Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells

To exclude bacteria- or animal-derived factors from cultured fabrication of transplantable epithelial cell sheets, primary human oral mucosal epithelial cells were seeded on temperature-responsive culture inserts having submicron-scale pores. Supplying culture medium containing human autologous serum to both apical and basal sides of human epithelial cells allows these cells to grow to confluence. These proliferating cells created stratified epithelial layers even when 3T3 feeder layers and fetal bovine serum were eliminated from culture. Normal keratin expression profiles were obtained with these cells, and basal and midlayer cells expressed p63, a putative stem/progenitor marker. These results suggest that temperature-responsive culture inserts can be useful in clinical settings that require the exclusion of xenogeneic factors.     

A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations

Corneal epithelial cell sheets co-cultivated with feeder cells are used to reconstruct the ocular surface in stem cell-depleted eyes. The present study was conducted to investigate the optimal method of using feeder cells in the interest of preserving progenitor cells in cultivated sheets. We compared the phenotype and secondary colony forming efficiency (CFE) of cell sheets that were engineered using 3T3 feeder cells as a separate layer or as a contact layer. We also devised a novel "duplex feeder" system that consists of two separate layers of feeder cells. After cells reached confluence, cells were cultured at the air-liquid interface to allow full stratification. Stratified sheets were then analyzed using immunohistochemistry and secondary colony formation. Contact feeder cultures and duplex feeder cultures yielded epithelial sheets with small, cuboid basal cells with strong expression of keratin (K)3, K12, and K 15. Furthermore, only duplex feeder layers reproduced the basal K 15, suprabasal K12 limbal phenotype where epithelial stem cells reside. A similar effect was observed when cornea stroma-derived progenitor cells were used as feeder cells. Duplex feeder sheets also produced significantly more secondary colonies than cells dissociated from single layer sheets, suggesting that the duplex feeder system produces transplantable sheets with a higher yield of progenitor cells.     

Development of transplantable genetically modified corneal epithelial cell sheets for gene therapy

The purpose of this study was to establish a method for the fabrication of exogenous gene-transferred, transplantable corneal epithelial cell sheets. Corneo-limbal epithelial cells collected from USA eye bank eyes were transduced with an EGFP-expressing lentiviral vector at differential MOI. Multi-layered corneal epithelial cell sheets were fabricated by co-cultivation of transduced cells and mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes. These cultured epithelial cells could be harvested as intact sheets by simply lowering the temperature. The number of EGFP-positive cells was increased as the MOI raised, and at an MOI of 100, nearly 100% of the superficial cells showed strong EGFP expression. Histological analysis revealed that EGFP was expressed in all layers of the cell sheet of which cell source was transduced with the lentiviral vector at an MOI of 100. Immunofluorescence data showed that p63 was also expressed in the basal layer of the same cell sheet. These results suggest that this technique will likely be applicable to ex vivo gene therapies for various corneal disorders.     

N-Cadherin is expressed by putative stem/progenitor cells and melanocytes in the human limbal epithelial stem cell niche

Corneal epithelial stem cells are known to be localized to the basal layer of the limbal epithelium, providing a model system for epithelial stem cell biology; however, the mechanisms regarding the maintenance of these stem cells in their specialized niche remain poorly understood. N-cadherin is a member of the classic cadherin family and has previously been demonstrated to be expressed by hematopoietic stem cells. In the present study, we demonstrate that N-cadherin is expressed by putative stem/progenitor cells, as well as melanocytes, in the human limbal epithelial stem cell niche. In addition, we demonstrate that upon in vitro culture using 3T3 feeder layers, loss of N-cadherin expression occurs with cell proliferation. These results indicate that N-cadherin may be a critical cell-to-cell adhesion molecule between corneal epithelial stem/progenitor cells and their corresponding niche cells in the limbal epithelium.     

A new isolation method of human limbal progenitor cells by maintaining close association with their niche cells

In human corneal epithelium, self-renewal and fate decision of stem cells are highly regulated in a niche microenvironment called palisades of Vogt in the limbus. Herein, we discovered that digestion with dispase, which cleaves off the basement membrane, did not remove the entire basal epithelial progenitor cells. In contrast, digestion with collagenase isolated on cluster consisting of not only entire epithelial progenitor cells but also their closely associated mesenchymal cells because of better preservation of some basement membrane matrix. Collagenase isolated more basal epithelial progenitor cells, which were p63α+ and small in the size (8 μm in diameter), and generated significantly more holoclones and meroclones on 3T3 fibroblast feeder layers than dispase. Further, collagenase isolated more small pan-cytokeratin-/p63α-/vimentin+ cells with the size as small as 5 μm in diameter and heterogeneously expressing vimentin, Oct4, Sox2, Nanog, Rex1, Nestin, N-cadherin, SSEA4, and CD34. Maintenance of close association between them led to clonal growth in a serum-free, low-calcium medium, whereas disruption of such association by trypsin/EDTA resulted in no clonal growth unless cocultured with 3T3 fibroblast feeder layers. Similarly, on epithelially denuded amniotic membrane, maintenance of such association led to consistent and robust epithelial outgrowth, which was also abolished by trypsin/EDTA. Epithelial outgrowth generated by collagenase-isolated clusters was significantly larger in diameter and its single cells yielded more holoclones on 3T3 fibroblast feeder layers than that from dispase-isolated sheets. This new isolation method can be used for exploring how limbal epithelial stem cells are regulated by their native niche cells.     

