A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis

Understanding the molecular mechanisms that regulate the corneal epithelial stem cells (CESCs) in maintaining corneal homeostasis remains elusive largely due to the lack of a specific marker for their isolation. This study aims to enrich CESCs from human donor limbal epithelium and to evaluate the level of enrichment based on expression of ΔNp63α, a putative CESC marker. A two‐stage enrichment of CESCs was carried out. (a) The limbal basal epithelial cells were isolated by differential enzymatic treatment and five‐fold enrichment was achieved from 2% of CESCs present in the total limbal epithelium. The CESCs were quantified on the basis of two parameters—high expression of p63/ABCG2 and nucleus to cytoplasmic (N/C) ratio ≥0.7. (b) Cytospin smears of isolated basal cells were Giemsa stained and cells with N/C ratio ≥0.7 were separated by laser capture microdissection. This strategy resulted in an enrichment of CESCs to 78.57% based on two‐parameter analysis using p63 and 76.66% using ABCG2. RT‐PCR was carried out for ΔNp63 isoforms (α, β, and γ) and connexin‐43, with GAPDH for normalization. The expression of ΔNp63α was restricted to the enriched population of CESCs in contrast to its absence in limbal basal cells with N/C ratio <0.7 and CCECs. The unique expression of ΔNp63α and 5.9‐fold reduced connexin‐43 expression in the enriched population of CESCs indicates its high purity. Further analysis of these cells will help in elucidating the molecular mechanisms associated with stemness and also in identifying a specific marker for CESCs.     

Propagation of intercellular calcium waves in C6 glioma cells transfected with connexins 43 or 32

In this study, gap junction-deficient C6 glioma cells, transfected with either connexin 43 (Cx43) or 32 (Cx32), have been used to evaluate the ability of these connexins to pass intercellular Ca2+ waves. Ca2+ waves, observed with fluorescence imaging using fura-2 or fluo-3, were initiated by mechanical stimulation in the presence of a supra-perfusion of the extracellular fluid or by the non-contact technique of flash photolysis of intracellular caged-IP3. Following manual mechanical stimulation, the parental C6 glioma cells and cells expressing Cx43 and Cx32 gap junctions all propagated intercellular Ca2+ waves. Ca2+ waves in cells expressing Cx43 traveled approximately twice the distance as compared to waves in cells expressing Cx32 or parental cells. The cells expressing Cx43 were also about twice as sensitive to ATP as cells expressing Cx32. In the presence of a supra-perfusion of extracellular fluid, the Ca2+ waves in parental cells were almost abolished while the mechanically induced Ca2+ waves in the cells expressing Cx43 and Cx32 propagate similar but limited distances of several cells in a direction opposite to the fluid flow. The photolytic release of IP3, but not Ca2+, in cells expressing Cx43 or Cx32 resulted in the propagation of Ca2+ waves that traveled distances similar to those observed in the presence of supra-perfusion. Parental C6 glioma cells did not initiate intercellular Ca2+ waves when stimulated by photolysis. From these studies we conclude that (1) both Cx43 and Cx32 based gap junctions are permeable to IP3 and can serve to communicate Ca2+ waves, (2) that Ca2+ wave propagation via gap junctions was dependent on the diffusion of IP3 but not Ca2+, (3) that an extracellular messenger capable of communicating waves is released from only the stimulated cell, and (4) that simultaneous intracellular and extracellular signaling can occur to enhance the propagation of intercellular Ca2+ waves.     

Q-FIHC: quantification of fluorescence immunohistochemistry to analyse p63 isoforms and cell cycle phases in human limbal stem cells

Fluorescence microscopy has long been used for qualitative characterization of various parameters such as subcellular distribution of proteins, lipids, nucleic acids, and ions. However, quantification of these parameters is complicated by a variety of optical, biological, and physical factors. In the last decade, the progress achieved with powerful softwares and digital image processing systems has facilitated the development of fluorescence immunohistochemistry (FIHC) into a widely used quantitative assay (quantitative-FIHC or Q-FIHC). We describe here a rapid and sensitive Q-FIHC assay based on the use of a laser scanning confocal microscope and advanced image analysis softwares (Zeiss semi automatic LSM 510 and fully automatic Axiovision 4.4) for the detection and quantification of fluorescent intensity in human corneal tissues and cells obtained from small clinical samples. We have used this methodology to characterize and quantify the gene expression profile of p63 and its DeltaNalpha isoform, specific markers of human limbal stem cells. The validity of this method was evaluated through comparative studies with conventional approaches suggesting no significant differences and providing an alternative technique to traditional methods. Since Q-FIHC requires at least 20-fold less cells than traditional techniques, we have adopted it as the main quality control for our limbal cultures destined to clinical application.     

