a b s t r a c tCutaneous innervation is increasingly recognized as a major element of skin physiopathology through the neurogenic inflammation driven by neuropeptides that are sensed by endothelial cells and the immune system. To investigate this process in vitro, models of innervated tissue-engineered skin (TES) were developed, yet exclusively with murine sensory neurons extracted from dorsal root ganglions. In order to build a fully human model of innervated TES, we used induced pluripotent stem cells (iPSC) generated from human skin fibroblasts. Nearly 100% of the iPSC differentiated into sensory neurons were shown to express the neuronal markers BRN3A and b3-tubulin after 19 days of maturation. In addition, these cells were also positive to TRPV1 and neurofilament M, and some of them expressed Substance P, TrkA and TRPA1. When stimulated with molecules inducing neuropeptide release, iPSC-derived neurons released Substance P and CGRP, both in conventional monolayer culture and after seeding in a 3D fibroblastpopulated collagen sponge model. Schwann cells, the essential partners of neurons for function and axonal migration, were also successfully differentiated from human iPSC as shown by their expression of the markers S100, GFAP, p75 and SOX10. When cultured for one additional month in the TES model, iPSCderived neurons seeded at the bottom of the sponge formed a network of neurites spanning the whole TES up to the epidermis, but only when combined with mouse or iPSC-derived Schwann cells. This unique model of human innervated TES should be highly useful for the study of cutaneous neuroinflammation. Statement of SignificanceThe purpose of this work was to develop in vitro an innovative fully human tissue-engineered skin enabling the investigation of the influence of cutaneous innervation on skin pathophysiology. To reach that aim, neurons were differentiated from human induced pluripotent stem cells (iPSCs) generated from normal human skin fibroblasts. This innervated tissue-engineered skin model will be the first one to show iPSC-derived neurons can be successfully used to build a 3D nerve network in vitro. Since innervation has been recently recognized to play a central role in many human skin diseases, such as psoriasis and atopic dermatitis, this construct promises to be at the forefront to model these diseases while using patient-derived cells.

A 163-base pair enhancer in the CYP2B2 5 flank confers phenobarbital (PB) inducibility and constitutes a PB response unit (PBRU). By transfection of primary hepatocytes, we analyzed the function of elements comprising the PBRU and evaluated the role of the constitutive androstane receptor (CAR) in PB responsiveness. A 51-base pair PB-responsive enhancer module (PBREM) within the PBRU confers near-maximal PB response when fused to a tk promoter. However, replacing the PBRU with the PBREM in the CYP2B2 5 flank in the natural sequence context reduced PB responsiveness by approximately 4-fold. Mutational analysis also demonstrated that PBRU sequence elements outside the PBREM are essential for maximal PB responsiveness. The PBRU contains two putative nuclear receptor binding sites, NR1 and NR2. CAR binds to retinoic acid ␤2 response elements (␤RARE) and to the NR1 and NR2 sites of the PBRU and activates transcription of reporter genes in cell lines. However, conversion of NR1 into ␤RARE was the equivalent of an inactivating mutation, indicating that CAR does not activate PB-dependent transcription via NR1 in the natural sequence context. A ␤RARE؋2-tk reporter construct was inducible by all-trans-retinoic acid (at-RA) as expected and also responded to PB. The latter can be attributed to nuclear accumulation of CAR after PB exposure. Exogenous CAR increased both the basal and PB-induced response of ␤RARE؋2-tk but reduced PBRU-dependent PB response. Furthermore, exogenous CAR also reduced the at-RA response of the ␤RARE؋2-tk construct. Thus, CAR acts negatively on PB responsiveness mediated by the CYP2B2 PBRU just as it prevents maximal at-RA responsiveness mediated by ␤RARE. Hepatic cytochrome P450s (CYPs)1 play a critical role in the metabolism of hydrophobic xenobiotics, and many CYPs are selectively inducible by xenobiotic compounds. Recently progress has been made toward understanding the molecular mechanisms underlying phenobarbital (PB) inducibility of the homologous rat CYP2B2 (and CYP2B1) and mouse Cyp2b10 genes (Refs. 1-9; reviewed in Refs. 10 -13) and of the chicken CYP2H1 gene (14). We identified a 163-bp Sau3AI fragment at coordinates Ϫ2317/Ϫ2155 in the CYP2B2 5Ј-flank that confers PB inducibility on the heterologous tk promoter and has the properties of a transcriptional enhancer when cat reporter constructs are transfected into primary rat hepatocytes (1). The homologous region of the 5Ј-flank of the PB-inducible mouse Cyp2b10 gene was found to contain a 162-bp segment 92% identical to the rat CYP2B2 163-bp fragment (there is a 1-bp deletion in the mouse sequence with respect to the rat; see Fig. 1A) that also possesses the properties of a transcriptional enhancer and confers PB inducibility on the heterologous tk promoter in primary mouse hepatocytes (3). Further analysis of the rat 163-bp Sau3AI fragment led us to conclude that it is a multicomponent enhancer constituting a PB response unit or PBRU (4). The localization of the PBRU at coordinates Ϫ2317/ Ϫ2155 in the CYP2B2 5Ј-flank is in agreement wit...

Extraction of mouse spinal motor neurons from transgenic mouse embryos recapitulating some aspects of neurodegenerative diseases like amyotrophic lateral sclerosis has met with limited success. Furthermore, extraction and long-term culture of adult mouse spinal motor neurons and glia remain also challenging. We present here a protocol designed to extract and purify high yields of motor neurons and glia from individual spinal cords collected on embryos and adult (5-month-old) normal or transgenic mice. This method is based on mild digestion of tissue followed by gradient density separation allowing to obtain two millions motor neurons over 92% pure from one E14.5 single embryo and more than 30,000 from an adult mouse. These cells can be cultured more than 14 days in vitro at a density of 100,000 cells/cm2 to maintain optimal viability. Functional astrocytes and microglia and small gamma motor neurons can be purified at the same time. This protocol will be a powerful and reliable method to obtain motor neurons and glia to better understand mechanisms underlying spinal cord diseases.