Comparative Analysis of Human-Derived Feeder Layers with 3T3 Fibroblasts for the Ex Vivo Expansion of Human Limbal and Oral Epithelium

Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and Iβ1 and p63 (putative stem cell markers) in all culture conditions. The level of Iβ1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.     

Human amniotic epithelial cells as novel feeder layers for promoting ex vivo expansion of limbal epithelial progenitor cells

Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan-cytokeratins (pan-CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan-CK(+)/Vim(+) or pan-CK(+)/Vim(-) were maintained in serum-free media with high calcium. In contrast, all HAECs became pan-CK(+)/Vim(+) in serum-containing media, which also promoted HAEC proliferation for at least eight passages, especially supplemented with epidermal growth factor and insulin. Mitomycin C-arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC-supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43 but higher levels of stem cell-associated markers such as p63, Musashi-1, and ATP-binding cassette subfamily G2 than those of 3T3-supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63-positive epithelial progenitor cells as well as nestin-positive neuronal-like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus. Disclosure of potential conflicts of interest is found at the end of this article.     

Limbal epithelial side-population cells have stem cell-like properties, including quiescent state

Corneal epithelial (CE) stem cells are believed to reside in the basal layer of the limbal epithelium but remain poorly understood due to the lack of an accepted in vivo reconstitution assay as well as definitive markers for epithelial stem cells. It has been reported that side-population (SP) cells with the ability to efflux the DNA-binding dye Hoechst 33342 have stem cell-like properties and that the SP phenotype accurately represents a quiescent and immature stem cell population in the adult bone marrow. In the present study, we investigated whether SP cells isolated from the limbal epithelium have stem cell-like properties. SP cells, separated by fluorescence-activated cell sorting, comprise approximately 0.4% of all limbal epithelial cells and have markedly higher expression of the stem cell markers ABCG2, Bmi-1, and nestin but no expression of markers for differentiated CE cells compared with non-SP cells. Cell-cycle and telomerase activity analyses revealed that SP cells are growth arrested and reside in the quiescent state. Moreover, limbal epithelial SP cells did not demonstrate proliferative capabilities under typical in vitro epithelial cell culture conditions using 3T3 feeder layers. These findings present the possibility that quiescent limbal epithelial SP cells may represent an extremely immature stem cell population compared with currently defined epithelial stem or progenitor cells.     

Intrastromal invasion by limbal epithelial cells is mediated by epithelial-mesenchymal transition activated by air exposure

Corneal epithelial stem cells are located in the basal layer of the limbus between the cornea and the conjunctiva. Regulation of these limbal epithelial progenitor cells by the stromal niche dictates corneal surface health. To further characterize this process, limbal explants were cultured at the air-fluid interface, termed air-lifting, to stimulate the niche. As compared to submerged cultures, air-lifting significantly promoted epithelial stratification, migration, proliferation, and intrastromal invasion by limbal epithelial cells. Epithelial intrastromal invasion was noted when the limbal, but not corneal, epithelium was recombined with the limbal stroma containing live, but not dead, cells. Invading limbal basal cells displayed up-regulated nuclear expression of p63 and Ki67, down-regulated E-cadherin and cornea-specific keratin 3, and switched expression of beta-catenin from intercellular junctions to the nucleus and cytoplasm, indicating the activation of the Wnt/beta-catenin pathway. Invaded cells isolated by collagenase from the stroma of air-lifted, but not submerged, explants showed vivid clonal growth on 3T3 fibroblast feeder layers and complete epithelial-mesenchymal transition by expressing nuclear p63 and cytoplasmic S100A4. These findings collectively suggest that epithelial-mesenchymal transition via the Wnt/beta-catenin pathway influences the fate of limbal epithelial cells, likely to be progenitor cells, between regeneration and fibrosis when the stromal niche is activated.     