Brief Note: Isolation,culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85± 4.6%), and a correspondingly low proportion K3 positive cells (15 ± 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean ± SE, n=10). Cultures reached full confluency after 17.3 ± 1.2 days when the medium was supplemented with human EGF, while 21.7 ± 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.     

Morphological and molecular pathology of CCL4‐induced hepatic fibrosis in connexin43‐deficient mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl₄)‐induced hepatic fibrosis in mice. We administered CCl₄ to both Cx43‐deficient (Cx43^+/?) and wild‐type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real‐time PCR. We demonstrated that Cx43^+/? mice developed excessive liver fibrosis compared with wild‐type mice after CCl₄‐induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43^+/? mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43^+/? mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl₄‐treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Optimization of culture conditions for an efficient xeno‐feeder free limbal cell culture system towards ocular surface regeneration

Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever‐increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno‐diseases to human system. This study was aimed to establish an efficient xeno‐feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using “interactive measurements” of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno‐feeder free epithelial equivalents towards ocular surface reconstruction. Microsc. Res. Tech. 73:1045–1052, 2010. © 2010 Wiley‐Liss, Inc.     

Comparative analysis and application of fluorescent protein-tagged connexins

In order to examine connexin transport, assembly, and turnover in living cells, we tagged green fluorescent protein or its color variants to several members of the connexin family of proteins. When green fluorescent protein was tagged to the carboxyl terminal end of connexin43 (Cx43-GFP), the resulting fusion protein was transported and assembled into functional gap junctions. However, when GFP was tagged to the amino terminal end of Cx43 (GFP-Cx43), this chimera was biosynthesized, transported to the plasma membrane, but failed to form gap junction channels that could transfer Lucifer yellow. Single cells that expressed Cx43-GFP were capable of transporting this fusion protein to the cell surface in the absence of cell-cell contact. Imaging of Cx43-yellow (Y)FP (Cx43-YFP) was quite efficient; however, the low quantum yield Cx43-BFP and the requirement for ultraviolet excitation made this chimera less suitable for time-lapse imaging. Cx43-cyan C(FP) (Cx43-CFP) was more suitable for imaging than Cx43-blue (B)FP and could be effectively separated from Cx43-YFP. The versatility of tagging GFP to the carboxyl terminal end of other members of the connexin family was established when Cx32-GFP and Cx26-YFP were found to assemble into gap junctions capable of transferring Lucifer yellow. Finally, we are examining the effectiveness of using a new red fluorescent protein (DsRed) fused to connexins in combination with Cx-GFP to simultaneously examine the kinetics, transport and turnover of two connexins. Together, our studies suggest that tagging fluorescent proteins to the carboxyl terminal end of connexins is an effective and valuable approach for studying the life cycle and dynamics of connexins in living cells.     

Transplanted choroidal plexus epithelial cells can integrate with organotypic spinal cord slices into a new system

This study aimed to evaluate the integration of transplanted choroidal plexus epithelial cells with organotypic spinal cord slices. Organotypic spinal cord slices, normally cultured for 6 days, were divided into control group (Ctrl) and transplanted group (T). The choroidal plexus epithelial cells were dissociated and primary cultured (C group). The choroidal plexus epithelial cells cultured for 6–7 days were labeled by 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanineperchlorate (CM-Dil), and were identified by transthyretin (TTR) in immunocytochemistry. They were adjusted to the density of 0.5–1 × 10⁷/ml, then 2 μl cells suspension were transplanted to the spinal cord slices in the T group. The same amount of basal medium was dripped on the spinal cord slices in the Ctrl group. After 14 days of transplantation, the differentiations into neurons and astrocytes, and the synapses were identified by immunofluorescence histochemistry. At the same time, the ratios of cell differentiations and synapses in new system, and the changes of MAPK signaling pathway were tested by western blotting. The choroid plexus epithelial cells were well labeled by CM-Dil and were immune-stained by TTR in immunocytochemistry. The choroid plexus epithelial cells bodies were small when transplanted on the spinal cord slices, but big when transplanted on the polyester membrane inserts. The transplanted cells could differentiate into astrocytes, and possibly differentiate into neurons, and there were a large number of synaptophysin positive vesicles between transplanted cells and organotypic spinal cord slices in immunofluorescence histochemistry. The levels of GFAP, TUB-III and synaptophysin in the T group were higher than which in the Ctrl and C groups in western blotting (P < 0.05). And the ratios of p-JNK/JNK and p-P38/P38 in the T group were significantly lower than which in the Ctrl and C groups (P < 0.05). But the ratio of p-ERK/ERK in the three groups was of no significant difference. The transplanted choroidal plexus epithelial cells can integrate with organotypic spinal cord slices into a new system.     

