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The formation of hair follicles, a landmark of mammals, requires complex mesenchymal–epithelial interactions and it is commonly believed that embryonic epidermal cells are the only cells that can respond to hair follicle morphogenetic signals in vivo. Here, we demonstrate that epithelial stem cells of non-skin origin (e.g. that of cornea, oesophagus, vagina, bladder, prostate) that express the transcription factor Tp63, a master gene for the development of epidermis and its appendages, can respond to skin morphogenetic signals. When exposed to a newborn skin microenvironment, these cells express hair-follicle lineage markers and contribute to hair follicles, sebaceous glands and/or epidermis renewal. Our results demonstrate that lineage restriction is not immutable and support the notion that all Tp63-expressing epithelial stem cells, independently of their embryonic origin, have latent skin competence explaining why aberrant hair follicles or sebaceous glands are sometimes observed in non-skin tissues (e.g. in cornea, vagina or thymus)., Adult stem cells are thought to be fate restricted to lineages distinct to their tissue of origin. Here, the authors demonstrate that Tp63 expressing epithelial stem cells from several disparate tissues can respond to skin morphogenetic signals and contribute to hair follicles, sebaceous glands and/or epidermis.

Single-cell cloning is essential in stem cell biology, cancer research, and biotechnology. Regulatory agencies now require an indisputable proof of clonality that current technologies do not readily provide. Here, we report a one-step cloning method using an engineered pipet combined with an impedance-based sensing tip. This technology permits the efficient and traceable isolation of living cells, stem cells, and cancer stem cells that can be individually expanded in culture and transplanted.

Cryogels are macroporous materials that display remarkable properties such as high pore inter- connection, large surface to volume ratio and high mechanical stability. As a result they are used in many areas of science spanning from blood separation and filtering to biological scaffolds for cells. However, shaping such material remains challenging because of the harsh conditions of synthesis at temperatures as low as −80◦C. In this paper we propose a solution for the 3D print- ing of cryogels. A micro-fabricated dispensing tool is presented and allows the last-second mixing of the different components for the cryogel synthesis as well as the control in temperature of the material during printing. This dispensing tool is mounted on a 3D printer allowing the multi-layer printing of the cryogel. By controlling the temperature of the dispensing tool during the printing process, we show the possibility to actively and locally tune the pore size of the cryogel. The printed gel are then coated with collagen and seeded with cells. The change in pore size allowed selective cell colonization opening the door to cell patterning through pore size manipulation.

The skin is privileged because several skin-derived stem cells (epithelial stem cells from epidermis and its appendages, mesenchymal stem cells from dermis and subcutis, melanocyte stem cells) can be efficiently captured for therapeutic use. Main indications remain the permanent coverage of extensive third degree burns and healing of chronic cutaneous wounds, but recent advances in gene therapy technology open the door to the treatment of disabling inherited skin diseases with genetically corrected keratinocyte stem cells. Therapeutic skin stem cells that were initially cultured in research or hospital laboratories must be produced according strict regulatory guidelines, which ensure patients and medical teams that the medicinal cell products are safe, of constant quality and manufactured according to state-of-the art technology. Nonetheless, it does not warrant clinical efficacy and permanent engraftment of autologous stem cells remains variable. There are many challenges ahead to improve efficacy among which to keep telomere-dependent senescence and telomere-independent senescence (clonal conversion) to a minimum in cell culture and to understand the cellular and molecular mechanisms implicated in engraftment. Finally, medicinal stem cells are expansive to produce and reimbursement of costs by health insurances is a major concern in many countries. (C) 2012 Elsevier Ltd. All rights reserved.

Adult stem cells are essential for tissue renewal, regeneration, and repair, and their expansion in culture is of paramount importance for regenerative medicine. Using the whisker follicle of the rat as a model system, we demonstrate that ( i ) clonogenicity is an intrinsic property of the adult stem cells of the hair follicle; ( ii ) after cultivation for >140 doublings, these stem cells, transplanted to the dermo-epidermal junction of newborn mouse skin, form part or all of the developing follicles; ( iii ) the stem cells incorporated into follicles are multipotent, because they generate all of the lineages of the hair follicle and sebaceous gland; ( iv ) thousands of hair follicles can be generated from the progeny of a single cultivated stem cell; ( v ) cultured stem cells express the self-renewal genes Bmi1 and Zfp145 ;( vi ) several stem cells participate in the formation of a single hair bulb; and ( vii ) there are many more stem cells in whisker follicles than could be anticipated from label-retaining experiments.

International audience; There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy.

