μM Retinoic Acid Research Articles

Influence of retinoic acid on human gingival epithelial barriers.

The gingival epithelium plays an important role in the protection of oral tissues from microbial challenge. Oral keratinocytes form a barrier and show various cellular contacts, including tight junctions (TJ). To analyse the barrier function in vitro the transepithelial electrical resistance (TER) is commonly used. Retinoic acid (RA) is an important signalling molecule in most tissues, including epithelial differentiation. RA signalling is mediated through three RA receptors. The aim of the study was to investigate the influence of RA on human gingival barriers in vitro. Immortalized human gingival keratinocytes were seeded on culture plate inserts. The effect of RA with and without infection with Porphyromonas gingivalis W83 on the barrier was analysed by TER measurements. The expression of TJ proteins was investigated by western blot. During differentiation, mean TER increased from 16 (1 h), 43 (4 h) to 62 (6 h) Ohm × cm2 . Addition of 15 μm RA increased TER by +19 after 1 h, +25 after 4 h and +16 Ohm × cm2 after 6 h. The pan-RA receptor inhibitor BMS 493 resulted in TER values comparable to the control. The mean established TER of the control was approximately 110 Ohm × cm2 . Addition of 15 μm RA elevated TER to 127 Ohm × cm2 after 1 h, 150 Ohm × cm2 after 4 h and 189 Ohm × cm2 after 6 h (p ≤ 0.01). RA plus infection with P. gingivalis W83 further increased the TER increasing effect but could not prevent the destruction of TER induced by bacterial infection. The protein expression of the TJ proteins claudin 4 and occludin was enhanced while ZO-1 was downregulated after 1 h of RA incubation. RA provides barrier-positive elements to the gingival epithelial cell model that is accompanied by altered expression of TJ proteins.

Ouabain-Induced Signaling and Cell Survival in SK-N-SH Neuroblastoma Cells Differentiated by Retinoic Acid.

Ouabain stimulates activation of various signaling cascades such as protein kinase B (Akt) and Extracellular-signaling-regulated kinase 1/2 (ERK 1/2) in various cell lines. Retinoic acid (RA) is commonly used to induce neuroblastoma differentiation in cultures. Upon RA administration, human neuroblastoma cell line, SK-N-SH demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Here we report that ouabain-induced signaling is altered under the action of 1 μM RA in human neuroblastoma SK-N-SH cells. RA increased the expression of p110α subunit of phosphoinositide 3-kinase (PI3K), Akt and β1 subunit of Na+/K+-ATPase. Ouabain activated Akt and ERK 1/2 in differentiated SK-N-SH cells; this effect was not observed in non-differentiated SK-N-SH cells. Long-term incubation of non-differentiated SK-N-SH with 1 μM ouabain led to a decrease in the number of cells; this effect was reduced in differentiated SK-N-SH cells. Taken together, these results suggest that ouabain leads to cell death in neuroblastoma cells rather than neuronal cells due to the different response to ouabain manifested by activation of Akt and ERK 1/2. Highlights • RA increases the expression of p110α subunit of PI3K, Akt and β1 subunit of Na+/K+-ATPase• Ouabain induces activation of Akt and ERK 1/2 in differentiated SK-N-SH cells but not in non-differentiated cells• 1 μM ouabain leads to a decrease in the number of cells in non-differentiated SK-N-SH• Reduction of ouabain-induced cell death in differentiated SK-N-SH

Derivation of male germ cells from ram bone marrow mesenchymal stem cells by three different methods and evaluation of their fate after transplantation into the testis.

