuses, were cultured on mouse embryonic fibroblast (MEF) or STO feeder layers. PGCs in KSR-supplemented medium proliferated, maintaining round and typical stem celllike morphologies. In addition, the efficiency of EG cell lines in KSR medium were significantly higher than that cultured in FCS medium(P<0.05). The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FBS-based medium(P<0.05). The cultured cells have been continuously maintained undifferentiated state for 14 passages on MEF or STO feeder layer with serum-free culture medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro. The human embryonic germ cells(hEGCs) colonies were typically like mouse EG colonies in morphology, tight, compact, multilayer cells with large and prominent nuclei, and showed normal and stable diploid karyotype, expressing AP, SSEAs and glycoprotein markers such as SSEA-1, SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, and Oct-4, PCNA, C-kit, and with high telomerase activity. In contrast, mouse EGCs do not express SSEA-3, SSEA-4, TRA-1-60,TRA-1-81. Human EG cells can form simple and cystic embryoid bodies (EBs) that consist various cell types including all three embryonic germ layers, such as neural cells, epithelia-like, fibroblast-like, polygon cells and rhythmically beating myocardium-like cells, even sperm and oocyte-like cells. These germ like cells are positive for Oct4,C-kit and Scp3. So we built a appropriate serum-free culture system for the establishment of human EG cell lines and obtained pluripotent human EG cell lines derived from PGCs.

A defined, feeder-independent medium for human embryonic stem cell culture

Human embryonic stem cells (hESC) are derived from the epiblast tissue of the inner cell mass and are capable of giving rise to cells from all three germ layers - ectoderm, mesoderm, and endoderm. Although the basic techniques to create and culture hESC are well established, limitations remain in many of the procedures used for their culture. In particular, most existing hESC lines have been cultured with components derived from animal serum and using mouse embryonic fibroblast feeder layers. However, the use of serum or feeder layers induces variability in these culture systems, and limits their application in the clinical setting. Accordingly, there is considerable interest in the development of serum-free and feeder-independent hESC culture systems. mTeSR™1 is a fully-defined, serum-free medium for the derivation and maintenance of hESCs cultured on BD Matrigel™ hESC-qualified Matrix(Ludwig et al. Nat Methods 3(8):637-46). mTeSR™1 has been demonstrated to support the undifferentiated culture of multiple hESC lines derived by independent sources, with examples including H1, H9 CA1, and SA121. Cells grown for multiple passages in mTeSR™1 maintain characteristic undifferentiated colony morphology, and FACS analysis confirms high expression of pluripotency-associated markers (Oct4, SSEA-3, SSEA4, TRA-1-60, and TRA-1-81), and the absence of expression of the differentiation marker SSEA-1. RT-PCR analysis of cells cultured in mTeSR™1 also validate the continued expression of Oct4 and Nanog and the lack of expression of multiple differentiation markers. Furthermore, hESC cultures maintained in mTeSR™1 retain a high proliferative capacity and normal karyotype after extended culture. Finally, the teratoma formation assay verified the preserved potential of these cells to subsequently differentiate into tissues derived from all three germ layers. Together these results establish mTeSR™1 as an optimal, standardized medium for researchers culturing hESC.

Long-term culture of keratinocyte-like cells derived from mouse embryonic stem cells

A number of epithelial lineages have been derived from mouse embryonic stem cells during the past decades, but the long lasting culture has never been reported. In this paper, we report when mouse embryonic stem cells were dispersed into small clumps containing approximately 50 to 100 cells and grown on mitotically inactivated mouse embryonic fibroblast feeder layers for up to 10 d to form epithelial-like colonies. Through subsequent cultivation without mouse embryonic fibroblast feeder layers, a serially subcultured keratinocyte-like cell lineage was established under these conditions. Pan cytokeratin, cytokeratin 14, and cytokeratin 18 were observed in these proliferating cells using immunocytochemistry and flow cytometry. E-cadherin, Involucrin, and keratin mRNAs were determined by a semi-quantitative and a quantitative real time reverse transcription-polymerase chain reaction (RT-PCR). These results confirmed the establishment of a keratinocyte-like cell lineage derived from mouse embryonic stem cells. In this paper also, we describe a method by which mouse embryonic stem cells can be differentiated into cells with some characteristics of epidermal keratinocytes and kept these cells in long-term culture. Potential applications of this method are the in vitro differentiation of cells of interest from embryonic stem (ES) cells of mice during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments or better understanding the mechanisms for epithelial differentiation of embryonic stem cells.

A new technique for primary hepatocyte expansion in vitro

The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell-cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell-cell interactions in the modulation of hepatic proliferation and function.

