Gut-liver microphysiological systems revealed potential crosstalk mechanism modulating drug metabolism.

The small intestine and liver play important role in determining oral drug's fate. Both organs are also interconnected through enterohepatic circulation, which imply there are crosstalk through circulating factors such as signaling molecules or metabolites that may affect drug metabolism. Coculture of hepatocytes and intestinal cells have shown to increase hepatic drug metabolism, yet its crosstalk mechanism is still unclear. In this study, we aim to elucidate such crosstalk by coculturing primary human hepatocytes harvested from chimeric mouse (PXB-cells) and iPSc-derived intestinal cells in a microphysiological systems (MPS). Perfusion and direct oxygenation from the MPS were chosen and confirmed to be suitable features that enhanced PXB-cells albumin secretion, cytochrome P450 (CYP) enzymes activity while also maintaining barrier integrity of iPSc-derived intestine cells. Results from RNA-sequencing showed significant upregulation in gene ontology terms related to fatty acids metabolism in PXB-cells. One of such fatty acids, arachidonic acid, enhanced several CYP enzyme activity in similar manner as coculture. From the current evidences, it is speculated that the release of bile acids from PXB-cells acted as stimuli for iPSc-derived intestine cells to release lipoprotein which was ultimately taken by PXB-cells and enhanced CYP activity.

Enhanced restoration of in situ-damaged hairs by intradermal transplantation of trichogenous dermal cells.

We developed a nude rat model for determining the capacity of trichogenous cells to restore in vivo-damaged hair follicles (HFs). A surgical scalpel was inserted into the rat's dermis to generate the in vivo-damaged pelage HFs, the HFs whose lower parts were lost, but the upper parts containing sebaceous and bulge regions remained intact. Dermal papilla cells (DPCs) and dermal sheath cells (DSCs) from EGFP transgenic rat vibrissae were propagated in culture, and each alone (DPC or DSC) or a mixture (DPC/DSC) was transplanted into the intradermal path made by a scalpel. It was found that the in vivo-damaged HFs had hair self-restoration ability, and the transplanted trichogenic dermal cells prominently enhanced this ability, DPC/DSC transplants being more effective in enhancement than DPC or DSC alone. The restored bulbs contained EGFP-positive cells, shed their original straight shafts, generated new shafts, and further developed into hairs with a sebaceous gland and bulge structures by ~6 weeks post-transplantation. Compared to the preceding animal models, this model is less invasive, requires fewer donor cells and allows repeated operations with higher reproducibility and accuracy. The present study suggests that conditions causing in situ-damaged HFs, such as androgenic alopecia, in which HFs are damaged and miniaturized, can be restored by functional trichogenous dermal cell transplantation therapy. Copyright © 2015 John Wiley & Sons, Ltd.

Development of the Heart Endocardium at an Early Stage of Chick Embryos Evaluated at Light- and Electron-Microscopic Levels.

Development of the endocardium in the heart of 4 to 4·1/2-day-incubated chick embryos was observed light and electron microscopically, and these results were evaluated by immunohistochemistry for desmin, FLK1 (VEGFR-2) or CD31, and by in situ hybridization assays for flk1-mRNA expression. At this developmental stage, the atrium and the ventricle were already discriminated by formation of the atrio-ventricular junction. The cardiac wall consisted of three layers; the inner endocardium, the middle myocardium, and the outer epicardium. The developing endocardium was seen as a chain of single-layered endocardial cells. Along its inner surface, numerous clusters of blood corpuscles were distributed, which seemed to contain some undifferentiated endocardial cells estimated from their characteristic ultrastructure and histological topography. Several blood corpuscles were in directly contact with the myocardium at the missing portions of the developing endocardial cell-chains. Differentiating endocardial cells individually showed roundish, small and large crescent, or flat in shapes. Such a prominent change of cell shapes appeared to be in parallel with their secretory activity during the transformation from the undifferentiated cells to the endocardial cells. Furthermore, immunohistochemistry for FLK1 or CD31, and in situ hybridization assays for flk1-mRNA labeled the cells composing developing endocardial cell-chains. Though these expressional analyses could not document clearly the transition of precursor cells into endocardial cells, the present study provided for the first time some important information regarding the morphological transition process toward endocardial cells at ultrastructural levels. Anat Rec, 299:1080-1089, 2016. © 2016 Wiley Periodicals, Inc.

Restorative effect of hair follicular dermal cells on injured human hair follicles in a mouse model.

No model is available for examining whether in vivo-damaged human hair follicles (hu-HFs) are rescued by transplanting cultured hu-HF dermal cells (dermal papilla and dermal sheath cells). Such a model might be valuable for examining whether in vivo-damaged hu-HFs such as miniaturized hu-HFs in androgenic alopecia are improvable by auto-transplanting hu-HF dermal cells. In this study, we first developed mice with humanized skin composed of hu-keratinocytes and hu-dermal fibroblasts. Then, a 'humanized scalp model mouse' was generated by transplanting hu-scalp HFs into the humanized skin. To demonstrate the usability of the model, the lower halves of the hu-HFs in the model were amputated in situ, and cultured hu-HF dermal cells were injected around the amputated area. The results demonstrated that the transplanted cells contributed to the restoration of the damaged HFs. This model could be used to explore clinically effective technologies for hair restoration therapy by autologous cell transplantation.

