Low temperature and mTOR inhibition favor stem cell maintenance in human keratinocyte cultures.

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.

Capturing epidermal stemness for regenerative medicine.

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.

Oligopotent stem cells are distributed throughout the mammalian ocular surface.

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; treatments range from local medications to corneal transplants, and more recently to stem cell therapy. The corneal epithelium is a squamous epithelium that is constantly renewing, with a vertical turnover of 7 to 14 days in many mammals. 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; 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. 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) 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.

Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin.

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.

Traceable Impedance-Based Dispensing and Cloning of Living Single Cells.

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.

Tp63-expressing adult epithelial stem cells cross lineages boundaries revealing latent hairy skin competence.

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).

A single epidermal stem cell strategy for safe ex vivo gene therapy.

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.

Stem cell niches in mammals.

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.

Long-term renewal of hair follicles from clonogenic multipotent stem cells.

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.