IFN-α Enhances Poly-IC Responses in Human Keratinocytes by Inducing Expression of Cytosolic Innate RNA Receptors: Relevance for Psoriasis

Keratinocytes play a key role in innate immune responses of the skin to bacterial and viral pathogens. Viral double-stranded RNA and its synthetic analogue polyriboinosinic-polyribocytidylic acid (poly-IC) are recognized via multiple pathways involving the receptors Toll-like receptor 3 (TLR3), protein kinase R (PKR), and the recently described cytosolic RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We show that preincubation of human keratinocytes with IFN-alpha enhances the proinflammatory responses to poly-IC. Kinetic studies suggest that this is mediated via upregulation of the receptors TLR3, PKR, RIG-I, and MDA5. Interestingly, expression of RIG-I, MDA5, and PKR was significantly increased in lesional skin from patients with psoriasis, a chronic inflammatory skin disease that is characterized by high IFN-alpha levels. These results suggest that psoriatic keratinocytes show increased sensitivity to viral RNA intermediates, thereby leading to excessive proinflammatory responses and maintenance of the inflammatory skin phenotype. Here, we provide early evidence that point toward a role for the recently described cytosolic innate RNA receptors in non-viral chronic inflammatory diseases.

Cloned Embryos from Semen. Part 1:In VitroProliferation of Epithelial Cells on Embryonic Fibroblasts after Isolation from Semen by Gradient Centrifugation

Although epithelial-like somatic cells have been previously isolated from semen, cell proliferation rates were low. Culture of whole semen samples resulted in loss of potentially valuable spermatozoa. The aims of the present study were to: (1) isolate somatic cells from semen, while preserving sperm viability, and (2) optimize in vitro culture conditions for semen-derived epithelial cells. Density gradient centrifugation of washed ejaculates of two rams (Ovis aries) (n = 24) and one eland bull (Taurotragus oryx) (n = 4) was performed using a three-layer discontinuous Percoll column consisting of 90% (P-90), 50% (P-50), and 20% (P-20) Percoll. In vitro culture and Trypan Blue staining indicated that live somatic cells settled in the P-20 layer. Nonmotile spermatozoa were recovered at the P-50 and P-90 interfaces, whereas motile spermatozoa were collected in the pellet from the P-90 layer. Subsequently, somatic cells isolated from the P-20 layer were plated either on inactivated 3T3 mouse embryonic fibroblast feeder layers, collagen-coated plates with 3T3 feeder cell inserts, or on collagen-coated plates. Initial somatic cell plating was similar among treatments, but proliferation significantly increased when cocultured with 3T3 cells (feeder or insert). Furthermore, two different types of epithelial cells were obtained. The exact origin of the cells in the male reproduction system is uncertain and probably variable. The present method of cell isolation and in vitro culture may be of value for preserving endangered species. Specifically, cells isolated and cultured from cryopreserved semen of nonliving males could be used for producing embryos by somatic cell nuclear transfer.

Isolation and characterization of embryonic stem‐like cells derived from in vivo‐produced cat blastocysts

Embryonic stem (ES)-like cells were isolated from in vivo-produced cat embryos. Total of 101 blastocysts were collected from female cats. The inner cell mass (ICM) were mechanically isolated and cultured on mitomycin-C-treated cat embryonic fibroblast feeder layers in medium supplemented with knockouttrade mark Serum Replacement (KSR-medium) or fetal bovine serum (FBS-medium). Putative ES-like cell colonies developed in both KSR- and FBS-medium conditions, but formed domed and flat colonies, respectively. ICM cell attachment and ES-like cell colony formation were significantly higher in KSR-medium, but subsequent cell proliferation was significantly lower than in FBS-medium. For passaging, 32 and 18 colonies in KSR- and FBS-medium were separated by enzymatic dissociation or mechanical disaggregation. Enzymatic dissociation resulted in cell differentiation; however, mechanical disaggregation generated cells that remained undifferentiated over more than four passages and yielded two cat ES-like cell lines that continued to grow for up to eight passages in FBS-medium. These cells had typical stem cell morphology, expressed high levels of alkaline phosphatase activity, and were positive for the ES cell-markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-3, and SSEA-4. These cells formed embryoid bodies (EBs) in suspension culture after extended suspension culture. When simple EBs were cultured on tissue culture plates, they differentiated into several cell types, including epithelium-like and neuron-like cells. In addition, EBs were positive for mesoderm marker, desmin. After prolonged in vitro culture, some colonies spontaneously differentiated into beating myocardiocytes, and were positive for alpha-actinin. These observations indicate that cat ES-like cells were successfully isolated and characterized from in vivo-produced blastocysts.

