Effects of Thioureas, Inhibitors of Melanogenesis, on Lens Transdifferentiation of Cultured Chicken Embryonic Retinal Pigment Cells

The effects of phenylthiourea (PTU) and its analogues on chick embryonic pigmented epithelial cells (PECs) in culture were studied to elucidate the correlation between inhibition of melanogenesis of PECs and enhancement of their transdifferentiation into lens cells.
Both 0.25–0.5 mM PTU and 0.1 mM alpha-naphthylthiourea (ANTU) effectively inhibited melanogenesis of PECs and stimulated their transdifferentiation into lens cells at the same time. Thiourea (TU) also inhibited melanogenesis at a much higher concentration (4 mM), but did not stimulate the lens transdifferentiation at all. Methylthiourea (MTU), on the other hand, did not inhibit melanogenesis, but stimulated the lens transdifferentiation. Testicular hyaluronidase effectively amplified the above-mentioned stimulating effects of thioureas without their altering optimum concentrations, although this enzyme itself never enhanced the lens transdifferentiation of PECs but suppressed their melanogenesis at a concentration of 100 U/ml medium, onward.
These results suggest that the suppression of melanogenesis of PECs by PTU or its analogues does not directly correlate with their transdifferentiation into lens cells. The possible mode of thiourea actions on the lens transdifferentiation of PECs cultured in vitro is discussed.     

Cellular and molecular background of Wolffian lens regeneration

Based on studies of wolffian lens regeneration in the newt, in which the lens can be regenerated from the iris pigmented epithelium, we have shown by cell culture studies that the capacity of lens transdifferentiation is not limited to the newt cells, but widely conserved in pigmented epithelial cells (PECs) of chick and quail embryos and even of human fetuses. Recently, we have established a unique in vitro model system of chick embryonic PECs. In this culture system we are able to control each step of transdifferentiation from PECs into lens cells by regulating culture conditions and to produce a homogeneous cell population with potential for synchronous differentiation into either lens or pigment cell phenotype. These multipotent (at least bipotent) cells showed cellular characteristics resembling neoplastic cells in many ways. They did not express both lens and pigment cell specific genes analyzed so far, except δ-crystallin gene, which is expressed in developing lens of chick embryos. It has been proved by application of cell culture procedures of the system that PECs dissociated from fully-grown human eyes readily transdifferentiated into lens phenotypes in the manner observed in chick embryo PECs. In addition, we could predict that molecules detected in either cell surface or intercellular space stabilized the differentiated state of PECs in the newt and that the loss of these molecules might be one of the key steps of lens regeneration from the iris epithelium.     

In VitroCulture System for Iris-Pigmented Epithelial Cells for Molecular Analysis of Transdifferentiation

Dissociated cells of the iris-pigmented epithelium (IPE) from a 1-day-old chick grew in monolayer culture and stably maintained their differentiated state when cultured with standard culture medium. After replacement of the control medium by EdFPH medium, which is effective in inducing dedifferentiation of retinal pigmented epithelium (RPE) cells, all cells rapidly lost pigment granules, proliferated intensively, and dedifferentiated. By further addition of ascorbic acid, dedifferentiated cells accumulated and formed a large number of lentoids. This system provides a useful opportunity for analyzing cellular and molecular mechanism involved in each step of transdifferentiation. Furthermore, Northern blot data indicates that the up-regulation of pax-6 gene could be an important event during lens regeneration as well as during normal lens development.     

The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells

A monoclonal antibody (MC/1) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that MC/1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomal matrix. A single protein band with an apparent molecular weight of 115,000 was labelled by MC/1 in Western blotting. The 115 kDa polypeptide identified by MC/1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable.     

Gene expression during lens transdifferentiation from pigmented epithelial cells

Pigmented epithelial cells of chicken and human dedifferentiate in the medium containing phenylthiourea and testicular hyaluronidase, and then trans-differentiate into lens cells in vitro. To understand the molecular mechanisms of transdifferentiation, gene expression during lens transdifferentiation was analyzed. As the first step, pigment cell and lens specific genes were isolated and expression of these gene was analyzed by Northern blotting . These results clearly shown that lens transdifferentiation proceeds via neutral cell state in which both pigment and lens specific genes are repressed. Oncogene expression was also analyzed. An elevated expression of the c-myc gene was observed during dedifferentiation process. It is expected that elevated expression of c-myc gene might prevent the cells from entering the G0 phase and thus lead to dedifferentiated state.     

