The genus Anacharis Dalman, 1823 comprises parasitoid wasps that target early instars of brown lacewing larvae (Neuroptera: Hemerobiidae). So far, five species were recognised from the Western Palaearctic region, of which four are reported from Northwestern Europe. In this study, we address the Northwestern European species diversity of the genus with an extended integrative taxonomy toolkit. A total of 700 specimens were examined for their external morphology, including the relevant type specimens. For 354 specimens, we obtained CO1 barcode sequences and applied three molecular species delimitation methods. All DNA barcode data are made publicly available via the German Barcode of Life (GBOL) and Barcode of Life Data system (BOLD) database. In addition, we examined images of Wing Interference Patterns (WIPs), examined the male genitalia and performed multivariate morphometric analyses. The analyses revealed two clusters which we describe as the immunis and eucharioides species groups based on differences in DNA barcode, external morphology, WIPs and size of the male genitalia. Furthermore, we complement the diagnosis of the genus Anacharis and describe three new species, Anacharis martinae Vogel, Forshage & Peters, sp. nov., Anacharis maxima Vogel, Forshage & Peters, sp. nov. and Anacharis minima Vogel, Forshage & Peters, sp. nov. Finally, we synonymise A. fergussoni Mata-Casanova & Pujade-Villar, 2018, syn. nov. with A. eucharioides (Dalman, 1818), and we reinstate A. ensifer Walker, 1835, stat. rev., A. typica Walker, 1835, stat. rev. and A. petiolata Zetterstedt, 1838, stat. rev. as valid species. In total, we recognise nine Northwestern European species to which we provide an identification key. The species of Anacharis are morphologically very variable. Morphometric analyses alone did not provide information sufficient to delimit species, neither did analyses of WIPs and male genitalia, with few notable exceptions. Analyses of molecular sequence data proved crucially helpful to reliably delimit species and to find morphological diagnostic characters in a reverse taxonomy approach. For delimiting species groups, all included analyses proved helpful, and we show that exploring an extended integrative taxonomy toolkit can be beneficial for a comprehensive characterisation of species. We acknowledge that a complete overview of species distributions, and characterisation of ecological niches & host records is still required to deeply understand the genus as a whole, yet our results already allow broad access to and inclusion of Anacharis species in downstream biodiversity research.

This protocol aims to provide standardisation for the necessary steps, specifically to a) produce standardised, artifact-free and high-quality WIP images, b) secure long-term-storage of the specimens, c) make the prepared wings re-traceable to the specimens they have been taken from d) secure reproducibility of the imaging even decades after preparation (i.e., easy cleaning of the wing specimens prior to imaging), and to e) allow reproduction of the protocol with minimal financial resources. In all these aspects, there is undeniably room for improvement.

There is increasing evidence for the impor- tance of events that govern and influence the interaction between the transformed cell and its host being ultimately responsible for the establishment of the cancer phenotype. To derive an animal model that will allow us to define some of these phenomena at the molecular level, we have chosen to induce the expression of a viral oncogene in all tissue types, with the hope of identifying sites that are more susceptible to malignant transformation. When the gene for simian virus 40 large tumor antigen (T antigen) was placed under the control of a major histocompatibility complex class I gene enhancer, the resulting transgenic mice not only developed choroid plexus papillomas, as seen with wild-type simian virus 40, but also lymphoid hyperplasia and multiple endocrine neoplasias. The develop- ment of lymphoid hyperplasia was preceded by an elevated level of expression of T antigen in these tissues at an early age. Surprisingly, the striking thymic hyperplasia has not been observed to progress toward malignancy. The multiple endo- crine neoplasias developed later in life and involved the pancreas, pituitary, thyroid, adrenals, and testes. While not preceded by an elevated level of expression of T antigen, once endocrine tumors appeared they quickly progressed toward malignant growth. Although other tissues also exhibited a basal level of expression of the viral oncogene similar to that detected in endocrine tissues, they rarely developed tumors. This transgenic mouse model seems particularly suitable for a molecular understanding of events responsible for certain tissue types being so much more susceptible to neoplastic conversion, with others being so refractory.

The perivascular microenvironment helps in maintaining stem cells in many tissues. We sought to determine whether there is a perivascular niche for hair follicle stem cells. The association of vessels and follicle progenitor cells began by embryonic day 14.5, when nascent hair placodes had blood vessels approaching them. By birth, a vascular annulus stereotypically surrounded the keratin 15 negative (K15-) stem cells in the upper bulge and remained associated with the K15- upper bulge throughout the hair cycle. The angiogenic factor Egfl6 was expressed by the K15- bulge and was localized adjacent to the vascular annulus, which comprised post-capillary venules. Although denervation altered the phenotype of upper bulge stem cells, the vascular annulus persisted in surgically denervated mouse skin. The importance of the perivascular niche was further suggested by the fact that vascular annuli formed around the upper bulge of de novo-reconstituted hair follicles before their innervation. Together, these findings demonstrate that the upper bulge is associated with a perivascular niche during the establishment and maintenance of this specialized region of hair follicle stem cells.

Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200(+) CD45(-) BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200(+) CD45(-) BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200(+) CD45(-) cells, representing ~1,500-fold enrichment. CD200(-) CD45(-) BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC.

Primary human squamous cell carcinomas (SCCas) are heterogeneous invasive tumors with proliferating outer layers and inner differentiating cell masses. To determine if tumor-initiating cells (TICs) are present in SCCas, we utilized newly developed reliable in vitro and in vivo xenograft assays that propagate human SCCas, and demonstrated that a small subset of SCCa cells (∼1%) expressing Prominin-1 (CD133) in the outer layers of SCCas were highly enriched for TICs (∼1/400) compared with unsorted SCCa cells (TICs ∼1/106). Xenografts of CD133+ SCCas recreated the original SCCa tumor histology and organizational hierarchy, whereas CD133- cells did not, and only CD133+ cells demonstrated the capacity for self-renewal in serial transplantation studies. We present a model of human SCCas in which tumor projections expand with outer leading edges that contain CD133+ TICs. Successful cancer treatment will likely require that the TICs identified in cancers be targeted therapeutically. The demonstration that TICs are present in SCCas and are enriched in a CD133- expressing subpopulation has not been, to our knowledge, previously reported.

