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Supplementary Table 1 from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

Supplementary Methods and Materials from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

Supplementary Figure 1 from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

Supplementary Figure Legends 1-2 from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

Supplementary Figure 2 from Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

Cell replacement therapy in the nervous system has a rich history, with ∼40 years of research and ∼30 years of clinical experience. There is compelling evidence that appropriate cells can integrate and function in the dysfunctioning human nervous system, but the clinical results are mixed in practice. A number of factors conspire to vary patient outcome: the indication, cell source, patient selection, and team performing transplantation are all variables that can affect efficacy. Most early clinical trials have used fetal cells, a limited cell source that resists scale and standardization. Direct fetal cell transplantation creates significant challenges to commercialization that is the ultimate goal of an effective cell therapy. One approach to help scale and standardize fetal cell preparations is the expansion of neural cells in vitro. Expansion is achieved by transformation or through the application of mitogens before cryopreservation. Recently, neural cells derived from pluripotent stem cells have provided a scalable alternative. Pluripotent stem cells are desirable for manufacturing but present alternative concerns and manufacturing obstacles. All cell sources require robust and reproducible manufacturing to make nervous system cell replacement therapy an option for patients. Here, we discuss the challenges and opportunities for cell replacement in the nervous system. In this review, we give an overview of completed and ongoing neural cell transplantation clinical trials, and we discuss the challenges and opportunities for future cell replacement trials with a particular focus on pluripotent stem cell-derived therapies.

Uncaging promoter and enhancer dynamics In order to understand cellular differentiation, it is important to understand the timing of the regulation of gene expression. Arner et al. used cap analysis of gene expression (CAGE) to analyze gene enhancer and promoter activities in a number of human and mouse cell types. The RNA of enhancers was transcribed first, followed by that of transcription factors, and finally by genes that are not transcription factors. Science , this issue p. 1010

Mouse and human somatic cells can either be reprogrammed to a pluripotent state or converted to another lineage with a combination of transcription factors suggesting that lineage commitment is a reversible process. Here we show that only one factor, the active intracellular form of Notch1, is sufficient to convert mature pigmented epidermal-derived melanocytes into functional multipotent neural crest (NC) stem-like cells. These induced NC stem cells (iNCSCs) proliferate as spheres under stem cell media conditions, re-express NC-related genes, and differentiate into multiple NC-derived mesenchymal and neuronal lineages. Moreover, iNCSCs are highly migratory and functional in vivo. These results demonstrate that mature melanocytes can be reprogrammed toward their primitive NC cell precursors through the activation of a single stem cell-related pathway. Reprogramming of melanocytes to iNCSCs may provide an alternate source of NCSCs for neuroregenerative applications.

Human multipotent dermal stem cells (DSCs) have been isolated and propagated from the dermal region of neonatal foreskin. DSCs can self-renew, express the neural crest stem cell markers NGFRp75 and nestin, and are capable of differentiating into a wide variety of cell types including mesenchymal and neuronal lineages and melanocytes, indicative of their neural crest origin. When placed in the context of reconstructed skin, DSCs migrate to the basement membrane zone and differentiate into melanocytes. These findings, combined with the identification of NGFRp75-positive cells in the dermis of human foreskin, which are devoid of hair, suggest that DSCs may be a self-renewing source of extrafollicular epidermal melanocytes. In this review, we discuss the properties of DSCs, the pathways required for melanocyte differentiation, and the value of 3D reconstructed skin to assess the behavior and contribution of DSCs in the naturalized environment of human skin. Potentially, DSCs provide a link to malignant melanoma by being a target of UVA-induced transformation.

The use of adherent monolayer cultures have produced many insights into melanoma cell growth and differentiation, but often novel therapeutics demonstrated to act on these cells are not active in vivo. It is imperative that new methods of growing melanoma cells that reflect growth in vivo are investigated. To this end, a range of human melanoma cell lines passaged as adherent cultures or induced to form melanoma spheres (melanospheres) in stem cell media have been studied to compare cellular characteristics and protein expression. Melanoma spheres and tumours grown from cell lines as mouse xenografts had increased heterogeneity when compared with adherent cells and 3D-spheroids in agar (aggregates). Furthermore, cells within the melanoma spheres and mouse xenografts each displayed a high level of reciprocal BRN2 or MITF expression, which matched more closely the pattern seen in human melanoma tumours in situ, rather than the propensity for co-expression of these important melanocytic transcription factors seen in adherent cells and 3D-spheroids. Notably, when the levels of the BRN2 and MITF proteins were each independently repressed using siRNA treatment of adherent melanoma cells, members of the NOTCH pathway responded by decreasing or increasing expression, respectively. This links BRN2 as an activator, and conversely, MITF as a repressor of the NOTCH pathway in melanoma cells. Loss of the BRN2-MITF axis in antisense-ablated cell lines decreased the melanoma sphere-forming capability, cell adhesion during 3D-spheroid formation and invasion through a collagen matrix. Combined, this evidence suggests that the melanoma sphere-culture system induces subpopulations of cells that may more accurately portray the in vivo disease, than the growth as adherent melanoma cells.

SummaryMelanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knockdown of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation.

Cells with stem-cell markers and features have recently been identified in melanoma tissues and cell lines. Melanoma stem-like cells possess many traits of tumor-initiating or tumor stem cells including self-renewal capacity, high tumorigenicity, and differentiation into various mesenchymal lineages, including melanocytic cells. Four subpopulations of melanoma-initiating cells have been distinguished: CD20+, CD133+, label-retaining or slow-cycling cells, and side-population cells with high efflux activities. Whether these are distinct or overlapping populations is currently under investigation. Ongoing studies are dissecting and characterizing the hierarchy of these subpopulations within a malignant lesion. Understanding these and the dynamics of clonal dominance will aid in the development of novel therapeutic strategies.

Although multiple genetic lesions and dysregulated signaling pathways have been identified in melanoma as potential pharmacological targets, currently no treatments exist that can effectively combat disseminated melanoma, suggesting that perhaps the proper targets are not being recognized. Recently, a small subpopulation of stem-like cells has been isolated from melanomas that posess many attributes of cancer stem cells, exhibiting unlimited self-renewal capacity, differentiation into various cell types and induction of tumor formation with small cell numbers. Thus, considerable interest lies in determining whether melanomas are derived from transformed melanocyte stem or progenitor cells, or from the transformation of more mature melanocytes that have acquired stem cell properties. Questions have also arisen as to whether these melanoma cancer stem cells should be specifically targeted for therapy. It is probable that, the development of new strategies is required that target both the main population of ...

Human multipotent dermal stem cells (DSCs) have been isolated and propagated from the dermal region of neonatal foreskin. DSCs can self-renew, express the neural crest stem cell markers NGFRp75 and nestin, and are capable of differentiating into a wide variety of cell types including mesenchymal and neuronal lineages and melanocytes, indicative of their neural crest origin. When placed in the context of reconstructed skin, DSCs migrate to the basement membrane zone and differentiate into melanocytes. These findings, combined with the identification of NGFRp75-positive cells in the dermis of human foreskin, which are devoid of hair, suggest that DSCs may be a self-renewing source of extrafollicular epidermal melanocytes. In this review, we discuss the properties of DSCs, the pathways required for melanocyte differentiation, and the value of 3D reconstructed skin to assess the behavior and contribution of DSCs in the naturalized environment of human skin. Potentially, DSCs provide a link to malignant melanoma by being a target of UVA-induced transformation.

Melanocytes are neural crest-derived pigment-producing cells that reside in the inner ear, in the uveal tract, in hair follicles, and in the skin. The main function of melanocytes is to provide pigmentation through melanin production and secretion to the immediate surrounding area. Although much is known about mature melanocyte function and regulation, particularly in the skin, little is known with regard to the signals and gene expression patterns that ensue upon melanocyte development and differentiation from embryonic precursors. The ability to examine these patterns in an in vitro specified setting through the use of embryonic stem cells holds great potential for understanding melanocyte biology. In this chapter, we outline our procedures for the differentiation of human embryonic stem cells toward mature pigment-producing melanocytes that express the appropriate melanocytic markers and home to the epidermal basal layer in 3D skin reconstructs.

The importance of mitogen-activated protein kinase signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf, yet clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions showed that Notch activity is significantly higher in melanomas than their nontransformed counterparts. The use of a constitutively active, truncated Notch transgene construct (NIC) was exploited to determine if Notch activation is a “driving” event in melanocytic transformation or instead a “passenger” event associated with melanoma progression. NIC-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma, such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. NIC-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth, suggesting that Notch alone is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene. This new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease. [Cancer Res 2009;69(13):5312–20]

Melanocytes are neural crest-derived pigment-producing cells that reside in the inner ear, in the uveal tract, in hair follicles, and in the skin. The main function of melanocytes is to provide pigmentation through melanin production and secretion to the immediate surrounding area. Although much is known about mature melanocyte function and regulation, particularly in the skin, little is known with regard to the signals and gene expression patterns that ensue upon melanocyte development and differentiation from embryonic precursors. The ability to examine these patterns in an in vitro specified setting through the use of embryonic stem cells holds great potential for understanding melanocyte biology. In this chapter, we outline our procedures for the differentiation of human embryonic stem cells toward mature pigment-producing melanocytes that express the appropriate melanocytic markers and home to the epidermal basal layer in 3D skin reconstructs.