饲养层细胞体系及白血病抑制因子对维持小鼠胚胎干细胞未分化状态的作用

目的建立并筛选最适合的小鼠胚胎干(ES)细胞饲养层培养体系。方法建立7种经过不同处理的鼠胚成纤维细胞作为饲养层培养体系,分别进行小鼠胚胎干细胞的培养,观察7种饲养层培养体系对小鼠ES细胞增殖能力及未分化状态的维持情况。结果小鼠胚胎细胞在3代内均可用于ES细胞的培养。与其他各组比较,第3代培养鼠胚成纤维细胞经丝裂霉素C处理2h后加外源白血病抑制因子(IF,10μg/ml)用作饲养层的克隆形成率和碱性磷酸酶(AKP)染色阳性度最高。结论外源性白血病抑制因子是抑制小鼠ES细胞分化的主要因素。     

A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture

Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.     

Monolayer culture of normal human esophageal epithelial cells

Summary A method is described for the isolation of epithelial cells from normal human esophagus and the establishment of the epithelial cells in monolayer cell culture using irradiated 3T3 fibroblast feeder layers as a substrate. The monolayer cultures were subsequently used to study the effects of a tumor promoter and carcinogen on human esophageal epithelial cells in vitro.     

Subcutaneous transplantation of autologous oral mucosal epithelial cell sheets fabricated on temperature-responsive culture dishes

The oral mucosa is an attractive cell source for autologous transplantation in human patients who require regenerative therapies of various epithelia. However, the time-course of cellular changes in transplanted oral mucosal epithelia at ectopic sites remains poorly understood. By applying a rat model, we analyzed phenotypic changes in oral mucosal epithelial cell sheets after harvest from temperature-responsive culture dishes and subsequent autologous subcutaneous transplantation. We used monoclonal antibodies to identify epithelial-specific cytokeratins 4, 10, 13, and 14, the stem/progenitor cell marker p63, and proliferating cell nuclear antigen, within the regenerated tissues. Transplanted oral mucosal epithelial cell sheets proliferated during the first week after grafting in conjunction with host inflammation, but then began to degenerate afterward with complete disappearance after 3 weeks. Our findings suggest that host subcutaneous tissues support proliferation and differentiation of the oral mucosal epithelial cell sheets, but are unable to promote maintenance of stem and progenitor cells and therefore cannot produce long-term survivability.     

Growth and differentiation of human distal airway epithelial cells in culture. Effects of small amounts of serum in defined medium

We herein describe a new simple method for culturing primary epithelial cells derived from the distal airway of adult human lung. Peripheral lung tissue obtained from surgical materials was cultured as explants in Ham's F12 medium supplemented with hormones and growth factors. From days 10 to 14, outgrowth of epithelial cells started on the dish surface, and even after removal of explants at days 14 to 16, these cells continued to replicate during the 3rd and 4th week of culture, and eventually formed epithelial cell foci (10 to 20-fold increase in population). They then ceased to replicate and terminally differentiated in the 5th week. Addition of 1% serum to the culture medium enhanced the initial outgrowth of epithelial cells, whereas small amounts of serum had no effect on proliferation of cells after explant removal. On the other hand, serum modulated the differentiation phenotypes of epithelial cells. In the presence of 1% serum, numerous ciliated and secretory cells appeared, whereas the cells underwent epidermoid differentiation in the absence of serum. When replated onto 3T3 fibroblast feeder layers, the epithelial cells from earlier cultures showed a great replicative capability and formed colonies at higher frequencies (colony-forming efficiency, 8 to 27% at days 14 to 21), but the replicative capability was significantly reduced at the confluent stage (colony-forming efficiency, 0.44 to 2.0% at day 28). Morphologic examinations of explants strongly suggested that the primary cells were derived from the bronchiolar epithelium. We conclude that this new culture system provides an excellent model for studying the growth, differentiation, and function of human bronchiolar epithelial cells in vitro.     

Selective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer

The main drawback of the selective culture of human mammary epithelial cells from primary breast cancer is the overgrowth of tumor-associated stromal fibroblasts. This drawback may be overcome by using, in primary culture, lethally irradiated 3T3 cells which act as a feeder layer to maintain tumor-derived epithelial cell proliferation. These 3T3 cells, exposed to 60 Gy at confluence, form a specific cellular substrate which constitutes an obstacle to fibroblast attachment. Enzyme-disaggregated breast cells from six primary breast carcinomas were cocultured over lethally irradiated but living 3T3 cells. The method led to the purification of tumor-derived epithelial cells from all six cancer samples, and long-term culture was obtained in one. The epithelial nature of these purified tumor-derived epithelial cells was demonstrated by their general morphology and by the expression of cytokeratins and Epithelial Membrane Antigen. These results confirm the stimulatory effect of a this stromal feeder layer on breast epithelial cell growth and show that this stromal feeder layer can also control the fibroblast overgrowth. Our results provide an alternative approach in the selective culture of epithelial cells from primary breast carcinoma.     