SummaryWe describe two familial cases of dominant dystrophic epidermolysis bullosa (DDEB) that are heterozygous for deletions in COL7A1 that alter splicing, despite intact consensus splice-site sequences. One patient shows a 28-bp genomic deletion (6081del28) in exon 73 associated with the activation of a cryptic donor splice site within this exon; the combination of both defects restores the phase and replaces the last 11 Gly-X-Y repeats of exon 73 by a noncollagenous sequence, Glu-Ser-Leu. The second patient demonstrates a 27-bp deletion in exon 87 (6847del27), causing in-frame skipping of this exon; consensus splice sites, putative branch sites, and introns flanking exons 73 and 87 showed a normal sequence. Keratinocytes from the probands synthesized normal and shortened type VII collagen polypeptides and showed intracellular accumulation of type VII procollagen molecules. This first report of genomic deletions in COL7A1 in DDEB suggests a role for exonic sequences in the control of splicing of COL7A1 pre-mRNA and provides evidence that shortened type VII collagen polypeptides can alter, in a dominant manner, anchoring-fibril formation and can cause DDEB of differing severity.

SummaryWe have characterized 21 mutations in the type VII collagen gene (COL7A1) encoding the anchoring fibrils, 18 of which were not previously reported, in patients from 15 unrelated families with recessive dystrophic epidermolysis bullosa (RDEB). COL7A1 mutations in both alleles were identified by screening the 118 exons of COL7A1 and flanking intron regions. Fourteen mutations created premature termination codons (PTCs) and consisted of nonsense mutations, small insertions, deletions, and splice-site mutations. A further seven mutations predicted glycine or arginine substitutions in the collagenous domain of the molecule. Two mutations were found in more than one family reported in this study, and six of the seven missense mutations showed clustering within exons 72–74 next to the hinge region of the protein. Patients who were homozygous or compound heterozygotes for mutations leading to PTCs displayed both absence or drastic reduction of COL7A1 transcripts and undetectable type VII collagen protein in skin. In contrast, missense mutations were associated with clearly detectable COL7A1 transcripts and with normal or reduced expression of type VII collagen protein at the dermo/epidermal junction. Our results provide evidence for at least two distinct molecular mechanisms underlying defective anchoring fibril formation in RDEB: one involving PTCs leading to mRNA instability and absence of protein synthesis, the other implicating missense mutations resulting in the synthesis of type VII collagen polypeptide with decreased stability and/or altered function. Genotype-phenotype correlations suggested that the nature and location of these mutations are important determinants of the disease phenotype and showed evidence for interfamilial phenotypic variability.

Stem cells safeguard tissue homeostasis and guarantee tissue repair throughout life. The decision between self-renewal and differentiation is influenced by a specialized microenvironment called stem cell niche. Physical and molecular interactions with niche cells and orientation of the cleavage plane during stem cell mitosis control the balance between symmetric and asymmetric division of stem cells. Here we highlight recent progress made on the anatomical and molecular characterization of mammalian stem cell niches, focusing particularly on bone marrow, tooth and hair follicle. The knowledge of the regulation of stem cells within their niches in health and disease will be instrumental to develop novel therapies that target stem cell niches to achieve tissue repair and re-establish tissue homeostasis.

Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell–like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest–derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.

Keratinocyte stem cells together with hematopoietic stem cells have the longest record in cell therapy. Howard Green and colleagues pioneered stem cell therapy of the skin in the early 1980s, and gene therapy of hereditary disabling skin diseases using genetically corrected keratinocyte stem cells has recently become a reality. Since Green and colleagues' seminal publication in 1984, thousands of extensively burned patients have been transplanted worldwide with cultured autologous keratinocyte stem cells. The emergency situation of extensive third degree burn wounds necessitates a massive ex vivo expansion of autologous keratinocyte stem cells that must be achieved in the shortest time possible from a small skin biopsy. Successful stem cell engraftment has permitted the lives of many patients to be saved by permanently reconstituting a functional epidermal barrier. Nevertheless, many challenges remain. Stem cell engraftment is variable for unknown reasons; the functionality and the aesthetic appearance of the regenerated skin must improve, possibly through the use of composite grafts associating dermal and keratinocyte stem cells. Sweat glands and hair follicles must also be regenerated and the pigmentation of the skin controlled. It is thus essential to thoroughly comprehend the cellular and molecular mechanisms involved in skin morphogenesis and fetal wound healing, in epidermal renewal and dermal remodeling. In parallel, it is crucial to master costs to make keratinocyte stem cell-based products more affordable. Only then will stem cell therapy become a major therapeutic option in dermatology and reconstructive surgery.

The upper region of the outer root sheath of vibrissal follicles of adult mice contains multipotent stem cells that respond to morphogenetic signals to generate multiple hair follicles, sebaceous glands, and epidermis, i.e., all the lineages of the hairy skin. At the time when hair production ceases and when the lower region of the follicle undergoes major structural changes, the lower region contains a significant number of clonogenic keratinocytes, and can then respond to morphogenetic signals. This demonstrates that multipotent stem cells migrate to the root of the follicle to produce whisker growth. Moreover, our results indicate that the clonogenic keratinocytes are closely related, if not identical, to the multipotent stem cells, and that the regulation of whisker growth necessitates a precise control of stem cell trafficking.

