Traceable impedance-based single-cell pipetting, from a research set-up to a robust and fast automated robot: DispenCell-S1.

Single-cell isolation is a truly transformative tool for the understanding of biological systems. It allows single-cell molecular analyses and considers the heterogeneity of cell populations, which is of particular relevance for the diagnosis and treatment of evolving diseases and for personalized medicine. Single-cell isolation is also a key process in cell line development, where it is used to obtain stable and high producing clonally-derived cell lines, thus contributing to the efficiency, safety and reproducible quality of the drug produced.

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.

Impedance-Based Single-Cell Pipetting.

Many biological methods are based on single-cell isolation. In single-cell line development, the gold standard involves the dilution of cells by means of a pipet. This process is time-consuming as it is repeated over several weeks to ensure clonality.

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.

Automated collective motion analysis validates human keratinocyte stem cell cultures.

Identification and quality assurance of stem cells cultured in heterogeneous cell populations are indispensable for successful stem cell therapy. Here we present an image-processing pipeline for automated identification and quality assessment of human keratinocyte stem cells. When cultivated under appropriate conditions, human epidermal keratinocyte stem cells give rise to colonies and exhibit higher locomotive capacity as well as significant proliferative potential.

Chronic inflammation imposes aberrant cell fate in regenerating epithelia through mechanotransduction.

Chronic inflammation is associated with a variety of pathological conditions in epithelial tissues, including cancer, metaplasia and aberrant wound healing. In relation to this, a significant body of evidence suggests that aberration of epithelial stem and progenitor cell function is a contributing factor in inflammation-related disease, although the underlying cellular and molecular mechanisms remain to be fully elucidated. In this study, we have delineated the effect of chronic inflammation on epithelial stem/progenitor cells using the corneal epithelium as a model tissue.

Cell motion predicts human epidermal stemness.

Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement.

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.

Clonal, self-renewing and differentiating human and porcine urothelial cells, a novel stem cell population.

Although urothelial progenitor-like cells have been described in the human urinary tract, the existence of stem cells remains to be proven. Using a culture system that favors clonogenic epithelial cell growth, we evaluated and characterized clonal human urothelial cells. We isolated human urothelial cells that were clonogenic, capable of self-renewal and could develop into fully differentiated urothelium once re-implanted into the subcapsular space of nude mice.

Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation.

The epidermal growth factor (EGF) receptor/ligand system stimulates multiple pathways of signal transduction, and is activated by various extracellular stimuli and inter-receptor crosstalk signaling. Aberrant activation of EGF receptor (EGFR) signaling is found in many tumor cells, and humanized neutralizing antibodies and synthetic small compounds against EGFR are in clinical use today. However, these drugs are known to cause a variety of skin toxicities such as inflammatory rash, skin dryness, and hair abnormalities.

Actin filament dynamics impacts keratinocyte stem cell maintenance.

Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion.

Corneal endothelial cell fate is maintained by LGR5 through the regulation of hedgehog and Wnt pathway.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a target of Wnt signaling, is reportedly a marker of intestine, stomach, and hair follicle stem cells in mice. To gain a novel insight into the role of LGR5 in human corneal tissue, we performed gain- and loss-of-function studies. The findings of this study show for the first time that LGR5 is uniquely expressed in the peripheral region of human corneal endothelial cells (CECs) and that LGR5((+)) cells have some stem/progenitor cell characteristics, and that in human corneal endothelium, LGR5 is the target molecule and negative feedback regulator of the Hedgehog (HH) signaling pathway.

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.

Sox10 promotes the formation and maintenance of giant congenital naevi and melanoma.

Giant congenital naevi are pigmented childhood lesions that frequently lead to melanoma, the most aggressive skin cancer. The mechanisms underlying this malignancy are largely unknown, and there are no effective therapies. Here we describe a mouse model for giant congenital naevi and show that naevi and melanoma prominently express Sox10, a transcription factor crucial for the formation of melanocytes from the neural crest.

Generation and characterization of a Notch1 signaling-specific reporter mouse line.

Signaling through the Notch1 receptor is essential for the control of numerous developmental processes during embryonic life as well as in adult tissue homeostasis and disease. Since the outcome of Notch1 signaling is highly context-dependent, and its precise physiological and pathological role in many organs is unclear, it is of great interest to localize and identify the cells that receive active Notch1 signals in vivo. Here, we report the generation and characterization of a BAC-transgenic mouse line, N1-Gal4VP16, that when crossed to a Gal4-responsive reporter mouse line allowed the identification of cells undergoing active Notch1 signaling in vivo.

'Hearts and bones': the ups and downs of 'plasticity' in stem cell biology.

More than a decade ago, 'plasticity' suddenly became a 'fashionable' topic with overemphasized implications for regenerative medicine. The concept of 'plasticity' is supported by old transplantation work, at least for embryonic cells, and metaplasia is a classic example of plasticity observed in patients. Nevertheless, the publication of a series of papers showing rare conversion of a given cell type into another unrelated cell raised the possibility of using any unaffected tissue to create at will new cells to replace a different failing tissue or organ.

Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells.

The thymus develops from the third pharyngeal pouch of the anterior gut and provides the necessary environment for thymopoiesis (the process by which thymocytes differentiate into mature T lymphocytes) and the establishment and maintenance of self-tolerance. It contains thymic epithelial cells (TECs) that form a complex three-dimensional network organized in cortical and medullary compartments, the organization of which is notably different from simple or stratified epithelia. TECs have an essential role in the generation of self-tolerant thymocytes through expression of the autoimmune regulator Aire, but the mechanisms involved in the specification and maintenance of TECs remain unclear.

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.

Wound repair and regeneration.

The repair of wounds is one of the most complex biological processes that occur during human life. After an injury, multiple biological pathways immediately become activated and are synchronized to respond. In human adults, the wound repair process commonly leads to a non-functioning mass of fibrotic tissue known as a scar.

Control of thrombin signaling through PI3K is a mechanism underlying plasticity between hair follicle dermal sheath and papilla cells.

In hair follicles, dermal papilla (DP) and dermal sheath (DS) cells exhibit striking levels of plasticity, as each can regenerate both cell types. Here, we show that thrombin induces a phosphoinositide 3-kinase (PI3K)-Akt pathway-dependent acquisition of DS-like properties by DP cells in vitro, involving increased proliferation rate, acquisition of ;myofibroblastic' contractile properties and a decreased capacity to sustain growth and survival of keratinocytes. The thrombin inhibitor protease nexin 1 [PN-1, also known as SERPINE2) regulates all those effects in vitro.

Hes1 regulates corneal development and the function of corneal epithelial stem/progenitor cells.

Hes1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of Hes1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in Hes1-null mice.

The MRL mouse repairs both cryogenic and ischemic myocardial infarcts with scar.

Objective: The MRL mouse strain shows extraordinary wound healing capacities. Some years ago, Leferovich et al. (Proc Natl Acad Sci U S A 2001;98:9830-35) have reported the absence of scar formation after cryogenically-induced right ventricular myocardial infarcts in adult MRL mice.