Stratum corneum thickness was estimated from water concentration profiles of the skin measured by a confocal Raman spectrometer. Stratum corneum apparent thickness (SCAT) was defined as the depth where the water content reached an almost constant value. Site variations were determined using 15 healthy Japanese subjects (6 males, 9 females), and age variations at the cheek and forearm were examined using 27 female Japanese subjects. There were marked site variations in mean SCAT; 16.8 microm for cheek, 22.6 microm for volar forearm, 29.3 microm for back of the hand, and 173.0 microm for palm. These variations were similar to reported stratum corneum thickness values obtained in biopsy tissues. The SCAT tended to become age-dependently thicker at the forearm, but not at the cheek. In addition, SCAT was increased up to two-fold by hydration for 90 min, while lesser increases were seen with shorter hydration periods.

The molecular mechanisms behind phenotypic modulation of smooth muscle cells (SMCs) remain unclear. In our recent paper, we reported the establishment of novel culture system of gizzard SMCs (Hayashi, K., H. Saga, Y. Chimori, K. Kimura, Y. Yamanaka, and K. Sobue. 1998. J. Biol. Chem. 273: 28860–28867), in which insulin-like growth factor-I (IGF-I) was the most potent for maintaining the differentiated SMC phenotype, and IGF-I triggered the phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB(Akt)) pathway. Here, we investigated the signaling pathways involved in de-differentiation of gizzard SMCs induced by PDGF-BB, bFGF, and EGF. In contrast to the IGF-I–triggered pathway, PDGF-BB, bFGF, and EGF coordinately activated ERK and p38MAPK pathways. Further, the forced expression of active forms of MEK1 and MKK6, which are the upstream kinases of ERK and p38MAPK, respectively, induced de-differentiation even when SMCs were stimulated with IGF-I. Among three growth factors, PDGF-BB only triggered the PI3-K/PKB(Akt) pathway in addition to the ERK and p38MAPK pathways. When the ERK and p38MAPK pathways were simultaneously blocked by their specific inhibitors or an active form of either PI3-K or PKB(Akt) was transfected, PDGF-BB in turn initiated to maintain the differentiated SMC phenotype. We applied these findings to vascular SMCs, and demonstrated the possibility that the same signaling pathways might be involved in regulating the vascular SMC phenotype. These results suggest that changes in the balance between the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways would determine phenotypes of visceral and vascular SMCs. We further reported that SMCs cotransfected with active forms of MEK1 and MKK6 secreted a nondialyzable, heat-labile protein factor(s) which induced de-differentiation of surrounding normal SMCs.

Abstract-The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one is critically involved in the development and progression of atherosclerosis. Although many cytokines and growth factors have been reported as atherogenic factors, the critical pathogens for inducing atherosclerosis remain unknown, largely because proper examining systems of them have not been developed. We recently established primary culture systems for visceral SMCs and VSMCs in which both SMCs, when cultured on laminin with insulin-like growth factor-I, show a differentiated phenotype, as indicated by a spindle-like shape, ligand-induced contractility, and a high level of SMC differentiation marker gene expression. In this study, we searched for critical dedifferentiation factors for these SMCs using our culture system. We found that polar lipids extracted from human serum markedly induced VSMC dedifferentiation, and this activity was solely present in the lysophosphatidic acid (LPA) fraction. Among several LPA species detected in human serum lipids, unsaturated LPAs were identified as major contributors to the induction of VSMC dedifferentiation. Signaling and phenotype analyses revealed that unsaturated LPA-induced VSMC dedifferentiation is mediated through the coordinated activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. Thus, this report demonstrates the first finding that unsaturated LPAs, but not saturated LPAs, specifically induce VSMC phenotypic modulation, suggesting that these molecules could function as atherogenic factors. T he phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one, which results in cell proliferation and migration, is a hallmark of the development and progression of atherosclerosis. Numerous studies have reported the possible involvement of cytokines and growth factors in the phenotypic modulation of VSMCs. 1,2 The critical factors affecting the VSMC phenotype, however, remain unclear because proper in vitro assay systems have not yet been available. Under conventional culture conditions, VSMCs in primary culture rapidly display a phenotypic change. 3,4 Dedifferentiated VSMCs obtained after passaging cannot revert to a differentiated phenotype, even under quiescent culture conditions. Therefore, studies using passaged VSMCs have not been able to investigate adequately the molecular mechanisms underlying the phenotypic modulation of VSMCs. To overcome this obstacle, we recently established primary culture systems of visceral SMCs and VSMCs in which both types of SMCs can maintain a differentiated phenotype for a long time, when cultured on laminin with insulin-like growth factor-I (IGF-I). 5,6 Using our culture system, we investigated the signaling pathways affecting the visceral SMC phenotype and found that the IGF-I-stimulated phosphoinositide 3-kinase (PI3-K)/protein kinase B (PKB[Akt]) pathway plays a vital role in maintaining a different...