Contact between dermal papilla cells and dermal sheath cells enhances the ability of DPCs to induce hair growth.

We previously showed that cultured rat dermal papilla cells (DPCs) retain their hair-inducing capacity on afollicular epidermal cell (EPCs). Here, we examined the hair growth-inducing capacity of differently subcultured DPCs by transplanting them, along with rat EPCs, onto the backs of nude mice (graft chamber assay). DPCs at passage (p) 6 (DPCs(p6) or, more generally, low-passage DPCs) induced hair formation. However, DPCs(p>30) (high-passage DPCs) had no such activity and induced only subepidermal hair follicles (HFs) that were not encapsulated by the dermal sheath (DS). Thus, we examined the effect of DS cells (DSCs(p=1)) on the ability of DPCs(p=60) to induce hair growth by testing a mixture of these two cell types (cotransplant) in the graft chamber assay, in which DSCs(p=1) and DPCs(p=60) were labeled with enhanced green fluorescent protein (EGFP) and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), respectively. These cotransplants generated hairs as actively as did DPCs(p=6) transplants. Their HFs were encapsulated with EGFP(+)-DS and had DPs consisting largely of EGFP(+)-DPCs (47%) and DiI(+)-DPCs (43%), indicating a major contribution of DSC(p=1)-derived DPCs to HF induction. In addition, the results of in vitro coculture of DPCs(p=60) and DSCs(p=1) suggest that high-passage DPCs stimulate the expression of certain trichogenic genes in DSCs.

In vitro reconstruction of a three-dimensional middle ear mucosal organ and its in vivo transplantation.

CONCLUSIONS: These experimental findings suggest the feasibility of artificial middle ear mucosa grafting as an effective treatment for achieving mucosal regeneration after middle ear surgery. OBJECTIVES: Postoperative mucosal regeneration of tympanic cavity and mastoid cavity is of great importance after middle ear surgery. We reconstructed in vitro a three-dimensional middle ear mucosal organ, and assessed its feasibility for regenerative medicine of middle ear-related diseases. MATERIALS AND METHODS: Epithelial cells and fibroblasts were isolated from the middle ear mucosa of rats and propagated by subculturing. An artificial middle ear mucosal organ was reconstructed by overlaying the middle ear epithelial cells on three-dimensional lattices of a collagen gel that had been repopulated with the fibroblasts. In addition, the artificial organ was implanted in the middle ear cavity of rats. RESULTS: The artificial middle ear mucosa consisted of the single layer of epithelial cells, the basal membrane, and the underlying connective tissue. Electron microscopy revealed the presence of tight junctions and adherence junctions on the apical side, and adhesion complexes made of desmosomes. The reconstituted mucosa expressed genes of mucin, strongly suggesting that the artificial middle ear mucosa was capable of secreting mucus proteins. The DiI-labeled artificial middle ear mucosa implanted into the middle ear cavity was well engrafted and associated with host tissues.

Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms.

Hair induction in the adult glabrous epidermis by the embryonic dermis was compared with that by the adult dermis. Recombinant skin, composed of the adult sole epidermis and the embryonic dermis containing dermal condensations (DC), was transplanted onto the back of nude mice. The epidermis of transplants formed hairs. Histology on the induction process demonstrated the formation of placode-like tissues, indicating that the transplant produces hair follicles through a mechanism similar to that underlying hair follicle development in the embryonic skin. An isolated adult rat sole skin piece, inserted with either an aggregate of cultured dermal papilla (DP) cells or an intact DP between its epidermis and dermis, was similarly transplanted. The transplant produced hair follicles. Histology showed that the epidermis in both cases surrounded the aggregates of DP cells. The epidermis never formed placode-like tissues. Thus, it was concluded that the adult epidermal cells recapitulate the embryonic process of hair follicle development when exposed to DC, whereas they get directly into the anagen of the hair cycle when exposed to DP. The expression pattern of Edar and Shh genes, and P-cadherin protein during the hair follicle development in the two types of transplants supported the above conclusion.

Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase.

This study developed an enzymatic method for high-throughput mapping of phosphoproteins on two-dimensional (2-D) polyacrylamide gels. Proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant lambda protein phosphatase and the other was not treated with the enzyme. The two aliquots were then subjected to 2-D electrophoresis. Phosphoproteins could be mapped on the 2-D gel of the nontreated aliquot by comparing the gels of the two aliquots, because the phosphoproteins in the treated aliquot shifted to more basic positions on the gel. This technique revealed that approximately 5% of the detectable proteins were phosphorylated. Fourteen phosphoproteins were identified by mass spectrometry, including proteasome component C8 and small glutamine-rich tetratricopeptide repeat-containing protein. Furthermore, the extent of phosphorylation of two actin modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. The method developed by this study can generally be applied to all biological materials and is useful for high-throughput mapping of phosphoproteins in proteome research.