Efficient expansion of clinical-grade human fibroblasts on microcarriers: Cells suitable for ex vivo expansion of clinical-grade hESCs

Human embryonic stem cells hold considerable potential for cell-based treatments of a variety of degenerative diseases, including diabetes, ischemic heart failure, and Parkinson's disease. However, advancing research to provide clinical-grade product requires scale-up to therapeutic quantities of stem cells and their differentiated progeny. Most human embryonic stem cell culture platforms require direct support by a fibroblast feeder layer or indirect support using fibroblast conditioned medium. Accordingly, large numbers of clinically compliant fibroblasts will be requisite for stem cell production. Published platforms for feeder production are insufficient for stem cell scale-up, being costly to operate and requiring considerable effort to prepare, maintain and harvest. Here we describe the expansion of cGMP-grade, FDA-approved human foreskin fibroblasts using cGMP-grade reagents and polystyrene-based cationic trimethyl ammonium-coated microcarriers in spinner flasks. Fibroblasts attach rapidly to the microcarriers ( T 1/2 =75 min), and expand with a maximum doubling time of 22.5 h. Importantly, microcarrier-expanded fibroblasts and their conditioned medium support pluripotent stem cell growth through >5 passages, enabling extended self-renewal and expansion while retaining full differentiation potential. In summary, the method described is an economical and cGMP-compliant means of producing human fibroblast cells in support of cGMP human embryonic stem cell culture.

405. Isolation and preliminary characterisation of putative sheep embryonic stem cells

The establishment of true, fully characterised embryonic stem (ES) cells from livestock, (eg sheep) has yet to be reported. Such cells could make a significant impact on assisted reproductive technologies, vaccine delivery, and animal health and well being in the livestock industries. To date in sheep, there is a single report of putative ES cells which were maintained in an undifferentiated state for only 2 passages. Here we report the isolation and culture of pluripotent ES-like cells from in vivo derived, vitrified, sheep blastocysts. The inner cell mass of blastocysts were isolated by immunosurgery, cultured in Stem Cell Sciences' (SCS) novel inhibitor-based media, on a feeder layer of mouse embryonic fibroblasts (MEFs), resulting in putative ovine ES cells proliferating to at least passage 5. One cell line, BMCOV002, was established out of four thaw-recovered embryos. The colonies formed compact, near homogenous, small cell, multilayered, and well defined dome shaped masses that were morphologically similar to both mouse and human ES cell colonies. A peripheral halo of filamentous differentiated cells was detected in selected colonies from passage 3 to passage 5. The putative ovine ES-like cells were passaged by mechanical excision between days 6–7, and these colonies stained positive for alkaline phosphatase at both passage 3 and passage 5. Expression levels of genes encoding the pluripotent transcription factors OCT4, SOX2, REX1 and NANOG are shown using RT PCR in cells from passage 3. An important first step in studying the properties of ovine ES-like cells is the ability shown here to isolate and culture cells. Our attention now is focussed on maintaining these cells for some months in an undifferentiated state, and on being able to successfully cryopreserve and regenerate these cell lines.

Isolation and Culture of Rabbit Primordial Germ Cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.

Progress in the derivation, culture of human embryonic stem cells

Human embryonic stem (hES) cells are considered to be a valuable resource for research in regenerative medicine, drug screening, and developmental studies. However, hES cells are usually established and maintained on mouse embryonic fibroblast feeder layers, and the risk of animal origin contamination from feeder layer generally excludes the clinical use of these hES cells. The main emphasis over the last several years has been in finding defined serum-and feeder layer-free system for derivation and culture of hES cells to enable the clinical use of hES cell for cell transplantation.

Epithelial-Mesenchymal Transition Events during Human Embryonic Stem Cell Differentiation

Epithelial-mesenchymal transition (EMT) occurs during embryonic development and may also be associated with the metastatic spread of epithelial tumors. During EMT, E-cadherin is down-regulated and this correlates with increased motility and invasion of cells. We show that differentiation of human embryonic stem (ES) cells in monolayer culture is associated with an E- to N-cadherin switch, increased vimentin expression, up-regulation of E-cadherin repressor molecules (Snail and Slug proteins), and increased gelatinase (matrix metalloproteinases; MMP-2 and MMP-9) activity and cellular motility, all characteristic EMT events. The 5T4 oncofetal antigen, previously shown to be associated with early human ES cell differentiation, is also part of this process. Abrogation of E-cadherin-mediated cell-cell contact in undifferentiated ES cells using neutralizing antibody (nAb) SHE78.7 resulted in increased cellular motility, altered actin cytoskeleton arrangement and a mesenchymal phenotype together with presentation of the 5T4 antigen at the cell surface. nAb-treated ES cells remained in an undifferentiated state, as assessed by OCT-4 protein expression, and did not express EMT-associated transcripts. Removal of nAb from ES cells resulted in the restoration of cell-cell contact, absence of cell surface 5T4, decreased mesenchymal cellular morphology and motility, and enabled the differentiation of the cells to the three germ layers upon their removal from the fibroblast feeder layer. We conclude that E-cadherin functions in human ES cells to stabilize the cortical actin cyoskeletal arrangement and this prevents cell surface localization of the 5T4 antigen. Furthermore, human ES cells represent a useful model system with which to study EMT events relevant to embryonic development and tumor cell metastasis.