Extrusion of colonic epithelial cells in vitro

Rat colonic mucosae fixed in situ in Ussing chambers provided a model of the extrusion of absorptive enterocytes and less commonly of goblet and enteroendocrine cells. The cells were lost at extrusion zones midway between crypt mouths. Even in mucosae in which the number of extruding cells was large, epithelial continuity was maintained as evaluated morphologically and electrophysiologically. Beneath points of remaining contact between desquamating cells and the epithelial sheet, microfilaments of the terminal web formed band-like structures linking adjacent junctional complexes. Freeze-fracture replicas disclosed extensive macular regions of tight junction strands in the plasma membranes of desquamating cells. Tight junctions between newly neighboring cells were often irregular and often occurred beneath the terminal web region. Dithio-threitol enhanced cell loss and increased basal epithelial conductance, but histological continuity was maintained and the mucosae continued to respond typically to bradykinin. These observations suggest that during the loss of senescent enterocytes, tight junctions are maintained; old junctional elements are lost, and tight junctions are formed between remaining adjacent cells. This model offers a means to study the synthesis and turnover of tight junctions and the maintenance of the colonic epithelial barrier.     

ACTH and adrenocortical gap junctions

Since the initial identification of gap junctions in the adrenal gland, it has been proposed that a system involving direct cell-cell communication might be involved in adrenal cortical functions. Gap junction channels do, in fact, provide pathways for direct intercellular exchange of small molecules (<1,000 Da), many of which have the potential to influence a wide range of cellular activities. Gap junctions are composed of proteins called connexin which, in the adrenal cortex, have proven to be remarkably consistent in both type and zonal distribution with connexin 43 (Cx43) as the predominant component in mammalian adrenal glands thus far evaluated. Only the inner two zones of the cortex (zonae fasciculata and reticularis) exhibit significant amounts of Cx43 and functional coupling. Adrenocorticotropin (ACTH) has been shown to increase Cx43 protein in vivo and in vitro, and a strong correlation has been noted between the presence of gap junctions and certain adrenal cortical functions, especially steroidogenic capacity and cell proliferation. This review summarizes evidence of the Cx43 expression in adrenal cortical cells and the likely role of Cx43 in steroidogenesis and cell proliferation. It is concluded that control of gap junction expression in the adrenal gland is hormonally dependent and is functionally linked to adrenal gland zonation.     

Improvement of transfection with reprogramming factors in urinederived cells

Human-induced pluripotent stem cells (iPSCs) are an accessible source of adult-derived, patient-specific pluripotent stem cells for use in basic research, drug discovery, disease modeling, and stem cell therapy. Improving the accessibility of methods to obtain iPSCs regardless of the cell source can enhance their clinical application. Therefore, our purpose is to report a simple protocol to obtain iPS-like cells from urine-derived renal epithelial cells (RECs) using different extracellular matrices and transfection reagents. In this study, we began by culturing urine-derived cells from healthy donors to establish a primary culture of renal epithelial cells, followed by their characterization. Subsequently, we generated iPS-like cells by transfecting renal epithelial cells (RECs) with vectors expressing Oct4, Sox2, L-Myc, Lin-28, and Klf4, and we compared the efficacy of different extracellular matrices and transfection reagents. The resultant iPS-like cells showed a human embryonic stem cell-like morphology and expressed the specific pluripotency markers Oct3/4, Nanog, Lin28, and Klf4. We concluded that Lipofectamine Stem Cell transfection reagent is more effective than FuGENE in obtaining iPS-like cells under the conditions tested. Moreover, the three matrices are similar in their efficiency of obtaining iPS-like cells. This report provides an experimental protocol for obtaining and generating iPS-like cells from urine samples for further cell therapy research on different human diseases.     

