Despite increasing knowledge regarding melanoma-initiating cells (MICs), questions persist regarding the number and phenotypic nature of cells with tumor-generating capability. Evidence for a phenotypically distinct human MIC has been found in NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice. However, a phenotypically distinct human MIC was not found in the NOD/SCIDIl2rg−/− (NSG) mouse model. The demonstration of a distinct population of human melanoma cells responsible for tumorigenesis and tumor cell self-renewal would provide an important target for new melanoma therapies. In this study, we show a 100-fold range in MIC frequency in human melanoma (1 in 18,000 to 1 in 1,851,000 cells) in the NOD/SCID mouse. In this model, human melanoma cells with high aldehyde dehydrogenase (ALDH) activity were enriched 16.8-fold in tumorigenic cells over unfractionated (UNF) cells, such that 1 in 21,000 cells was a MIC. In the NSG mouse, the ALDH expressing cell population was enriched 100-fold in tumorigenic cells over UNF cells, such that one in four cells was a MIC. Xenograft melanomas that developed from ALDH+ cells displayed robust self-renewal, whereas those from ALDH− cells showed minimal self-renewal in vitro. Thus, ALDH+ melanoma cells have enhanced tumorigenicity over ALDH− cells and superior self-renewal ability.

X-linked adrenoleukodystrophy (ALD) is a devastating inherited neurodegenerative disease caused by defects in the ABCD1 gene and affecting peripheral and central nervous system myelin. ABCD1 encodes a peroxisomal transmembrane protein required for very long chain fatty acid (VLCFA) metabolism. We show that zebrafish (Danio rerio) Abcd1 is highly conserved at the amino acid level with human ABCD1, and during development is expressed in homologous regions including the central nervous system and adrenal glands. We used TALENs to generate five zebrafish abcd1 mutant allele lines introducing premature stop codons in exon 1, as well as obtained an abcd1 allele from the Zebrafish Mutation Project carrying a point mutation in a splice donor site. Similar to patients with ALD, zebrafish abcd1 mutants have elevated VLCFA levels. Interestingly, we found that CNS development of the abcd1 mutants is disrupted, with hypomyelination in the spinal cord, abnormal patterning and decreased numbers of oligodendrocytes, and increased cell death. By day of life five abcd1 mutants demonstrate impaired motor function, and overall survival to adulthood of heterozygous and homozygous mutants is decreased. Expression of human ABCD1 in oligodendrocytes rescued apoptosis in the abcd1 mutant. In summary, we have established a zebrafish model of ALD that recapitulates key features of human disease pathology and which reveals novel features of underlying disease pathogenesis.

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin α subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

The neural crest is a transient cell population that travels long distances through the embryo to form a wide range of derivatives. The extensive migration of the neural crest is highly unusual and incompletely understood. We examined the ability of neural crest cells (NCCs) to migrate under different conditions in vitro. Unlike most motile cell types, avian NCCs migrate efficiently on a wide range of fibronectin concentrations. Strikingly, the migration of NCCs on laminin depends on the axial level from which the crest is derived. On high concentrations of laminin, cranial NCCs migrate at approximately twice the rate of trunk NCCs and show greater persistence, a higher percentage of migratory cells, and a less organized cytoskeleton. The difference in migration between cranial and trunk neural crest is not due to transcriptional differences in integrin mRNA, but rather to differences in posttranslational regulation. Overexpression of a single integrin is sufficient to significantly slow the migration velocity of cranial neural crest cultured on high laminin densities. These results demonstrate that neural crest cells accommodate a wide range of ECM concentrations in vitro and suggest that differences in integrin regulation along the anterior-posterior axis may contribute to differences in neural crest migration and cell fate.

As one of the most proliferative tissues in adult mammals, the epidermis is a good example of the precise regulation necessary between stem cell self-renewal and differentiation. The epidermis is derived from ectodermal progenitor cells and contains three distinct classes of cells: epidermal stem cells which are capable of infinite rounds of cell division; their immediate descendants, transient amplifying cells, which are capable of numerous but finite rounds of cell division; and finally, non-dividing, differentiating cells (Aberdam in Cell and Tissue Research 331:103-107, 2008). This proliferative hierarchy must be tightly regulated both temporally and spatially during epidermal development and homeostasis in order to prevent uncontrolled growth leading to hyperproliferative states and/or tumorigenesis. Historically, the most basic unit of epidermal proliferation has been described as the epidermal proliferation unit (EPU). The EPU, as originally characterized by Christophers, Potten and Mackenzie, is a proliferation unit consisting of approximately 10 basal cells with a clonogenic cell in the center and overlaid by the suprabasal and corneocyte progeny (reviewed in Potten, C. S. (1974). The epidermal proliferative unit: the possible role of the central basal cell. Cell and Tissue Kinetics, 7(1), 77-88). Numerous researchers have identified this classical EPU structure, consisting of approximately 20 cells, in a variety of mammalian skin sources. Recently however, lineage analyses have provided evidence for much larger clonal epidermal units consisting of hundreds to thousands of cells. Furthermore, cutaneous mosaicism as well as a variety of cutaneous pathologies indicate that clonal areas extend to whole patches of mammalian skin many centimeters across. In this review we revisit four decades of experimental evidence and put forward a model of clonal units derived from multiple classes of epidermal progenitors ranging from the largest and most primitive units, clonal ectodermal units, to epidermal stem cell units, and finally, to the most basic structural unit, the EPU.

