Owing to their location at the interface with the external environment, stem cells of the epidermis are particularly exposed to environmental variations such as fluctuating temperature. However, little is known about how temperature affects epidermal stem cell metabolism and behavior. Here we demonstrate that cultured human epidermal stem cells (HESCs) respond to a small drop in temperature through thermos-sensitive TRP channels connected to mTOR signaling. Exposure of HESCs to rapamycin or a small drop in temperature induces the nuclear translocation of mTOR with an impact on gene expression. We also demonstrate by single-cell analysis that long-term inhibition of mTORC1 favors the maintenance of human epidermal stemness. Taken together our results demonstrate that HESCs can adapt to environmental changes (e.g, small variations in temperature) through mTOR signaling, and constant inhibition of mTORC1 favors stem cell maintenance, a finding of paramount importance for regenerative medicine applications.

Adult autologous human epidermal stem cells can be extensively expanded ex vivo for cell and gene therapy. Identifying the mechanisms involved in stem cell maintenance and defining culture conditions to maintain stemness is critical, because an inadequate environment can result in the rapid conversion of stem cells into progenitors/transient amplifying cells (clonal conversion), with deleterious consequences on the quality of the transplants and their ability to engraft. Here, we demonstrate that cultured human epidermal stem cells respond to a small drop in temperature through thermoTRP channels via mTOR signaling. Exposure of cells to rapamycin or a small drop in temperature induces the nuclear translocation of mTOR with an impact on gene expression. We also demonstrate by single-cell analysis that long-term inhibition of mTORC1 reduces clonal conversion and favors the maintenance of stemness. Taken together, our results demonstrate that human keratinocyte stem cells can adapt to environmental changes (e.g., small variations in temperature) through mTOR signaling and constant inhibition of mTORC1 favors stem cell maintenance, a finding of high importance for regenerative medicine applications.

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.

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).

We present a 3D-printing technology allowing free-form fabrication of centimetre-scale injectable structures for minimally invasive delivery. They result from the combination of 3D printing onto a cryogenic substrate and optimisation of carboxymethylcellulose-based cryogel inks. The resulting highly porous and elastic cryogels are biocompatible, and allow for protection of cell viability during compression for injection. Implanted into the murine subcutaneous space, they are colonized with a loose fibrovascular tissue with minimal signs of inflammation and remain encapsulation-free at three months. Finally, we vary local pore size through control of the substrate temperature during cryogenic printing. This enables control over local cell seeding density in vitro and over vascularization density in cell-free scaffolds in vivo. In sum, we address the need for 3D-bioprinting of large, yet injectable and highly biocompatible scaffolds and show modulation of the local response through control over local pore size. STATEMENT OF SIGNIFICANCE:This work combines the power of 3D additive manufacturing with clinically advantageous minimally invasive delivery. We obtain porous, highly compressible and mechanically rugged structures by optimizing a cryogenic 3D printing process. Only a basic commercial 3D printer and elementary control over reaction rate and freezing are required. The porous hydrogels obtained are capable of withstanding delivery through capillaries up to 50 times smaller than their largest linear dimension, an as yet unprecedented compression ratio. Cells seeded onto the hydrogels are protected during compression. The hydrogel structures further exhibit excellent biocompatibility 3 months after subcutaneous injection into mice. We finally demonstrate that local modulation of pore size grants control over vascularization density in vivo. This provides proof-of-principle that meaningful biological information can be encoded during the 3D printing process, deploying its effect after minimally invasive implantation.

Cryogels are macroporous materials that display remarkable properties, such as high pore interconnection, large surface to volume ratio, and high mechanical stability, making them good candidates for 3D cell culture. However, shaping cryogels remains challenging because of the harsh conditions of synthesis at temperatures as low as −80 °C. In this paper, a solution for the 3D printing of functionalized cryogels is proposed. A microfabricated dispensing probe allowing the last second mixing of cryogel precursors as well as control of the temperature of the extruded material during printing is presented. This dispensing tool allows multilayer 3D printing of cryogels with on demand local pore size change through the control in temperature of the dispensed solution. Moreover, thanks to advanced functionalization of the scaffold, cells can be cultured in 3D within the printed scaffold and exhibited spreading. The ability to tune the pore size of the printed cryogels allows to select during printing where cells will get seeded.