Unique features of the basal cells of human prostate epithelium

The prostate gland is globally composed of epithelium and stroma. The epithelium plays an important role in the development of both benign and malignant disorders while the stroma is involved in benign prostatic hyperplasia. While the prostatic epithelium of the majority of laboratory animals is well recognized as a pseudostratified columnar, the classification of the human prostatic epithelium is controversial. Moreover, the role of the basal cells of the human prostatic epithelium is still uncertain. These cells have been described as undifferentiated cells, precursors of luminal cells, reserve and myoepithelial cells. The objective of the present study was to assess the similarities and/or differences between the epithelium of the human prostate and that of other laboratory animals and thus derive information about the potential functions of basal cells in the human prostate. In the human, basal cells form a continuous layer of cells resting on the basement membrane and upon which rests a layer of luminal cells. This results in a stratified columnar epithelium of two layers of cells, unlike the sporadic appearance of basal cells observed in other species where it results in a pseudostratified epithelium. In addition, the ratio of basal to luminal cells in the human is about 1:1, while the average ratio in the other animal species examined is about 1:7. Furthermore, the gap junctional proteins connexin 26 and 43, are present between basal and luminal cells in the human, thus suggesting that these cells communicate directly with each other. In addition, the ultrastructure of the human basal cells shows morphological evidence of differentiated but not of undifferentiated cells. Moreover, the presence of junction-like structures between adjacent basal cells suggests that these cells form a blood-prostate barrier. In this way, basal cells could prevent substances derived from the blood from directly coming in contact with the luminal cells. Human basal cells could thus regulate functions of the luminal cells by being part of a two-cell mechanism somewhat analogous to thecal and granulosa cells in the ovary.     

Light-microscopic demonstration of myoid fibrils in renal epithelial, mesangial and interstitial cells

In kidney sections treated with the tannic acid-phosphomolybdic acid-Levanol fast cyanine 5RN procedure, myoid fibrils were observed in tubular and glomerular cells. The myoid fibrils in tubular epithelial cells were delicate in normal kidneys, but became conspicuous under pathological conditions. Under certain conditions myoid fibrils were found in capsular epithelium and mesangial cells of glomeruli; in some cases podocytes also contained prominent myoid fibrils. There seemed to be a continuous system of myoid fibrils from the media of the afferent arteriole into the mesangium and through the capsular into the tubular epithelium. These myoid fibrils could not be identified with certainty in sections treated with conventional staining methods. Correlations of light-microscopic observations with electron-microscopic data indicate that the myoid fibrils in renal epithelial cells visualized by the TP-Levanol fast cyanine 5RN stain correspond to the myofilamentous system described in electron-microscopic studies by Pease (1968a). The myoid fibrils in tubular and capsular epithelial cells resembled the long forgotten ‘Basalreifen’ described around the turn of the century. A review of the literature shows that structures corresponding to the basal myoid fibrils were known to early histologists and that contractile functions had been suggested.     

Modified enzymatic collagen digestion-mediated isolation of osteocytes

This study established a method for isolating large numbers of high-purity osteocytes from high-density bone. Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times, and the digested cells and bone chips (BC) were cultured, digested, and passaged when cells were fully grown. The types of cells obtained were identified by morphology, viable cell counts, alkaline phosphatase staining, and biochemical activity analyses, and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain reaction. Our results showed that among the cells obtained from the third digestion (fractions 7–9) of wild mice tibias and femurs and the remaining BCs, 85%–90% of the cells were osteocytes. Moreover, their morphology was approximately one-tenth to one-fifth the size of osteoblasts, star-shaped or polygonal, with a dendritic structure, negative for alkaline phosphatase staining, and showed a high expression of dmp1 and sclerostin. Ninety percent of the cells in fractions 1–3 were osteoblasts, and were fusiform or polygonal shape. The activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction, while the expression of osteocyte-specific dmp1 and sclerostin was not detected. In the second portion (fractions 4–6), a large number were osteoblasts, mixed with a small number of osteocytes, and had high alkaline phosphatase activity and osteocyte mRNA levels, a specific level of the osteocyte marker dmp1, and no sclerostin was detected. Osteocytes in daβcat^ot mice were also successfully isolated by this method, and we found that Wnt signaling increased the proliferation of these osteocytes. The proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone, which provides an avenue for the study of osteocyte function in vitro.     