Mesenchymal stem cells (MSCs) have the capacity to differentiate into germ cells (GCs). This research, for the first time, has evaluated the fate of in vitro MSC-derived GCs generated by three different induction methods and compared them after transplantation into the testes of rams. Passage-3 ram bone marrow (BM)-MSCs were divided into three treatment groups: (1) 14-d treatment with 10 μM retinoic acid (RA; RA14), (2) 21-d treatment with 10 μM RA (RA21), and (3) 21-d treatment with 10 ng/ml transforming growth factor beta-1 (TGFb1). After confirmation of the existence of germ-like cells in the culture, the treated cells were labeled and transplanted into the testes of ram lambs. After 2 mo, we conducted histological evaluations of the rams' testes. Results showed that in vitro-derived GCs from all treatment groups survived in the testes. Some of these GCs homed at the basement membrane of seminiferous tubules and formed colonies. The homed cells and cell colonies were similar to testicular native spermatogonia and expressed PGP9.5. TGFb1 exhibited the highest efficiency for in vitro production of GCs as well as the highest capability for homing and colony formation in the testes. RA21 was less efficient than TGFb1, particularly in colony formation. RA14 was the weakest group. No further differentiation of the transplanted GCs was observed. From our results, it could be concluded that a 21-d treatment period of BM-MSCs with TGFb1 is the most efficient method for in vitro generation of spermatogonia-like cells that survive, home, and form colonies in the testes.

Ex vivo culture of human fetal gonads: manipulation of meiosis signalling by retinoic acid treatment disrupts testis development.

What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads? RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures. One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo. This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation. Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay. A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P < 0.05) and DMRT1-positive oogonia initiating meiosis (P < 0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P < 0.05), a reduced percentage of proliferating gonocytes (P < 0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology. The number of samples included in this study was relatively small due to the limited availability of human fetal tissue. The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects. This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and Aase & Ejnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest.

Application of retinoic acid improves form and function of tissue engineered corneal construct

ABSTRACTRetinoic acid has recently been shown to control the phenotype and extracellular matrix composition of corneal stromal cells cultured in vitro as monolayers. This study set out to investigate the effects of retinoic acid on human corneal keratocytes within a 3D environment. Human corneal keratocytes were encapsulated in collagen gels, which were subsequently compressed under load, and cultured in serum-free media supplemented with 10 µM retinoic acid or DMSO vehicle for 30 days. Cell proliferation was quantified on selected days, while the expression of several important keratocytes markers was evaluated at day 30 using RT-PCR and immunoblotting. The weight and size of the collagen constructs were measured before and after hydration and contraction analyses. Retinoic acid enhanced keratocyte proliferation until day 30, whereas cells in control culture conditions showed reduced numbers after day 21. Both gene and protein expressions of keratocyte-characteristic proteoglycans (keratocan, lumican and decorin), corneal crystallins and collagen type I and V were significantly increased following retinoic acid supplementation. Retinoic acid also significantly reduced the expression of matrix metalloproteases 1, 3 and 9 while not increasing α-smooth muscle actin and fibronectin expression. Furthermore, these effects were also correlated with the ability of retinoic acid to significantly inhibit the contractility of keratocytes while allowing the build-up of corneal stromal extracellular matrix within the 3D constructs. Thus, retinoic acid supplementation represents a promising strategy to improve the phenotype of 3D-cultured keratocytes, and their usefulness as a model of corneal stroma for corneal biology and regenerative medicine applications.

Analysis of the amyloid precursor protein role in neuritogenesis reveals a biphasic SH-SY5Y neuronal cell differentiation model.

The existence of an intrinsic programme controlling neuritogenesis and activated during early neuronal differentiation and regeneration stages is well established. However, the identity and role of each molecular player and event, as well as how such a programme is modified by environmental signals, remain a focus of research. The amyloid precursor protein (APP) is a neuromodulator of the developing and mature nervous system, although in a highly complex manner which is far from clear. To study APP-induced neuritogenesis, the retinoic acid (RA)-induced SH-SY5Y cell differentiation model was first minutely characterized in terms of RA dose, morphological outputs and relevant biochemical markers. The findings reported here unveiled two differentiation phases for the 10 μM RA dose: 1-4 (4 days excluded) and 4-8 days, clearly defined by fold increases in the ratio between APP and acetylated Tubulin. Moreover, we describe, for the first time, a unique peak of secreted APP (sAPP)/APP ratio in the first phase. Subsequent APP and sAPP modulations confirmed that a high sAPP/APP ratio potentiates the elongation of smaller processes at the earlier neuritogenic phase. This sAPP/APP ratio drops in the second phase, as holoAPP levels increase to assist the maintenance of the longer neurites, potentially via their stabilization.