Cell cycle changes during neural crest cell differentiation in vitro.

Chick trunk neural tubes containing neural crest cells were cultured in vitro. Cell outgrowth from these neural tube explants consists primarily of a small stellate cell population. After 3 days in culture the small stellate cell population undergoes a remarkable change in morphology that is characterized by a more refractile appearance in the phase contrast microscope. Subsequent to this change in morphology, pigment granules become visible in the cytoplasm after 4 days in culture. After 6 days in culture, virtually all of the small stellate cells are pigmented. The cell cycle parameters of the small stellate cell population are: S = 4.4 ± 1.2 hr (SD). G2 = 1.5 ± 1.0 hr (SD). M = 1.7 ± 0.6 hr (SD). and Gl = 3.8 ± 1.0 hr (SD). Continuous label experiments demonstrate that (G1+G2+M) increases from 7 hr in Day 4 cells, as yet unpigmented, to 12 hr in Day 5 cells that have become pigmented. This change is consistent with an increase in G1 and/or G2 that is closely correlated with the appearance of pigment granules. It is of interest that this cell cycle change is correlated with a rather late event in the developmental program of these neural crest cells rather than with the earlier morphological change observed after 3 days in culture.     

Alterations in pH and Serum Concentration have Contrasting Effects on Normal and "Foreign" Pathways of Differentiation in Cultures of Embryonic Chick Neuroretinal Cells

Nine-day chicken embryo neuroretinal cells transdifferentiate into both lens and pigment cells after 3–4 weeks when cultured in MEM medium containing 10% foetal calf serum at pH 7.4. At pH 6.8. the appearance of lens crystallins is retarded and cholineacetyltransferase (CAT) activity persists for longer, whereas at pH 8.0 crystallins appear earlier and CAT activity declines more rapidly. Cell survival and culture growth are about 10% lower at pH 6.8 than at pH 8.0. If the concentration of foetal calf serum (FCS) is increased from 10% to 25% (at pH 7.4), cell survival and growth are both promoted, crystallins appear slightly earlier and CAT activity declines more rapidly. Converse effects are observed with 5 % serum, accumulation of crystallins being greatly inhibited and CAT activity prolonged. Crystallin production in cultures with 10% or 25% chicken serum (CS) is much less extensive than in similar FCS cultures, but in cultures with 5 % CS, crystallins appear more rapidly, reaching higher levels than in 5 % FCS cultures. However, the pattern of CAT activity in response to different serum levels is similar for both CS and FCS. This might imply the presence of some factor(s) able to stimulate transdifferentiation in FCS, whereas CS can apparently inhibit this process.     

Birth of piglets derived from porcine zygotes cultured in a chemically defined medium.

We evaluated the in vitro development of porcine zygotes that were cultured in a novel culture medium, porcine zygote medium (PZM), under different conditions and compared to in vivo development. The viability of these zygotes to full term after culture was also evaluated by embryo transfer to recipients. Porcine single-cell zygotes were collected from gilts on Day 2 after hCG injection. Culture of zygotes in PZM containing 3 mg/ml of BSA (PZM-3) produced better results in terms of proportion of Day 6 blastocysts, Day 8 hatching rate, and numbers of inner cell mass (ICM) cells and total cells in Day 8 embryos than that in North Carolina State University (NCSU)-23 medium. In culture with PZM-3, embryo development was optimized in an atmosphere of 5% CO2:5% O2:90% N2 compared to 5% CO2 in air. The ICM and total cell numbers in Day 6 embryos cultured in PZM-3 or in PZM-3 in which BSA was replaced with 3 mg/ml of polyvinyl alcohol (PZM-4) were also greater than those of NCSU-23 but less than those developed in vivo. However, no difference was found in the ratio of ICM to total cells among embryos developed in PZM-3, PZM-4, or in vivo. When the Day 6 embryos that developed in PZM-4 (99 embryos) or in vivo (100 embryos) were each transferred into six recipients, no difference was found in the farrowing rate (83.3% for both treatments) and in the number of piglets born (33 and 42 piglets, respectively). Our results indicate that porcine zygotes can develop into blastocysts in a chemically defined medium and to full term by transfer to recipients after culture.     

