ORIGINAL RESEARCH REPORT Isolation of Pluripotent Neural Crest-Derived Stem Cells from Adult Human Tissues by Connexin-43 Enrichment Daniel Pelaez, 1,2 Chun-Yuh Charles Huang, 2 and Herman S. Cheung 1,2 Identification and isolation of pluripotent stem cells in adult tissues represent an important advancement in the fields of stem cell biology and regenerative medicine. For several years, research has been performed on the identification of biomarkers that can isolate stem cells residing in neural crest (NC)-derived adult tissues. The NC is considered a good model in stem cell biology as cells from it migrate extensively and contribute to the formation of diverse tissues in the body during organogenesis. Migration of these cells is modulated, in part, by gap junction communication among the cell sheets. Here we present a study in which, selection of connexin 43 (Cx43) expressing cells from human adult periodontal ligament yields a novel pluripotent stem cell population. Cx43 + periodontal ligament stem cells express pluripotency-associated transcription factors OCT4, Nanog, and Sox2, as well as NC-specific markers Sox10, p75, and Nestin. When injected in vivo into an immunodeficient mouse model, these cells were capable of generating teratomas with tissues from the three embryological germ layers: endoderm, mesoderm, and ectoderm. Furthermore, the cells formed mature structures of tissues normally arising from the NC during embryogenesis such as eccrine sweat glands of the human skin, muscle, neuronal tissues, cartilage, and bone. Immunohistochemical analysis confirmed the human origin of the neoplastic cells as well as the ectodermal and endodermal nature of some of the structures found in the tumors. These results suggest that Cx43 may be used as a biomarker to select and isolate the remnant NC pluripotent stem cells from adult human tissues arising from this embryological structure. The isolation of these cells through routine medical procedures such as wisdom teeth extraction further enhances their applicability to the regenerative medicine field. Introduction C ellular potency, or the potential for a cell to dif- ferentiate down various tissue lineages, is the defining characteristic as to the viability of the cell source for a given application. With the establishment of embryonic stem cell lines, the idea of utilizing pluripotent cell sources for regen- erative medical applications was introduced as a promising alternative to traditional medical practices. However, there are ethical dilemmas as well as immunological concerns arising from the use of embryonic cell sources in clinical set- tings. The advent of induced pluripotent stem cells (iPSCs) attempted to circumvent both of these issues and brought the concept of patient-specific pluripotent stem cell attainment to the forefront of the field [1]. This iPSC technology, however, is not without its drawbacks; still in its early stages, the scientific community is trying to decipher the mechanisms underlying the induction of pluripotency [2] and overcome the aberrant epigenetic changes arising from the implementation of the technique [3]. Nevertheless, the potential of obtaining plu- ripotent stem cells from adult tissues is of significant impor- tance and remains as the ultimate goal in overcoming the limitations presently found with other postnatal stem cell populations. Researchers have worked on the identification and isola- tion of remnant embryonic-like cells from adult tissues [4–6]. One of the main focuses has been on tissues arising from the neural crest (NC) [7,8]. The embryonic NC is a transient structure originating at the dorsal region of the neural pri- mordium and serves as a good model in stem cell biology. Cells from the NC migrate extensively, populate various organs and tissues, and give rise to cells from all three em- bryological germ layers [7–10]. Modulation of NC cell plur- ipotency and their highly migratory nature has been an active subject of research for years [9]. NC cells are derived from the dorsal neuroepithelium by an epithelium to mes- enchymal cell transformation [9,11] and have been shown to migrate as sheets and streams rather than single or aggre- gated cell clusters [11–15]. In prospective migratory NC cells, there is a transition from tight junctions to gap junctions [16]. 1 Geriatric Research, Education and Clinical Center. Miami VA Healthcare System, Miami, Florida. 2 Department of Biomedical Engineering. College of Engineering. University of Miami, Coral Gables, Florida. STEM CELLS AND DEVELOPMENT Volume 22, Number 21, 2013 Ó Mary Ann Liebert, Inc. DOI: 10.1089/scd.2013.0090 1