Differentiation of Wharton's jelly mesenchymal stem cells into neurons in alginate scaffold.

Alginate scaffold has been considered as an appropriate biomaterial for promoting the differentiation of embryonic stem cells toward neuronal cell lineage. We hypothesized that alginate scaffold is suitable for culturing Wharton's jelly mesenchymal stem cells (WJMSCs) and can promote the differentiation of WJMSCs into neuron-like cells. In this study, we cultured WJMSCs in a three-dimensional scaffold fabricated by 0.25% alginate and 50 mM CaCl2 in the presence of neurogenic medium containing 10 μM retinoic acid and 20 ng/mL basic fibroblast growth factor. These cells were also cultured in conventional two-dimensional culture condition in the presence of neurogenic medium as controls. After 10 days, immunofluorescence staining was performed for detecting β-tubulin (marker for WJMSCs-differentiated neuron) and CD271 (motor neuron marker). β-Tubulin and CD271 expression levels were significantly greater in the WJMSCs cultured in the three-dimensional alginate scaffold than in the conventional two-dimensional culture condition. These findings suggest that three-dimensional alginate scaffold cell culture system can induce neuronal differentiation of WJMSCs effectively.

RA induces differentiation of multipotent P19 cells towards male germ cell.

Generating male germ cells in vitro from multipotent stem cells is still a challenge for stem cell biologists. The difficulty is caused by the lack of knowledge about spermatogenesis molecular-controlling mechanisms. In vivo, PGCs differentiate into male germ cells in a very complicated environment through many middle steps. In this study, we use the pluripotent p19 cells to test their responses to different retinoic acid (RA) concentrations by evaluating markers for stem cells (bmp4, egr3), primordial germ cells (ddx4), spermatogonia (c-kit), premeiotic cells (stra8), and male germ cells (dazl and plzf). We have found that cyp26b1, which will catalyze RA, increases dramatically in p19 cells 1 d after RA treatment. Bmp3, egr3, and stra8 are stimulated after 1 d of RA treatment and then recover to normal after 3 d of RA treatment. C-kit keeps being expressed when treated with 10 nM-4 μM RA. Dazl and plzf are gained after 3 d of stimulation. The morphology of RA (100 nM-4 μM)-treated cells changes distinctively, and cell colonies are formed. Typical neural cell-like and germ cell-like morphologies appear in the 100 nM and 4 μM RA groups, respectively. We conclude that 100-500 nM RA can cause responses in p19 cells, but a high concentration of RA (1-4 μM) can drive these pluripotent cells' differentiation towards male germ cells. However, high concentrations of RA are also toxic. Some colonies that survived from 4 μM RA begin to express ddx4 and c-kit. Selection of the c-kit(+), dazl(+), and ddx4(+) cells after RA stimulation and creating a special culture medium for their propagation might benefit successful spermatogenesis induction in vitro.

Treatment with retinoic acid and lens epithelial cell-conditioned medium in vitro directed the differentiation of pluripotent stem cells towards corneal endothelial cell-like cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have extensive self-renewal capacity and the potential to differentiate into all tissue-specific cell lineages, including corneal endothelial cells (CECs). They are a promising prospect for the future of regenerative medicine. The method of derivation of CECs from ESCs and iPSCs, however, remains to be elucidated. In this study, mouse ESCs and iPSCs were induced to differentiate into CECs using CEC embryonic development events as a guide. All-trans retinoic acid (RA) treatment during the embryoid body (EB) differentiation step was used to promote neural crest (NC) cell differentiation as first step and was followed by a second induction in CEC- or lens epithelial cell (LEC)-conditioned medium (CM) to ultimately generate CEC-like cells. During the corresponding differentiation stages, NC developmental markers and CEC differentiation markers were detected at the protein level using immunocytochemistry (ICC) and at the mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). During the first stage, the data indicated that 4 days of treatment with 1 μM RA starting on day 4 of EB formation favored NC cell differentiation and that plating on gelatin-coated plates led to cell migration out of the EBs. The second-stage differentiation results showed that the CM, particularly the LEC-CM, enhanced the yield of polygonal cells with CEC-specific marker expression shown by ICC and RT-qPCR. This study demonstrates that mouse ESCs and iPSCs were induced and expressed CEC differentiation markers when subjected to a two-step inducement process, suggesting that they are a promising resource for corneal endothelium failure replacement therapy in the future.