Neoplastic populations with stem cell potential have been most recently identified in human cutaneous melanoma, and initially characterized for their phenotypic profile. Being melanoma stem cells (MSC) the most desirable target of therapeutic intervention, we asked whether they express the epigenetically-regulated cancer testis antigens (CTA) on which melanoma immunotherapy is increasingly focusing. Reverse transcription-PCR analyses identified the presence of the large majority of investigated CTA (i.e., MAGE, GAGE, NY-ESO, and SSX families) in different MSC populations. MSC expressed MAGE-A proteins as detected by western blot; noteworthy, the distribution of MAGE-A proteins was highly homogeneous within given MSC populations as shown by confocal immunofluorescence. Promoter methylation studies unveiled a homogeneously-demethylated MAGE-A3 promoter that paired MAGE-A3 expression in MSC. Altogether these findings demonstrate that MSC can be efficiently targeted by CTA-directed immunotherapeutic approaches, and suggest that epigenetic patterns most likely drive the expression of CTA in MSC as previously shown for melanoma cells.

The herpes simplex virus type 1 immediate-early protein, ICP4, activates the transcription of viral early and late genes and is essential for viral growth. It has been shown to bind DNA and interact with components of the general transcription machinery to activate or repress viral transcription, depending upon promoter context. Since early and late gene promoters have different architectures and cellular metabolism may be very different at early and late times after infection, the cellular requirements for ICP4-mediated activation of early and late genes may differ. This hypothesis was tested using tk and gC as representative early and late promoters, respectively. Nuclear extracts and phosphocellulose column fractions derived from nuclear extracts were able to reconstitute basal and ICP4-activated transcription of both promoters in vitro. When examining the contribution of the general transcription factors on the ability of ICP4 to activate transcription, the fraction containing the general transcription factor TFIIA was not essential for ICP4 activation of the gC promoter, but it was required for efficient activation of the tk promoter. The addition of recombinant TFIIA restored the ability of ICP4 to efficiently activate the tk promoter, but it had no net effect on activation of the gC promoter. The dispensability of TFIIA for ICP4 activation of the gC promoter required an intact INR element. In addition, microarray and Northern blot analysis indicated that TFIIA abundance may be reduced at late times of infection. This decrease in TFIIA expression during infection and its dispensability for activation of late but not early genes suggest one of possibly many mechanisms for the transition from viral early to late gene expression.

The start site regions of late genes of herpes simplex virus type 1 are similar to the eukaryotic initiator sequence (Inr), have been shown to affect the levels of expression, and may also play a role in transcription activation by the viral activator ICP4. A series of linker-scanning mutations spanning the start site of transcription and several downstream mutations in the true late gC promoter were analyzed in reconstituted in vitro transcription reactions with and without ICP4, as well as in the context of the viral genome during infection. The nucleotide contacts previously found to be important for Inr function were also found to be important for optimal induction by ICP4. While the Inr had a substantial effect on the accumulation of gC RNA during infection, no other sequence downstream of the TATA box to +124 had a significant effect on levels of expression during infection. Therefore, these studies suggest that TATA box and the Inr are the only cis -acting elements required to achieve optimal expression of gC, and that the high levels of late-gene transcription may be largely due to the induction by ICP4, functioning through the Inr element.

Tumor stem or initiating cells have been proposed to exist for melanoma. Stem-like cells have been propagated from melanoma cell lines and specimens. Additionally, classical stem cell markers, including ABCG2 and CD133, have been identified in clinical melanomas. However, definitive markers for the purification and further characterization of melanoma-initiating cells remained elusive. Recently, Schatton et al. provided solid evidence that the doxorubicin-resistant ATP-binding cassette transporter ABCB5 marks primitive cells capable of recapitulating melanomas in xenotransplantation models. The identification of melanoma-initiating cells has far-reaching implications, as new therapeutic strategies can be envisioned that specifically target these cells.