In vitro dedifferentiation of melanocytes from adult epidermis

In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation.     

Phenotypic modulation of parietal epithelial cells of Bowman's capsule in culture

The origin of cobblestone-like polygonal cells (the most numerous in renal glomerular cell culture) remains controversial; they could be either dedifferentiated podocytes or parietal epithelial cells (PECs) of Bowman's capsule. Poor cellular outgrowth from glomeruli devoid of Bowman's capsule (decapsulated glomeruli) hinders podocytes being obtained without contamination of PECs in culture. Since podocytes are easily damaged during the isolation of glomeruli by the conventional sieving method, we devised a gentle isolation method without forced sieving, resulting in substantial numbers of arborized cells growing out from decapsulated glomeruli. The cells were distinctly different from cobblestone-like polygonal cells in their irregular and often arborized shape and extended long cytoplasmic processes that often crossed over adjacent cells. The arborized cells from decapsulated glomeruli showed intense staining for a podocyte-specific marker, podocalyxin, but no staining for markers specific to PECs (pan cadherin), mesangial cells (Thy-1) or endothelial cells (von Willebrand factor, RECA-1), indicating their podocyte origin. Polygonal cells growing out from encapsulated glomeruli were negative for podocalyxin and positive for pan cadherin at the peripheral cell-cell contact. Thus, the cell population from decapsulated glomeruli is distinctly different from that from encapsulated glomeruli, supporting the idea that polygonal cells originate from PECs, although immunocytochemical markers specific to podocytes in vivo such as WT1, synaptopodin, HSP27 and P-31 antigen were expressed significantly in the polygonal cells. Occasionally, large irregular-shaped cells appeared at the periphery of the outgrowths from encapsulated glomeruli. They were similar in shape to the arborized cells from decapsulated glomeruli but were identical in antigenic properties to cobblestone-like polygonal cells and thus may be named "pseudo-arborized cells". We conclude that PECs in culture modulate their phenotype to resemble podocytes.     

In vitro prostaglandin release from and platelet-activating factor accumulation in isolated endometrial cells from pregnant and pseudopregnant rabbits.

Prostaglandin (PG) release from and platelet-activating factor (PAF) accumulation by enzymatically isolated endometrial epithelial and stromal cells from Day 6 pregnant and Day 6 pseudopregnant rabbits were studied in vitro, using RIA for PG measurement and a platelet aggregation assay for PAF measurement. On the first day of culture in serum-free media, PGF release into the medium was significantly higher from epithelial cells from Day 6 of pregnancy than from stromal cells from Day 6 of pregnancy or pseudopregnancy. PGE release did not differ significantly among these cell types. The addition of indomethacin (10(-5) M) to similar cultures inhibited release of both PGs from both cell types, but to a much greater extent from stromal than from epithelial cells. Significant stimulation of PG release by A23187 was achieved under all conditions on the fifth day of culture; PGE release was significantly greater than PGF release from stromal cells from Day 6 of pregnancy and pseudopregnancy, and release of both PGs from stromal cells was significantly greater from Day 6 of pregnancy than from Day 6 of pseudopregnancy. PG release from similar cells, cultured in medium containing 10% calf serum, was highest on the first or second day of culture and then, especially for PGF, declined with continued culture. PGE release was significantly higher than PGF release from stromal cells on the third and fourth days of culture. The ratios of PGF/PGE release from epithelial cells were significantly higher than those from stromal cells over the 5-day culture period for both reproductive stages. These ratios indicate the differential release of PGE and PGF from rabbit endometrial cell subpopulations and indicate a preferential release of PGE from stromal and of PGF from epithelial cells. Under basal conditions, PAF was not detected in epithelial or stromal cells cultured for 2 or 4 days, or in the associated culture media. If PAF had been released into the medium, it would have rapidly metabolized. Short exposure to calcium ionophore A23187 (10(-5) M) was able to stimulate PAF accumulation in epithelial and stroma cells in serum-free media, probably via the remodeling pathway. PAF was not detected in the medium. Intracellular PAF accumulation after exposure to A23187 (10(-5) M) for 5 min was significantly greater on the second day of culture than on the fourth day in epithelial and stromal cells from Day 6 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