Corneal regeneration by transplantation of corneal epithelial cell sheets fabricated with automated cell culture system in rabbit model

We have performed clinical applications of cell sheet-based regenerative medicine with human patients in several fields. In order to achieve the mass production of transplantable cell sheets, we have developed automated cell culture systems. Here, we report an automated robotic system utilizing a cell culture vessel, cell cartridge. The cell cartridge had two rooms for epithelial cells and feeder layer cells separating by porous membrane on which a temperature-responsive polymer was covalently immobilized. After pouring cells into this robotic system, cell seeding, medium change, and microscopic examination during culture were automatically performed according to the computer program. Transplantable corneal epithelial cell sheets were successfully fabricated in cell cartridges with this robotic system. Then, fabricated cell sheets were transplanted onto ocular surfaces of rabbit limbal epithelial stem cell deficiency model after 6-h transportation using a portable homothermal container to keep inner temperature at 36 °C. Within one week after transplantation, normal corneal epithelium was successfully regenerated. This automatic cell culture system would be useful for industrialization of tissue-engineered products for regenerative medicine.     

A novel culture technique for human embryonic stem cells using porous membranes

We have developed a novel culture technique for human embryonic stem cells (hESCs) using a porous membrane with feeder cells. The feeder cells were seeded and attached to the bottom of a porous membrane and, subsequently, hESCs were cultured on the top of the membrane. This porous membrane technique (PMT) allowed hESCs to be successfully cultured and to be effectively and efficiently separated from the feeder cell layer without enzyme treatment. hESCs being cultured by PMT were observed to interact with feeder cells through pores of membrane, where the interaction was dependent on the pore size of the membrane used. It was also revealed that the number of attached hESC colonies depended on the concentration of feeder cells on the bottom of the membrane. On the other hand, hESC colonies did not attach to porous membrane, as feeder cells were in the presence of culture dish, not the porous membrane. The hESCs cultured on porous membranes not only exhibited expression of several undifferentiated markers and a normal karyotype, but they also formed teratomas consisting of three germ layers in in vivo study. Compared with the mechanical isolation technique conventionally used, PMT significantly decreased mouse vimentin gene expression in cultured hESCs. Thus, a PMT for hESC culture would be a useful tool to exclude enzyme treatment and to reduce contamination from feeder cells simultaneously. Disclosure of potential conflicts of interest is found at the end of this article.     

Tissue engineered epithelial cell sheets for the creation of a bioartificial trachea

To successfully engineer a bioartificial tracheal replacement, it is believed that the regeneration of a functional epithelial lining is a key requirement. In the present study, rabbit tracheal epithelial cells were cultured on temperature-responsive culture dishes, under normal culture conditions at 37 degrees C. By simple temperature reduction to 20 degrees C, the cultured epithelial cells were noninvasively harvested as intact sheets, without the use of any proteolytic enzymes. Support Dacron grafts that had been subcutaneously implanted for 4 weeks to allow for host tissue and vessel infiltration were then opened, and the tracheal epithelial cell sheets were transplanted to the luminal surface without sutures. These fabricated constructs were then used as tracheal replacements, in a rabbit model. Four weeks after transplantation, results showed that the tracheal grafts were covered by a mature, pseudostratified columnar epithelium. In contrast, control constructs that did not receive cell sheet transplantation demonstrated only a thin, immature epithelium at the center of the replacement graft. These results therefore demonstrate that these tracheal epithelial cell sheets can create an epithelial lining on the luminal surface of a bioartificial trachea.     

Three-dimensional nanofibrillar surfaces promote self-renewal in mouse embryonic stem cells

The regulation of mouse embryonic stem cell (mESC) fate is controlled by the interplay of signaling networks that either promote self-renewal or induce differentiation. Leukemia inhibitory factor (LIF) is a cytokine that is required for stem cell renewal in mouse but not in human embryonic stem cells. However, feeder layers of embryonic fibroblasts are capable of inducing stem cell renewal in both cell types, suggesting that the self-renewal signaling pathways may also be promoted by other triggers, such as alternative cytokines and/or chemical or physical properties of the extracellular matrix (ECM) secreted by feeder fibroblasts. We have recently used a synthetic polyamide matrix (Ultra-Web) whose three-dimensional (3D) nanofibrillar organization resembles the ECM/basement membrane. Growth of mESCs on this nanofibrillar surface greatly enhanced proliferation and self-renewal in comparison with growth on tissue culture surfaces without nanofibers, despite the presence of LIF in both systems. Enhanced proliferation and self-renewal of the stem cells on nanofibrillar surfaces were correlated with the activation of the small GTPase Rac, the activation of phosphoinositide 3-kinase (PI3K) pathway, and the enhanced expression of Nanog, a homeoprotein required for maintenance of pluripotency. Inhibitors of PI3K reduced the expression level of Nanog in mESCs cultured on 3D nanofibrillar surfaces. These results provide support for the view that the three-dimensionality of the culture surface may function as a cue for the activation of Rac and PI3K signaling pathways, resulting in stem cell proliferation and self-renewal.