Observations of human corneal epithelium by tandem scanning confocal microscope

We applied the tandem scanning confocal microscope (TSCM) to 30 healthy human corneas of 3 normal volunteers and 27 patients with cataract and retinal detachment to observe normal corneal epithelial cells in vivo. All corneas were normal under slit lamp microscopic examination. The superficial and basal epithelial cells close to the basal lamina in the central cornea were recorded on videotape and analyzed by a computer-assisted digitizer. The mean cell areas of superficial cells exposed at the surface and basal cells at the horizontal section were 624 ± 109 μm² and 66 ± 5 μm², respectively. The ratio of superficial to basal mean cell area was 11.0 ± 4.5. TSCM was thus useful in evaluating the relationship between superficial and basal cells in human corneal epithelium in vivo.     

Observations of human corneal epithelium by tandem scanning confocal microscope

We applied the tandem scanning confocal microscope (TSCM) to 30 healthy human corneas of 3 normal volunteers and 27 patients with cataract and retinal detachment to observe normal corneal epithelial cells in vivo. All corneas were normal under slit lamp microscopic examination. The superficial and basal epithelial cells close to the basal lamina in the central cornea were recorded on videotape and analyzed by a computer-assisted digitizer. The mean cell areas of superficial cells exposed at the surface and basal cells at the horizontal section were 624 ± 109 μm² and 66 ± 5 μm², respectively. The ratio of superficial to basal mean cell area was 11.0 ± 4.5. TSCM was thus useful in evaluating the relationship between superficial and basal cells in human corneal epithelium in vivo.     

Altered enteroendocrine cell expression in T cell receptor alpha chain knock-out mice

Mice lacking T cell receptor alpha chain (TCRalpha(-/-)) develop inflammation of the colon. We have examined the effect of this inflammation on the colonic epithelium by studying markers of epithelial cuff, enteroendocrine, and immune cell differentiation. Using immunohistochemical techniques, colons were compared in normal C57/BL6 and murine TCR alpha(-/-) mice aged 2 and 3 weeks and 3-11 months. TCR alpha(-/-) mice aged 3-11 months had histologic evidence of inflammation with increased expression of CD45, CD4+, CD8+, and B220+ cells and a decrease in expression of IgA+ cells. There was a decrease in the number of cholecystokinin, serotonin, and neurotensin enteroendocrine expressing cells in the colon of TCR alpha(-/-) mice. These changes were not present in 2-3-week-old suckling/weaning mice. In contrast, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1, and gastrin expression did not change and small intestinal enteroendocrine cells remained unaltered. The change in colonic enteroendocrine cell expression appears to be a specific response, since only a subset of these cells was altered, and the epithelium was intact by histologic analysis. The absence of functional T cells in TCR alpha(-/-) colon has a marked effect on differentiation of a specific subpopulation of enteroendocrine cells, prior to loss of integrity of the epithelium.     

Novel neurotrophic factor secreted by amniotic epithelial cells

By virtue of expressions of glial and neural surface markers and capability of neurotransmitter metabolism, amniotic epithelial cells are considered as candidate cell type for transplantation strategies to treat neurological disorders. Previously, we have reported neurotrophism exhibited by human amniotic epithelial cells when transplanted after spinal cord injury in bonnet monkeys. Amniotic epithelial cells were believed to secrete an “Epidermal Growth Factor (EGF) - like” factor and exact identification was not made. At this juncture, through the present study it was found that, chicken neural retinal cells when grown alone failed to survive and contrarily when either co-cultured with chicken amniotic epithelial cells / cultured in amniotic epithelial cell conditioned medium not only survived but also showed extensive differentiation. Fibroblast Growth Factor – 2 (FGF-2) plays a critical role in retinal development especially in chicken neural retinal development. However, immunoassay using western blot did not revealed the presence of any already known isoforms of FGF-2 in the medium. It is interesting to note that while factor secreted by amniotic epithelial cells resembles EGF and/or FGF-2 in its biological action, known isoforms of them were not detected. Considering the biological closeness between EGF and FGF-2, results indicate the possibility of a novel isoform of these growth factors secreted by amniotic epithelial cells. Further studies will establish the nature of this novel factor which will enhance the application of this interesting cell type for neural transplantations.