No abstract

The use of stem cells has enabled the successful generation of simple organs. However, anatomically complicated organs such as the kidney have proven more refractory to stem-cell-based regenerative techniques. Given the limits of allogenic organ transplantation, an ultimate therapeutic solution is to establish self-organs from autologous stem cells and transplant them as syngrafts back into donor patients. To this end, we have striven to establish an in vitro organ factory to build up complex organ structures from autologous adult stem cells by using the kidney as a target organ. Cultivation of human mesenchymal stem cells in growing rodent embryos enables their differentiation within a spatially and temporally appropriate developmental milieu, facilitating the first step of nephrogenesis. We show that a combination of whole-embryo culture, followed by organ culture, encourages exogenous human mesenchymal stem cells to differentiate and contribute to functional complex structures of the new kidney.organogenesis ͉ regeneration

Asymmetric cell division plays an indispensable role during corticogenesis for producing new neurons while maintaining a self-renewing pool of apical progenitors. The cellular and molecular determinants favouring asymmetric division are not completely understood. Here, we identify a novel mechanism for generating cellular asymmetry through the active transportation and local translation of Cyclin D2 mRNA in the basal process. This process is regulated by a unique cis-regulatory sequence found in the 3 0 untranslated region (3 0 UTR) of the mRNA. Unequal inheritance of Cyclin D2 protein to the basally positioned daughter cell with the basal process confers renewal of the apical progenitor after asymmetric division. Conversely, depletion of Cyclin D2 in the apically positioned daughter cell results in terminal neuronal differentiation. We demonstrate that Cyclin D2 is also expressed in the developing human cortex within similar domains, thus indicating that its role as a fate determinant is ancient and conserved.

The mammalian neocortex is characterized as a six-layered laminar structure, in which distinct types of pyramidal neurons are distributed coordinately during embryogenesis. In contrast, no other vertebrate class possesses a brain region that is strictly analogous to the neocortical structure. Although it is widely accepted that the pallium, a dorsal forebrain region, is specified in all vertebrate species, little is known of the differential mechanisms underlying laminated or non-laminated structures in the pallium. Here we show that differences in patterns of neuronal specification and migration provide the pallial architectonic diversity. We compared the neurogenesis in mammalian and avian pallium, focusing on subtype-specific gene expression, and found that the avian pallium generates distinct types of neurons in a spatially restricted manner. Furthermore, expression of Reelin gene is hardly detected in the developing avian pallium, and an experimental increase in Reelin-positive cells in the avian pallium modified radial fiber organization, which resulted in dramatic changes in the morphology of migrating neurons. Our results demonstrate that distinct mechanisms govern the patterns of neuronal specification in mammalian and avian pallial development, and that Reelin-dependent neuronal migration plays a critical role in mammalian type corticogenesis. These lines of evidence shed light on the developmental programs underlying the evolution of the mammalian specific laminated cortex.

Cornified envelopes (CEs), rigid and insoluble structures in the stratum corneum, which are assembled by crosslinking of several precursor proteins by transglutaminases, provide a hydrophobic foundation for barrier function; omega-hydroxyceramides are covalently attached to the outer surface of CE components, and onto this hydrophobic assembly, lamellar layers of intercellular lipids are organized. Morphologically irregular, fragile CEs are found in the deep layer of the stratum corneum or in certain disorders, such as psoriasis, whereas most CEs from healthy subjects are rigid and polygonal. We have established a staining method to characterize such fragile CEs as immature and less hydrophobic CEs, and employed it to examine regional differences in the properties of CEs, especially in relation to the barrier function of the skin. CEs from the outermost stratum corneum of the trunk and extremities of healthy subjects were relatively uniform in morphology with larger shape, and were homogeneous in hydrophobicity as judged from the use of an environment-sensitive fluorescent dye, Nile red. However, CEs from the face were strikingly heterogeneous, and consisted of both rigid and fragile CEs. Rigid CEs were Nile red-positive and little stained by anti-involucrin. In contrast, fragile CEs were Nile red-negative but strongly stained with anti-involucrin, as detected by indirect immunofluorescence. Thus, CEs from the face were stained with Nile red or involucrin in a mutually exclusive manner. Fragile CEs were stained with antibodies against other CE components, including loricrin, envoplakin, filaggrin, and isopeptides. Such fragile, involucrin-positive CEs were detected not only in the face, but also in the deep layer of the stratum corneum of the arm. In addition, experimental barrier disruption resulted in the appearance of involucrin-positive CEs in the outermost stratum corneum. These results suggest that involucrin-positive, fragile CEs are immature and less hydrophobic, and that their occurrence is closely related to impairment of the barrier function of the skin.