Specificity protein 1 regulates topoisomerase IIβ expression in SH-SY5Y cells during neuronal differentiation.

Topoisomerase IIβ (top IIβ) is a nuclear enzyme with an essential role in neural development. The regulation of top IIβ gene expression during neural differentiation is poorly understood. Functional analysis of top IIβ gene structure displayed a GC box sequence in its transcription promoter, which binds the nuclear transcription factor specificity protein 1 (Sp1). Sp1 regulates gene expression via multiple mechanisms and is essential for early embryonic development. This study seeks to determine whether Sp1 regulates top IIβ gene expression during neuronal differentiation. For this purpose, human neuroblastoma SH-SY5Y cells were induced to neuronal differentiation in the presence of all-trans retinoic acid (RA) for 5 days. After incubation with 10 μM RA for 3-5 days, a majority of the cells exited the cell cycle to become postmitotic neurons, characterized by the presence of longer neurite outgrowths and expression of the neuronal marker microtubule-associated protein-2 (MAP2). Elevated Sp1 and top IIβ mRNA and protein levels were detected and found to be positively correlated with the differentiation stage. Chromatin immunoprecipitation assay demonstrated an increased recruitment of Sp1 to the top IIβ promoter after RA treatment. Mithramycin A, a compound that interferes with Sp1 binding to GC-rich DNA sequences, downregulated the expression of top IIβ, resulting in reduced expression of MAP2 and decreased neurite length compared with the control group. Our results indicate that Sp1 regulates top IIβ expression by binding to the GC box of the gene promoter during neuronal differentiation in SH-SY5Y cells.

Effects of retinoic acid on proliferation and gene expression of cleft and non-cleft palatal keratinocytes

Retinoic acid (RA) is a key regulator of embryonic development and linked to several birth defects including cleft lip and palate (CLP). The aim was to investigate the effects of RA on proliferation and gene expression of human palatal keratinocytes (KCs) in vitro. KCs from children with and without CLP were cultured with 2 and 5 μM RA. Proliferation was measured by quantification of DNA after 2, 4, 6, and 8 days. In addition, we analysed the effects of RA on messenger RNA expression of genes for proliferation, differentiation, apoptosis, and RA receptors. RA similarly inhibited proliferation of palatal KC from cleft and non-cleft subjects. The proliferation of KCs from cleft subjects was reduced to 59.8±13.4% (2 μM) and 41.5±14.0% (5 μM, Day 6), while that of cells from age-matched non-cleft subjects was reduced to 66.9±12.1% (2 μM) and 33.9±10.1% (5 μM). RA treatment reduced the expression of several of the investigated genes; the proliferating cell nuclear antigen (PCNA) was reduced in CLP KCs only. Keratins 10 and 16 were downregulated in keratinocytes from both cleft and non-cleft subjects. P63, a master regulator for epithelial differentiation, was only downregulated in KCs from cleft subjects, as was the RXRa receptor. Two P63 target genes (GJB6 and DLX5) were strongly downregulated by RA in all cell lines. None of the apoptosis genes was affected. Overall, RA similarly inhibits proliferation of palatal KCs from cleft and non-cleft subjects and reduces the expression of specific genes.

