Enhanced hepatogenic differentiation of bone marrow derived mesenchymal stem cells on liver ECM hydrogel.

Bone marrow derived mesenchymal stem cells (BM-MSC) is a promising alternative cell source to primary hepatocytes because of their ability to differentiate into hepatocyte-like cells. However, their inability to differentiate efficiently and potential to turn into myofibroblasts restrict their applications. This study developed a plate coating from the liver extracellular matrix (ECM) and investigated its ability in facilitating the BM-MSCs proliferation, hepatic differentiation, and hepatocyte-specific functions during in vitro culture. After 28-day culture, BM-MSCs on the ECM coating showed hepatocyte-like morphology, and certain cells took up low-density lipoprotein. Synthesis of albumin, urea, and anti-alpha-fetoprotein, as well as expression of certain hepatic markers, in cells cultured on ECM were higher than cells cultured on non-coated and Matrigel-coated plates. mRNA levels of CYP3A4, albumin, CK18, and CYP7A1 in cells on ECM coating were significantly higher than cells cultured on the non-coating environment. In conclusion, viability and hepatogenic differentiation of BM-MSCs cultured on both Matrigel and ECM coating were significantly enhanced compared with those cultured on non-coated plates. Moreover, the liver ECM coating induced additional metabolic functions relative to the Matrigel coating. The liver ECM hydrogel preserves the natural composition, promotes simple gelling, induces efficient stem cell hepatogenic differentiation, and may have uses as an injectable intermedium for hepatocytes. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 829-838, 2018.

Extracts from salivary glands stimulate aggression and inositol-1, 4, 5-triphosphate (IP3) production in the vomeronasal organ of mice.

Mammals use chemical cues to coordinate social and reproductive behaviors. Chemical cues are detected by the VNO organ (VNO), which is a cartilage-encased elongated organ associated with the vomer bone in the rostral nasal cavity. The resident intruder paradigm was utilized to examine the ability of saliva and its feeder exocrine glands, the submaxillary, parotid, and sublingual glands to mediate aggression in mice. Saliva and extracts from submaxillary and parotid glands, but not extracts from sublingual glands of male CD-1 mice, induced a greater number of attacks and lower latencies to sniff and attack (p<0.05) and significantly increased IP(3) production (p<0.05) versus vehicle (PBS) in CD-1 male mice VNO. We further show that CD-1 male mouse saliva and submaxillary gland extract induced significantly more attacks and a lower latency to attack in lactating female CD-1 mice and produced significantly more inositol triphosphate (IP(3)), indicative of phospholipase C(beta) signaling which mediates pheromonal activity, in CD-1 female VNO compared to PBS. Castrated CD-1 male mouse saliva, and exocrine gland extracts induced significantly less IP(3) production in male VNO and less aggression by CD-1 males and lactating females compared to responses to normal CD-1 male mouse saliva and gland extracts. Thus, chemical cues present in saliva, submaxillary and parotid glands of CD-1 male mice are capable of stimulating aggression in male and female congenic mice which are correlated with significant production of IP(3) in the VNO. Additionally, these stimulations of aggression and IP(3) production are shown to be androgen-dependent.

Pregnancy block by MHC class I peptides is mediated via the production of inositol 1,4,5-trisphosphate in the mouse vomeronasal organ.

The vomeronasal organ (VNO) has evolved to link an animal's behavior to its environment in a highly species-specific fashion. In mice, it is thought to be the primary sensory system responsible for the detection of pheromones. Pheromones regulate a variety of responses including mate recognition in the context of selective pregnancy failure. MHC (major histocompatibility complex) class I peptides have been identified as compounds that elicit the pregnancy block effect via the VNO. However, the transduction cascade of these molecules is unknown and it is not known if the production of these compounds are androgen dependent. By using male urine and MHC peptides, we show that female mice treated with MHC peptides (in urine or PBS) and urine from castrated males or juvenile mice of different haplotypes respond to the Bruce Effect paradigm in a manner equivalent to female mice exposed to whole urine. In addition to providing new evidence that urine from castrated or juvenile males and MHC peptides can induce pregnancy block, we show correlation of the effect with an increase in inositol 1,4,5-trisphosphate.

Chemosensory cues from the lacrimal and preputial glands stimulate production of IP3 in the vomeronasal organ and aggression in male mice.

The social and reproductive behaviors of most mammals are modulated by chemosensory cues. The perception of some of these cues is mediated by the vomeronasal organ, which is a cartilage-encased elongated organ associated with the vomer bone in the rostral nasal cavity. Several studies have shown that chemosensory cues are present in urine, seminal fluid or vaginal secretions but only a few studies have focused on exocrine glands as a source of chemosensory cues. Here we show that chemosensory cues present in two exocrine glands, i.e., the preputial gland located at the caudal region and the lacrimal gland located at the rostral region, are capable of stimulating aggression in male mice. We further show that these extracts can stimulate the production of inositol-(1,4,5)-trisphosphate in the vomeronasal organ.

Attenuation of the production of inositol 1,4,5-trisphosphate in the mouse vomeronasal organ by antibodies against the alphaq/11 subfamily of G-proteins.