Effect of carotenoids and ascorbic acid of Japanese persimmons on cellular lipid peroxidation in HepG2 cells

In this experiment, we examine the functional property of carotenoids; beta-cryptoxanthin (Cry), zeaxanthin (Zea), beta-carotene (Car)) and ascorbic acid (AsA). The accumulation amounts of Cry, Zea and Car in HepG2 cells cultured in the high concentration medium were larger than that in a low concentration. Further those accumulation amounts in long incubation time within 24 hours were greater than that in a shorter time. When the added carotenoid concentration, with or without hydrogen peroxide, increased from 0 to 5 microM in the culture medium, the thiobarbituric acid reaction substance (TBARS) values in the HepG2 cells decreased significantly (p < 0.05). The decrease of TBARS values shows the antioxidative property of the carotenoids. When AsA and Tocopherol(Toc) were added to the medium from 0 to 20 microM, the TBARS values, with or without hydrogen peroxide, decreased significantly with increasing concentrations of AsA and Toc respectively (p < 0.05). The decreased amount of TBARS in 5 microM Cry compared with control(0 microM) was the largest among 6 antioxidants (Cry, Car, Zea, Retinol(Ret), AsA, Toc) used in this experiment.     

Development retardation in cultured preimplantation rabbit embryos

Day 3 to Day 5 preimplantation rabbit embryos were cultured for 24 h in chemically defined media which are widely used in early embryo culture (BSM II and Ham's F-10) supplemented with BSA or homologous serum. For the next 24 h, the embryos were left in the same culture medium, placed in freshly made medium, or cultured in medium which was supplemented with uterine flushings. In addition, 24-h cultured embryos were transferred into uteri of synchronous recipients for 1 day. After culture or transfer, development was assessed by cell proliferation evaluated by incorporation of tritiated thymidine. In comparison to non-cultured controls, thymidine incorporation demonstrated a considerably impaired cell proliferation after culture in defined media irrespective of medium, supplement, or replenishment with fresh medium. For Day 3 embryos, there was a developmental retardation amounting to about 1 day after 2 days in culture. Compared to Day 3 embryos, delay was clearly more pronounced in Day 4 and Day 5 blastocysts, i.e. in stages which had been retrieved from the uterus before culture. Supplementation with uterine flushings markedly promoted blastocyst cell proliferation (P less than 0.001). Incorporation data examined after transfer showed that impairment of cell proliferation caused by 1 day in culture had been compensated for to a large extent within 1 day in utero.     

Enhanced neuronal expression in reaggregating cells of mouse cerebellum cultured in the presence of poly-L-lysine

Both neuronal and glial cell differentiation occur in reaggregating cell cultures of mouse cerebellums, as evidenced by electron microscopy and immunofluorescence to the glial fibrillary acidic protein (GFA). However, after the initial 10 days in culture a process occurs in which the neuronal cells degenerate while glial cells predominate. We have found that when poly-l-lysine is added to the culture medium either for the entire culture period or during the latter days of culture, i.e., Days 4 through 10, the neuronal character is stabilized, as evidenced by acetylcholin-esterase levels and electron microscopy, while the gliosis is inhibited. Culturing reaggregating cells in poly-l-lysine containing medium from Days 0 through 4 has no inhibitory influence on the gliosis observed on Day 10. Cerebellar cells cultured as monolayers on plastic surfaces coated with poly-l-lysine express an intense immunofluoresence with antisera to GFA as do cells grown on uncoated flasks. The data suggest that poly-l-lysine in reaggregating cell cultures stabilizes the neuronal cells by some unknown mechanism. It is postulated that a stable neuronal population reduces the trend toward gliosis in cerebellar aggregates.     