Regulation of URG4/URGCP and PPARα gene expressions after retinoic acid treatment in neuroblastoma cells

Neuroblastoma (NB), originating from neural crest cells, is the most common extracranial tumor of childhood. Retinoic acid (RA) which is the biological active form of vitamin A regulates differentiation of NB cells, and RA derivatives have been used for NB treatment. PPARα (peroxisome proliferator-activated receptor) plays an important role in the oxidation of fatty acids, carcinogenesis, and differentiation. URG4/URGCP gene is a proto-oncogene and that overexpression of URG4/URGCP is associated with metastasis and tumor recurrence in osteosarcoma. It has been known that URG4/URGCP gene is an overexpressed gene in hepatocellular carcinoma and gastric cancers. This study aims to detect gene expression patterns of PPARα and URG4/URGCP genes in SH-SY5Y NB cell line after RA treatment. Expressions levels of PPARα and URG4/URGCP genes were analyzed after RA treatment for reducing differentiation in SH-SY5Y NB cell line. To induce differentiation, the cells were treated with 10 μM RA in the dark for 3-10 days. Gene expression of URG4/URGCP and PPARα genes were presented as the yield of polymerase chain reaction (PCR) products from target genes compared with the yield of PCR products from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. SH-SY5Y cells possess small processes in an undifferentiated state, and after treatment with RA, the cells developed long neurites, resembling a neuronal phenotype. PPARα gene expression increased in RA-treated groups; URG4/URGCP gene expression decreased in SH-SY5Y cells after RA treatment compared with that in the control cells. NB cell differentiation might associate with PPARα and URG4/URGCP gene expression profile after RA treatment.

Effects of retinoic acid exposure during zebrafish retinogenesis

Retinoic acid (RA) is an important morphogen involved in retinal development. Perturbations in its levels cause retinal malformations such as microphthalmia. However, the cellular changes in the retina that lead to this phenotype are little known. We have used the zebrafish to analyse the effects of systemic high RA levels on retinogenesis. For this purpose we exposed zebrafish embryos to 0.1μM or 1μM RA from 24 to 48h post-fertilisation (hpf), the period which corresponds to the time of retinal neurogenesis and initial retinal cell differentiation. We did not find severe alterations in 0.1μM RA treated animals, but the exposure to 1μM RA significantly reduced retinal size upon treatment, and this microphthalmia persisted through larval development. We monitored histology and cell death and quantified both the proliferation rate and cell differentiation from 48hpf onwards, focusing on the retina and optic nerve of normal and 1μM treated animals. Retinal lamination and initial neurogenesis are not affected by RA exposure, but we found widespread apoptosis after RA treatment that could be the main cause of microphthalmia. Proliferating cells increased their number at 3days post-fertilisation (dpf) but decreased significantly at 5dpf maintaining the microphthalmic phenotype. Retinal cell differentiation was affected; some cell markers do not reach normal levels at larval stages and some cell types present an increased number compared to those of control animals. We also found the presence of young axons growing ectopically within the retina. Moreover although the optic axons leave the retina and form the optic chiasm they do not reach the optic tectum. The alterations observed in treated animals become more severe as larvae develop.

PMA withdrawal in PMA‐treated monocytic THP‐1 cells and subsequent retinoic acid stimulation, modulate induction of apoptosis and appearance of dendritic cells

To analyse proliferation, differentiation and apoptosis in THP-1 cells after stimulation with phorbol 12-myristate 13-acetate (PMA) and retinoic acid (RA). PMA and RA were used in a three-step-procedure: (i) treatment with 6, 30, 60nm PMA, that induced initial, intermediate and advanced levels of monocyte-macrophage transition, respectively; (ii) recovery in PMA-free medium; (iii) incubation with 4μm RA. Cultures were characterized cytokinetically (flow cytometry/bromodeoxyuridine uptake) and immunocytochemically (static cytometry) for expression of CD14, CD11b (monocyte-macrophage) and DC-SIGN (dendritic cell: DCs) markers. Some treatments determined appearance of monocyte/macrophage, dendritic and apoptotic phenotypes, percentages of which were related to PMA dose used in step 1, and dependent on presence/absence of PMA and RA. PMA withdrawal induced dedifferentiation and partial restoration of proliferative activity, specially in 6 and 30nm PMA-derived cells. Recovery in the presence of serum (fundamental to DC appearance) indicated that depending on differentiation level, cell proliferation and apoptosis were inversely correlated. Treatment with 30nm PMA induced intermediate levels of monocytic-macrophagic differentiation, with expression of alternative means of differentiation and acquisition of DCs without using cytokines, after PMA withdrawal and RA stimulation. Our experimental conditions favoured differentiation, dedifferentiation and transdifferentiational pathways, in monocytic THP-1 cells, the balance of which could be related to both cell proliferation and cell death.