The social and reproductive behaviors of most mammals are modulated by pheromones, which are perceived by the vomeronasal organ (VNO). Vomeronasal transduction in vertebrates is activated through G-protein-coupled receptors, which in turn leads to the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) by the activity of phospholipase C. DAG has been shown to gate the transient receptor potential channel 2, whereas IP(3) may play a role in stimulating the release of calcium from the endoplasmic reticulum store. To investigate the role of the alpha subunits of G(q/11) in the transduction process, microvillar membranes from female mice VNO were preincubated with a selective C-terminal peptide antibody against Galpha(q/11) and then stimulated with adult male urine. Incubation of VNO membranes with antibodies against Galpha(q/11) blocked the production of IP(3) in a dose-dependent manner. We were also able to impair the production of IP(3) when we stimulated with 2-heptanone or 2,5-dimethylpyrazine in the presence of antibodies against the alpha subunit of G(q/11). 2-Heptanone is a known pheromone that has been linked to VIR receptors. Thus, our observations indicate that the alpha subunits of G(q/11) play a role in pheromonal signaling in the VNO.

Sex-specific responses to urinary chemicals by the mouse vomeronasal organ.

Social behaviors of most mammals are affected by chemical signals, pheromones, exchanged between conspecifics. Previous experiments have shown that behavioral responses to the same pheromone differ depending on the sex and endocrine status of the respondent. Although the exact mechanism of this dimorphism is not known, one possible contributor may be due to sexually dimorphic receptors or due to differences in central processing within the brain. In order to investigate the differences in response between male and female mice to the same pheromonal stimulus two urinary compounds (2-heptanone and 2,5-dimethylpyrazine) were used to stimulate the production of Inositol (1,4,5)-trisphosphate (IP(3)) in microvillar membrane preparations of the vomeronasal organ as an indirect measurement of pheromonal stimulation. Incubation of such membranes from prepubertal mice with urine from the same sex or opposite sex, results in an increase in production of IP(3). This stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore we found that 2-heptanone present in both male and female urine was capable of stimulating increased production of IP(3) in the female VNO but not the male VNO. Finally, 2,5-dimethylpyrazine present only in female urine was also only capable of stimulating increased production of IP(3) in the female VNO.

Involvement of G(q/11) in signal transduction in the mammalian vomeronasal organ.

Social behaviors of most mammals are profoundly affected by pheromones. Pheromones are detected by G-protein coupled receptors in the vomeronasal organ (VNO). To investigate the role of G alpha(q/11) in vomeronasal signal transduction pathways, microvillar membranes from murine VNO were prepared. Incubation of such membranes from prepubertal females with adult male urine results in an increase in production of inositol-(1,4,5)-trisphosphate (IP(3)). This stimulation is mimicked by GTP gamma S, blocked by GDP beta S and is tissue specific. Furthermore, use of bacterial toxins such as pertussis that lead to ADP-ribosylation of the G-protein alpha subunits of G(o) and G(i2) do not block the increase in IP(3) levels but U-73122, a PLC inhibitor, blocks the production of IP(3). Studies with monospecific antibodies revealed the presence of three G-proteins, G alpha(o), G alpha(i2) and G alpha(q/11)-related protein, in vomeronasal neurons, concentrated on their microvilli. Our observations indicate that pheromones in male urine act on vomeronasal neurons in the female VNO via a receptor-mediated, G alpha(q/11)-protein-dependent increase in IP(3) levels.

Impaired olfactory behavior in mice deficient in the alpha subunit of G(o).

The ability to respond to chemical signals is essential for the survival and reproduction of most organisms. Olfactory signaling involves odorant receptor-mediated activation of G(olf), a homologue of G(s), on the dendrites of olfactory neurons. Olfactory receptor cells, however, also express Galpha(i2) and Galpha(o) on their axons, with all neurons expressing G(o) and a subset G(i2). Despite their abundance, possible contributions of G(o) and G(i2) to chemoreception remain unexplored. We investigated whether homologous recombinant mice deficient in the alpha subunit of G(o) are able to respond to odorants, whether possible olfactory impairments are dependent on genetic background, and whether formation of glomeruli in their olfactory bulbs is compromised. In an olfactory habituation/dishabituation test, G(o)-/- mice were unresponsive when exposed to odorants. Analysis of variance shows that performance of G(o)+/- mice crossed into the CD-1 background is also diminished in this test compared to their G(o)+/+ counterparts. Following food deprivation, G(o)-/- mice in the 129 Sv-ter/C57BL/6 genetic background were unable to locate a buried food pellet until they were approximately 10 weeks of age after which they performed as well as their litter mate controls. However, CD-1 G(o)-/- mice could locate a buried food pellet even when tested immediately after weaning. Despite their compromised olfactory responsiveness, histological examination did not reveal gross alterations in the olfactory bulbs of G(o)-/- mice. Thus, Galpha(o) is necessary for the expression of olfactory behavior under normal conditions and dependent on genetic background, but is not essential for the formation and maintenance of glomeruli.