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.

SPINK5 (serine protease inhibitor Kazal-type 5) encodes the proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor). In skin, LEKTI expression is restricted to the stratum granulosum of the epidermis and the inner root sheath of hair follicles. Mutations that create premature termination codons in SPINK5 have been reported as the cause of Netherton syndrome (NS), a human autosomal recessive disorder characterized by congenital ichthyosis with defective cornification, a specific hair shaft defect known as trichorrexis invaginata or bamboo hair, and severe atopic manifestations, including atopic dermatitis and hayfever. Spink5 knockout mice display a similar phenotype, but die at birth due to severe dehydration. In order to follow the effects of Spink5 knockout in the skin over a longer period of time, Descargues, Barrandon and colleagues transplanted whole back skin from Spink5-/- newborn mice onto the back of nude mice. Surprisingly, psoriasis-like hyperplasia, basement membrane breakdown followed by invasion of spindle-shaped epidermal cells into the dermal compartment, and formation of numerous sweat gland-like structures were observed in the grafts. These observations suggested a new role for LEKTI on the proliferation and fate determination of keratinocyte stem cells. The work described in this thesis aims to investigate the mechanisms by which LEKTI can impact these biological processes, using clonogenic keratinocytes isolated from patients with NS, and Spink5-downregulated hair follicle multipotent stem cells of the rat. I show that membrane-bounded enzymes FURIN and TACE (tumor necrosis factor alpha converting enzyme or ADAM17) are potential targets of the LEKTI inhibitory activity. Moreover, I demonstrate that prominent cleavage of EDA (Ectodysplasin A) and TNFα (tumor necrosis factor alpha), proteins activated by FURIN and TACE, respectively, is observed in the absence of LEKTI. Altogether, my results thoroughly support the hypothesis that LEKTI may act as a regulator node in several signaling pathways involved in epidermal stem cell behavior.

Replying to: T.-T. Sun, S. C. Tseng & R. M. Lavker , 10.1038/nature08805 (2010) Our claim is not that there are no stem cells in the limbus, but that there is more to corneal renewal than the limbus and that the double-dome-shaped structure of the cornea and physical constraints have a crucial impact on cell dynamics https://www.nature.com/articles/nature08805 1.

Keratinocyte stem cells, along with hematopoietic stem cells, have the longest record in cell therapy. Following the seminal work by Green and colleagues in the early 1980s, hundreds of burned patients worldwide had their lives saved by the transplantation of cultured autologous keratinocyte stem cells. Reconstitution of a functional epidermal barrier, self-renewal of the epidermis, and the unexpected regeneration of a papillary dermis are major accomplishments. Gene therapy of hereditary, disabling skin diseases using genetically modified keratinocyte stem cells is down the road. Nevertheless, many more challenges lie ahead. The functionality, the mechanical properties and the aesthetic of the regenerated skin must improve. Sweat glands and hair follicles must be reconstructed, and the pigmentation of the skin better-controlled. It is then necessary to thoroughly comprehend the cellular and molecular mechanisms involved in skin morphogenesis, epidermal renewal, stem cell interactions, dermal remodeling, and fetal wound healing. Only then will stem cell therapy become a major therapeutic option in plastic and reconstructive surgery. Nevertheless, several recent developments are extremely promising, such as ex vivo keratinocyte stem cell gene therapy, the capacity to derive adult-like keratinocyte from embryonic stem cells, and the capacity to derive induced pluripotent stem cells from adult keratinocytes.

Reference EPFL-CONF-159747View record in Web of Science Record created on 2010-11-30, modified on 2017-05-12

Corneal stem cell niches It has been believed based on BrdU studies that the limbus is the niche for the stem cells responsible for the long-term renewal of the cornea. Majo et al . show instead that the renewal of the corneal epithelium is not different from other squamous epithelia, and is self-maintained without limbal contribution. The authors also show that the entire ocular surface of the pig, including the central cornea, contains stem cells that are oligopotent and can generate individual colonies of corneal and conjunctival cells. They propose that the limbus is not the sole niche for corneal stem cells.

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.

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.