AGE-DEPENDENT CHANGE IN THE TRANSDIFFERENTIATION ABILITY OF CHICK NEURAL RETINA IN CELL CULTURE*

We examined how the transdifferentiation ability of neural retinal cells into lens and/or pigment cells in call culture is changed with the development of the donor. Cells dissociated from neural retinas of chick embryos ranging from 3-day-old to the stage immediately before hatching and of 3-day-old chicks were cultured for about 60 days. The results clearly indicated that the transdifferentiation ability decreased with age. The latest developmental stage at which the differentiation of lens cells took place was in 18-day-old embryos. A gradual decrease in this ability was shown by the comparison of crystallin content in cultures prepared from embryos at different stages. The differentiation of pigment cells was recognized in cultures of neural retinas earlier than in 15-day-old embryos. Such loss of the ability of neural retinal cells to transdifferentiate into pigment cells earlier than into lens cells can be partially attributed to inhibitory factors accumulated in medium conditioned with many neuronal cells present in cultures.     

The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos

Undissociated tissue explants of the retina and retinal pigment epithelium (RPE) of 3,5-, 4-, 5- and 8-day-old chick embryos were cultured in vitro. After 7 days in culture, lentoids were observed in explants of either retina or RPE from 3,5-, 4- and 5-day-old embryos. As demonstrated by immunohistochemistry, these lentoids contained specific chick lens proteins (alpha-, beta- and delta-crystallins). No crystallin-containing cells were found in eye tissue explants from 8-day-old embryos. However, when 5-bromo-deoxyuridine (25 microM) was introduced into the medium at the beginning of culturing (for 12 h), large eosinophilic cells containing alpha-, beta- and delta-crystallins were detected in retinal explants of the 8-day old embryos. Thus, retina and RPE of 3,5-5-day-old chick embryos are capable of lens differentiation after explantation in vitro without dissociation into individual cells. This capacity is lost during development.     

Differentiation of newborn rat preadipocytes in culture: effects of insulin and dexamethasone

A preadipocyte cell population isolated from the inguinal tissue of 3-day-old rats converts at confluence into mature adipocytes when cultured with insulin (10(-9) M). Insulin is necessary only from Day 4 postplating. If the addition of insulin is further delayed, the proportion of cells which will undergo adipose conversion decreases. A loss of the differentiation competence is also observed when the cells are allowed to proliferate (seeding at a low density in a serum containing medium). A preexposure of the primary cells to dexamethasone during the insulin-insensitive period (Days 0-4) accelerates the subsequent "insulin-dependent" adipose conversion. In order to produce its effect, dexamethasone needs only to be present for 4 h on Day 2 postplating. The effect of dexamethasone is probably due neither to inhibition of cell proliferation nor to induction of the cell content of insulin receptors. The evolution of G3PDH enzyme activity as well as of G3PDH protein and mRNA was used as an indicator of the differentiation process. The enzyme accumulates to a low extent during culture in the absence of insulin. When insulin is present, the enzyme level is dramatically increased (maximum on Day 11). Dexamethasone pretreatment (Days 0-4, or 4 h on Day 2) accelerated the G3PDH enzyme activity increase as well as protein and mRNA accumulation. This was also true in cells maintained in insulin-free medium; however, in this case, the increase in the enzyme activity was limited to the first 8 days of culture and full differentiation did not take place. We conclude that: (1) the rat preadipocytes are committed to differentiate, requiring insulin as a sufficient physiological stimulus; (2) the differentiation program is progressively lost after greater than 4 days of culture without insulin and more rapidly if the cells are allowed to undergo divisions; and (3) dexamethasone accelerates the insulin-dependent adipose conversion but alone does not ensure the complete differentiation process.     

Three-Step Method for Proliferation and Differentiation of Human Embryonic Stem Cell (hESC)-Derived Male Germ Cells

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.