3D matrix microenvironment for targeted differentiation of embryonic stem cells into neural and glial lineages

The onset of neurodegenerative disorders is characterized by the progressive dysfunction and loss of subpopulations of specialized cells within specific regions of the central nervous system (CNS). Since CNS has a limited ability for self-repair and regeneration under such conditions, clinical transplantation of stem cells has been explored as an alternative. Although embryonic stem cells (ESCs) offer a promising therapeutic platform to treat a variety of neurodegenerative disorders, the niche microenvironment, which could regulate their differentiation into specialized lineages on demand, needs to be optimized for successful clinical transplantation. Here, we evaluated the synergistic role of matrix microenvironment (type, architecture, composition, stiffness) and signaling molecules (type, dosage) on murine ESC differentiation into specific neural and glial lineages. ESCs were cultured as embryoid bodies on either 2D substrates or within 3D scaffolds, in the presence or absence of retinoic acid (RA) and sonic hedgehog (Shh). Results showed that ESCs maintained their stemness even after 4 days in the absence of exogenous signaling molecules, as evidenced by Oct-4 staining. RA at 1 μm dosage was deemed optimal for neural differentiation and neurite outgrowth on collagen-1 coated substrates. Significant neural differentiation with robust neurite outgrowth and branching was evident only on collagen-1 coated 2D substrates and within 3D matrigel scaffolds, in the presence of 1 μm RA. Blocking α6 or β1 integrin subunits on differentiating cells inhibited matrigel-induced effects on neural differentiation and neurite outgrowth. Hydrogel concentration strongly regulated formation of neural and astrocyte lineages in 1 μm RA additive cultures. When RA and Shh were provided, either alone or together, 3D collagen-1 scaffolds enhanced significant motor neuron formation, while 3D matrigel stimulated dopaminergic neuron differentiation. These results suggest a synergistic role of microenvironmental cues for ESC differentiation and maturation, with potential applications in cell transplantation therapy.

Use of retinyl acetate, retinoic acid and insulin-like growth factor-I (IGF-I) to enhance goat embryo production

Experiments were carried out to investigate the beneficial effects of retinyl acetate (RAc) and retinoic acid (RA) on goat oocyte maturation as well as the effects of insulin-like growth factor-I (IGF-I), RAc and RA during embryo culture under chemically defined conditions. In Experiment 1, in vitro maturation (IVM) was performed in a chemically defined basic maturation medium (bMM) supplemented with 0.3 μM RAc or 0.5 μM RA. Presumptive zygotes and embryos (2-4 cells) were cultured in droplets of potassium simplex optimised medium (KSOM); however, none of the embryos reached the blastocyst stage. In Experiment 2, oocytes were matured in bMM + RAc or bMM + RA. Presumptive zygotes and 2- to 4-cell embryos were placed in fresh KSOM droplets supplemented with RAc, RA, IGF-I, RAc+IGF-I or RA+IGF-I. In Experiment 1, addition of RAc and RA to bMM increased (P < 0.05) the proportion of 2- to 4-cell embryos reaching the morula stage as compared to the control. In Experiment 2, supplementation of embryo culture media with retinoids and IGF-I increased (P < 0.05) the proportion of 2- to 4-cell stage embryos developing to the morula and blastocyst stage. Our data demonstrate that goat embryo production in chemically defined media could be improved by exogenous RAc or RA and by the interaction between retinoids and IGF-I, and that goat embryos can be produced in vitro from oocytes following protocols similar to those currently used for cattle.