Down‐regulation of Pax6 is associated with abnormal differentiation of corneal epithelial cells in severe ocular surface diseases

Pax6 is the universal master control gene for eye morphogenesis. Other than retina and lens, Pax6 also expressed in the ocular surface epithelium from early gestation until the postnatal stage, in which little is known about the function of Pax6. In this study, corneal pannus tissues from patients with ocular surface diseases such as Stevens-Johnson syndrome (SJS), chemical burn, aniridia and recurrent pterygium were investigated. Our results showed that normal ocular surface epithelial cells expressed Pax6. However, corneal pannus epithelial cells from the above patients showed a decline or absence of Pax6 expression, accompanied by a decline or absence of K12 keratin but an increase of K10 keratin and filaggrin expression. Pannus basal epithelial cells maintained nuclear p63 expression and showed activated proliferation, evidenced by positive Ki67 and K16 keratin staining. On 3T3 fibroblast feeder layers, Pax6 immunostaining was negative in clones generated from epithelial cells harvested from corneal pannus from SJS or aniridia, but positive in those from the normal limbal epithelium; whereas western blots showed that some epithelial clones expanded from pannus retained Pax6 expression. Transient transfection of an adenoviral vector carrying EGFP-Pax6 transgenes into these Pax6(-) clones increased both Pax6 and K12 keratin expression. These results indicate that Pax6 helps to maintain the normal corneal epithelial phenotype postnatally, and that down-regulation of Pax6 is associated with abnormal epidermal differentiation in severe ocular surface diseases. Reintroduction of activation of the Pax6 gene might be useful in treating squamous metaplasia of the ocular surface epithelium.

Advances in corneal stem-cell transplantation in rabbits with severe ocular alkali burns

To evaluate the efficacy of autologous corneal epithelial sheet implantation in restoring transparency of rabbit corneas severely injured by alkaline and the effect of photocoagulation in arresting corneal neovessel ingrowth. Ophthalmology Department, School of Biomedical Sciences, Universidad Austral, Buenos Aires, Argentina. Limbal stem-cell deficiency (LSCD) was induced in 14 rabbits by alkali burns. A limbal cell biopsy was done in the contralateral eye, and the cells were cultured on a fibroblast feeder layer grown on autologous clotted platelet-poor plasma or commercial fibrin for 21 days. Anterior keratectomy was followed by suturing corneal cell sheets over the stroma. If regrowth of vessels occurred, argon laser photocoagulation was applied to them. Rabbits were killed at 30, 60, 90, 180, and 360 days and the corneas processed for histopathology and inmunohistochemistry. A small (2.5 mm(2)) limbal biopsy achieved stem-cell replication in vitro. Corneal clarity and epithelial defects evolved with a trend toward improvement. There was a significant reduction in corneal neovascularization. Histology showed a multilayered stratified epithelium including several epithelial-like cells with clear cytoplasm in the deepest part. There were no signs of intraepithelial mucin cells on the implanted corneas. Immunohistochemical results showed expression of cytokeratins 3 and 12 in the central corneal epithelium and an absence of cytokeratin 19. Autologous limbal epithelial cell transplantation improved the corneal surface in eyes with LSCD. Photocoagulation of neovessel ingrowth was effective over the 1-year follow-up. Results may facilitate the application of this technique in patients.

A New System for Cultivation of Human Keratinocytes on Acellular Dermal Matrix Substitute with the Use of Human Fibroblast Feeder Layer

To improve the proliferative potential of human keratinocytes (HK) cultured on acellular dermal matrix (ADM), HK and mitomycin C-treated human fibroblasts (MMC-HF) were seeded onto ADM to form four types of composite skin: type I, HK were seeded onto the epidermal side of ADM; type II, both HK and MMC-HF were seeded onto the epidermal side; type III, MMC-HF were preseeded onto the dermal side of ADM, and then HK were seeded onto the epidermal side, and type IV, where MMC-HF were preseeded onto both sides, and then HK were seeded onto the epidermal side. Compared with type I and III, the proliferative potential of HK of type II and IV was significantly higher on day 3, 5, 7 and 9 in vitro. In type I and III, HK grew into one layer on day 7–9, while in type II and IV keratinocytes grew into a confluent monolayer by day 4–6. The adherence to ADM of HK in types II and IV was stronger than that in type I and III. The take rate of type II and IV composite skin was also significantly higher. In conclusion, when MMC-HF and HK were cocultured on the epidermal side of ADM, MMC-HF could serve as excellent feeder cells.