Marie-Unna hypotrichosis (MU) is a rare autosomal dominant congenital alopecia characterised by progressive hair loss starting in early childhood, often aggravated at puberty and leading to scarring alopecia of variable severity. We have studied three multigeneration families of Belgian, British and French descent. The human genome was screened with microsatellite markers spaced at 10-cM intervals and significant evidence for linkage to the disease was observed on chromosome 8p21, with a maximum two-point lod score of 8.26 for D8S1786 at a recombination fraction of 0. Recombinants narrowed the region of interest to a genetic interval of about 12 cM flanked by markers D8S280 and D8S1839. This interval contains the hairless gene which is mutated in autosomal recessive congenital atrichia. Sequencing of the entire coding region and intronic splice sites of the hairless gene in these three families and in two unrelated familial cases revealed several polymorphic changes but failed to identify causative mutations. Nine other genes located within this region and expressed in skin were also excluded by mutation analysis. Together with a recent linkage study performed in a Dutch and a British family by van Steensel et al these results provide evidence for the presence of a gene distinct from hairless in chromosomal region 8p21 playing an important role in hair follicle biology.

Epidermolysis bullosa simplex (EBS) arises from mutations within the keratin 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocytes cytoskeleton. The majority of these defects are missense mutations in the rod domain, whose locations influence the disease severity. We investigated a large family dominantly affected with the Dowling-Meara form of EBS (EBS-DM). Sequencing of amplified and cloned K5 cDNA from cultured keratinocytes revealed a 66 nucleotide deletion in one allele corresponding to the last 22 amino acid residues encoded by exon 1 (Val164 to Lys185). Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygous G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5. This mutation leads to the use of an exonic GT cryptic donor splice site, located 66 nucleotides upstream from the normal donor splice site of intron 1. The corresponding peptide deletion includes the last five amino acids of the H1 head domain and the first 17 amino acids of the conserved amino terminal end of the 1A rod domain, including the first two heptad repeats and the helix initiation peptide. The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein. This is the first splice site mutation to be reported as a cause of EBS-DM. Owing to the functional importance of the removed region, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.

We have examined the growth capacity of keratinocytes isolated from human scalp hair follicles. Like the keratinocytes of glabrous epidermis, most of the colony-forming cells are classified as holoclones or meroclones when analyzed in a clonal assay. Some of them have extensive growth potential, as they are able to undergo at least 130 doublings. Therefore, the hair follicle, like the epidermis, contains keratinocytes with the expected property of stem cells: an extensive proliferative capacity permitting the generation of a large amount of epithelium. We have also examined the distribution of clonogenic keratinocytes within the hair follicle. Several hundred colony-forming cells are concentrated at a region below the midpoint of the follicle and outside the hair bulb. This region lies deeper than the site of insertion of the arrector pili muscle, which corresponds with the position of the bulge when the latter can be identified. In contrast, few colony-forming cells are present in the hair bulb, where most of the mitotic activity is observed during the active growth phase of the follicle. Paraclones, which are present both in the midregion and in the bulb of hair follicles, are unlikely to be the transient amplifying cells expected from kinetic studies.

The Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is a life-threatening autosomal disease characterized by loss of dermal-epidermal adherence with abnormal anchoring fibrils (AF). We recently linked HS-RDEB to the type VII collagen gene (COL7A1) which encodes the major component of AF. We describe a patient who is homozygous for an insertion-deletion in the FN-4A domain of the COL7A1 gene. This defect causes a frameshift mutation which leads to a premature stop codon in the FN-5A domain, resulting in a marked diminution in mutated mRNA levels, with no detectable type VII collagen polypeptide in the patient. Our data suggest strongly that this null allele prevents normal anchoring fibril formation in homozygotes and is the underlying cause of HS-RDEB in this patient.

We describe here eleven different mutations in SPINK5, encoding the serine protease inhibitor LEKTI, in 13 families with Netherton syndrome (NS, MIM256500). Most of these mutations predict premature termination codons. These results disclose a critical role of SPINK5 in epidermal barrier function and immunity, and suggest a new pathway for high serum IgE levels and atopic manifestations.

The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. Forty- five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of loss of corneal transparency(1); treatments range from local medications to corneal transplants, and more recently to stem cell therapy(2). The corneal epithelium is a squamous epithelium that is constantly renewing, with a vertical turnover of 7 to 14 days in many mammals(3). Identification of slow cycling cells ( label- retaining cells) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long- term renewal of the cornea(4); hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in marked opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium(5,6). Here we show that the corneal epithelium of the mouse can be serially transplanted, is self- maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. Furthermore, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones)(7,8) with the capacity to generate individual colonies of corneal and conjunctival cells. Therefore, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. We propose a model that unifies our observations with the literature and explains why the limbal region is enriched in stem cells.