All-trans retinoic acid inhibits craniopharyngioma cell growth: study on an explant cell model

The ratio between FABP5 and CRABPII determines cellular response to physiological level of retinoic acid; tumor cells undergo proliferation with high level of FABP5 and apoptosis with high level of CRABPII. We intended to study FABP5 and CRABPII expression in craniopharyngiomas, to establish craniopharyngioma cell model using explants method, and to study the effect of pharmacological dose of retinoic acid on craniopharyngioma cells. Expression of FABP5 and CRABPII in craniopharyngioma tissue from 20 patients was studied using immunohistochemistry. Primary craniopharyngioma cell cultures were established using tissue explants method. Craniopharyngioma cells were treated using various concentrations of all-trans retinoic acid, and cell growth curve, apoptosis, expression of FABP5, CRABPII and NF-κB were assayed in different groups. FABP5/CRABPII ratio was significantly higher in adamatinomatous group than that in papillary group. Cell cultures were established in 19 cases (95 %). Pharmacological level retinoic acid inhibited cell growth and induced cellular apoptosis in dose dependent manner, and apoptosis rate cells treated with 30 μM retinoic acid for 24 h was 43 %. Also, retinoic acid increased CRABPII, and decreased FABP5 and NF-κB expression in craniopharyngioma cells. High FABP5/CRABPII ratio is observed in adamatinomatous craniopharyngioma. Retinoic acid at pharmacological level induced craniopharyngioma cell apoptosis via increasing FABP5/CRABPII ratio and inhibiting NF-κB signaling pathway. Our study demonstrated that all-trans retinoic acid might be a candidate for craniopharyngioma adjuvant chemotherapy in future.

Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage

Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted ∼23 and ∼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPN(RAA)-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties.

Comparison of the efficacy of three concentrations of retinoic acid for transdifferentiation induction in sheep marrow‐derived mesenchymal stem cells into male germ cells

Recent studies have shown the unique role of retinoic acid (RA) in the induction of transdifferentiation in mesenchymal stem cells (MSCs) into germ cells (GCs). This study is the first study that compares the efficacy of three different concentrations of RA for the production of male GCs in vitro. Male sheep marrow-derived MSCs (MMSCs) were treated with the following concentrations of RA: 1 μm (RA1), 5 μm (RA2) and 10 μm (RA3) for a period of 21 days. The production of male GCs was evaluated by the assessment of expressions of GC-specific markers (by RT-PCR, qRT-PCR and immunocytochemistry), morphological characteristics and changes in alkaline phosphatase (ALP) activity. All three concentrations created male GC features. RA treatment upregulated the expressions of VASA and beta1 INTEGRIN and downregulated PIWIL2 and OCT4. DAZL was not expressed by RA treatment. Interestingly, immunocytochemistry detected PGP 9.5 expression in all treatment groups, with the highest expression noted in the RA3 group (P < 0.05). GC-like cells along with increased ALP activity were observed in all treated cultures, too. Finally, results showed that 10 μm RA has the most efficiency for transdifferentiation induction in MMSCs and production of male GCs in vitro.

Effects of retinoic acid and Gbx1 on feather-bud formation and epidermal transdifferentiation in chick embryonic cultured dorsal skin

Retinoic acid, an active metabolite of retinol, is known to regulate cell proliferation, differentiation, and morphogenesis during normal development of many tissues. Using chick embryonic tarsometatarsal skin, we showed previously that the expression of Gbx1, a divergent homeobox gene, is increased in the epidermis through interaction with retinol-pretreated dermal fibroblasts followed by epidermal transdifferentiation to mucous epithelium. This present study was performed to elucidate the effects of retinoic acid and Gbx1 on feather-bud formation and epidermal transdifferentiation. We showed that Gbx1 was expressed in the chick embryonic dorsal epidermis as early as at placode stage (Hamburger and Hamilton stage 31) and increased in amount during feather-bud formation. Treatment with 1 μM retinoic acid for 24 hr inhibited feather-bud formation and induced the transdifferentiation of the epidermis to a mucosal epithelium with a concomitant increase in Gbx1 mRNA expression in the epithelium. Furthermore, transient transfection of the epidermis with Gbx1 cDNA by electroporation induced elongation of the feather bud, but did not result in transdifferentiation. These results indicate that Gbx1 was involved in the feather-bud formation and was one of target genes of retinoic acid and that other signals in addition to Gbx1 were required for epidermal mucous transdifferentiation.