Like for other somatic tissues, isolation of a pure population of stem cells has been a primary goal in epidermal biology. We isolated discrete populations of freshly obtained human neonatal keratinocytes (HNKs) using previously untested candidate stem cell markers aldehyde dehydrogenase (ALDH) and CD44 as well as the previously studied combination of integrin α6 and CD71. An in vivo transplantation assay combined with limiting dilution analysis was used to quantify enrichment for long-term repopulating cells in the isolated populations. The ALDH+CD44+ population was enriched 12.6-fold for long-term repopulating epidermal stem cells (EpiSCs) and the integrin α6hiCD71lo population was enriched 5.6-fold, over unfractionated cells. In addition to long-term repopulation, CD44+ALDH+ keratinocytes exhibited other stem cell properties. CD44+ALDH+ keratinocytes had self-renewal ability, demonstrated by increased numbers of cells expressing nuclear Bmi-1, serial transplantation of CD44+ALDH+ cells, and holoclone formation in vitro. CD44+ALDH+ cells were multipotent, producing greater numbers of hair follicle-like structures than CD44−ALDH− cells. Furthermore, 58% ± 7% of CD44+ALDH+ cells exhibited label-retention. In vitro, CD44+ALDH+ cells showed enhanced colony formation, in both keratinocyte and embryonic stem cell growth media. In summary, the CD44+ALDH+ population exhibits stem cell properties including long-term epidermal regeneration, multipotency, label retention, and holoclone formation. This study shows that it is possible to quantify the relative number of EpiSCs in human keratinocyte populations using long-term repopulation as a functional test of stem cell nature. Future studies will combine isolation strategies as dictated by the results of quantitative transplantation assays, in order to achieve a nearly pure population of EpiSCs.

A prevalent belief in epidermal biology is that stem cells are highly clonogenic; that is, they have the ability to produce many large colonies in vitro. However, it has been wellestablished in hematology, and recently suggested in epithelial biology, that short-term in vitro clonogenic assays may not be reliable predictors of long-term in vivo repopulating ability. Numerous groups have shown that rapid adhesion to collagen selects for highly clonogenic keratinocytes, but it has not been demonstrated whether this subpopulation is enriched in stem cells as defined by long-term repopulating ability in vivo. We found that although rapid adhesion to collagen (within 5 minutes) selected for cells with increased short-term colony forming ability in vitro, these cells were not enriched in long-term proliferative ability in vitro or in repopulating ability in vivo after 9 weeks. Conversely, keratinocytes that did not adhere to collagen (after 20 minutes) were less clonogenic in short-term assays but possessed equivalent long-term proliferative ability in vitro and superior longterm repopulating ability in vivo. Both the rapidly adherent cell and not rapidly adherent cell populations contained small, noncomplex basaloid cells, expressed integrin ␣2 (a collagen IV receptor), and expressed the putative epidermal stem cell phenotype integrin ␣6 hi CD71 lo . Our results indicate that the superior short-term colony forming ability of collagen-adherent murine keratinocytes does not correlate with long-term repopulating ability in vitro or in vivo and that proliferation in vitro is not a reliable surrogate for stem cell behavior in vivo. STEM CELLS 2008;26:235-243 Disclosure of potential conflicts of interest is found at the end of this article.

Maintenance, repair and renewal of the epidermis are thought to depend on a pool of dedicated epidermal stem cells. Like for many somatic tissues, isolation of a nearly pure population of stem cells is a primary goal in cutaneous biology. We used a quantitative transplantation assay, using injection of keratinocytes into subcutis combined with limiting dilution analysis, to assess the long-term repopulating ability of putative murine epidermal stem populations. Putative epidermal stem cell populations were isolated by FACS sorting. The CD133 + population and the subpopulation of CD133 + cells that exhibits high mitochondrial membrane potential (DΨm hi ) , were enriched for long-term repopulating epidermal stem cells vs. unfractionated cells (3.9 and 5.2-fold, respectively). Evidence for self-renewal capacity was obtained by serial transplantation of long-term epidermal repopulating units derived from CD133 + and CD133 + ΔΨm hi keratinocytes. CD133 + keratinocytes were multipotent and produced significantly more hair follicles than CD133 − cells. CD133 + cells were a subset of the previously described integrin α6 + CD34 + bulge cell population and 28.9±8.6% were label retaining cells. Thus, murine keratinocytes within the CD133 + and CD133 + ΔΨm hi populations contain epidermal stem cells that regenerate epidermis for the long-term, are self-renewing, multipotent, and label-retaining cells.