Bringing Neural Cell Therapies to the Clinic: Past and Future Strategies.

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.

Direct reprogramming of melanocytes to neural crest stem-like cells by one defined factor.

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.

Dermis-derived stem cells: a source of epidermal melanocytes and melanoma?

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.

A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.

Melanomas 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.

Embryonic stem cells as a model for studying melanocyte development.

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.

Active Notch1 confers a transformed phenotype to primary human melanocytes.

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 (N(IC)) was exploited to determine if Notch activation is a "driving" event in melanocytic transformation or instead a "passenger" event associated with melanoma progression. N(IC)-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. N(IC)-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.

Melanoma stem cells: the dark seed of melanoma.

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.

Learning the ABCs of melanoma-initiating cells.

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.

Stabilized binding of TBP to the TATA box of herpes simplex virus type 1 early (tk) and late (gC) promoters by TFIIA and ICP4.

We have recently shown that ICP4 has a differential requirement for the general transcription factor TFIIA in vitro (S. Zabierowski and N. DeLuca, J. Virol. 78:6162-6170, 2004). TFIIA was dispensable for ICP4 activation of a late promoter (gC) but was required for the efficient activation of an early promoter (tk). An intact INR element was required for proficient ICP4 activation of the late promoter in the absence of TFIIA. Because TFIIA is known to stabilize the binding of both TATA binding protein (TBP) and TFIID to the TATA box of core promoters and ICP4 has been shown to interact with TFIID, we tested the ability of ICP4 to stabilize the binding of either TBP or TFIID to the TATA box of representative early, late, and INR-mutated late promoters (tk, gC, and gC8, respectively). Utilizing DNase I footprinting analysis, we found that ICP4 was able to facilitate TFIIA stabilized binding of TBP to the TATA box of the early tk promoter. Using mutant ICP4 proteins, the ability to stabilize the binding of TBP to both the wild-type and the INR-mutated gC promoters was located in the amino-terminal region of ICP4. When TFIID was substituted for TBP, ICP4 could stabilize the binding of TFIID to the TATA box of the wild-type gC promoter. ICP4, however, could not effectively stabilize TFIID binding to the TATA box of the INR-mutated late promoter. The additional activities of TFIIA were required to stabilize the binding of TFIID to the INR-mutated late promoter. Collectively, these data suggest that TFIIA may be dispensable for ICP4 activation of the wild-type late promoter because ICP4 can substitute for TFIIA's ability to stabilize the binding of TFIID to the TATA box. In the absence of a functional INR, ICP4 can no longer stabilize TFIID binding to the TATA box of the late promoter and requires the additional activities of TFIIA. The stabilized binding of TFIID by TFIIA may in turn allow ICP4 to more efficiently activate transcription from non-INR containing promoters.