Epithelial cancers are the most common malignancies and the greatest cause of cancer mortality worldwide. The incidence of keratinocyte-derived (non-melanoma) skin cancers is increasing rapidly. Despite access to abundant tumor tissue and ease of observation, acceptance of non-melanoma skin cancers as model carcinomas has been hindered by the lack of a reliable xenograft model. Herein we describe conditions that allow routine xeno-engraftment of primary human squamous cell carcinoma (SCCa) cells. Tumor development required creation of an appropriate stromal bed before xenografting tumor tissue onto the backs of athymic nude mice. We also demonstrate that the stromal bed must be "humanized" if primary human SCCa is to be propagated from cell suspensions. SCCa xenografts recapitulated the histological grade and phenotype of the original tumors with considerable fidelity, even after serial passage, irrespective of the histological grade of the primary human SCCa. This model, which to our knowledge is previously unreported, can be used for drug testing, as well as for studies that are relevant to the biology of primary human SCCa and other epithelial cancers.

Appendix S1 Supplementary materials and methods, Tables S1–S6 and Figures S1–S5. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The efficient culture of stem cells from epithelial tissues such as skin and corneas is important for both experimental studies and clinical applications of tissue engineering. We now demonstrate that treatment of human-skin-derived keratinocytes with a Rho-associated protein kinase inhibitor Y-27632 for the initial 6 days of primary culture can increase the number of keratinocytes that possess stem cell properties to form colonies during in vitro culture of freshly isolated cells and subsequent passage (50-fold). Further, we show that Y-27632 treatment can increase the total number of prostate epithelial cells derived from human prostate specimens. Therefore, the use of Y-27632 during primary cultures offers a simple and effective way to prepare a large number of epithelial stem cells from various human epithelial tissues.

The use of bioengineered human skin as a bioreactor to deliver therapeutic factors has a number of advantages including accessibility that allows manipulation and monitoring of genetically modified cells. We demonstrate a skin gene therapy approach that can regulate blood pressure and treat systemic hypertension by expressing atrial natriuretic peptide (ANP), a hormone able to decrease blood pressure, in bioengineered human skin equivalents (HSE). Additionally, the expression of a selectable marker gene, multidrug resistance (MDR) type 1, is linked to ANP expression on a bicistronic vector and was coexpressed in the human keratinocytes and fibroblasts of the HSE that were grafted on to immunocompromised mice. Topical treatments of grafted HSE with the antimitotic agent colchicine select for keratinocyte progenitors that express both MDR and ANP. Significant plasma levels of human ANP were detected in mice grafted with HSE expressing ANP from either keratinocytes or fibroblasts, and topical selection of grafted HSE resulted in persistent high levels of ANP expression in vivo. Mice with elevated plasma levels of human ANP showed lower renin levels and, correspondingly, had lower systemic blood pressure than controls. Furthermore, mice with HSE grafts expressing human ANP did not develop elevated blood pressure when fed a high-salt diet. These findings illustrate the potential of this human skin gene therapy approach to deliver therapeutic molecules systemically for long-term treatment of diverse diseases.

Abstract In the cancer stem cell hypothesis, tumors are initiated and maintained by a small subset of tumor initiating cells (TIC) that possess stem cell properties of self-renewal and long-term repopulation. However, TIC have not been well characterized in human prostate cancer with previous reports describing enrichment of TIC in prostate cancer based on expression of CD133 and a2b1 integrin cell surface markers. A significant problem has been the relative lack of cell surface markers that could be used to define subsets of prostate cancer cells. Rather than rely on cell surface markers, we wanted to identify subsets based on the presence or absence of the activated Wnt, Notch, and Sonic Hedgehog (Hh) signaling pathways that have been associated with stem cell maintenance and self-renewal, and also implicated in several cancers. We hypothesized that these signaling pathways may be more highly activated in TIC and may allow for the identification and enrichment of human prostate cancer TIC. To detect these signaling pathways, we generated lentiviral vectors with reporters downstream of specific enhancer-promoter sequences that can detect activated Wnt, Notch, or Hh signaling. Since established in vivo models that can recapitulate human prostate cancer from freshly isolated cancer cells do not exist, we developed in vitro clonogenic sphere formation assays, both in a 3-dimensional matrix and on non-adherent plates, to detect prostate cancer TIC. We first established the prostate cancer cells origin of the cells by demonstrating their invasive capacity in a matrigel cell invasion assay; their lack of CD10 and CD13 expression; and their ability to form non-adherent spheres when compared to normal prostate cells. The transduction efficiency of the prostate cancer cells by the lentiviral vectors was approximately 70%, with activated Wnt, Notch, and Hh signaling detected in 39%, 61%, and 34% of the transduced cells, respectively. We found that human prostate cancer cells with activated Wnt signaling produced approximately 10-fold more clonogenic spheres than Wnt-negative cells indicating enrichment of prostate cancer TIC within the Wnt-positive fraction. In contrast, prostate cancer cells lacking activated Notch signaling formed 2-fold more clonogenic spheres than cells with activated Notch signaling. The presence or absence of activated Hh signaling did not affect clonogenic sphere formation. When prostate cancer cells were transduced with both Wnt and Notch lentiviral vectors, the Wnt-postive/Notch-negative populations gave rise to 6-fold more clonogenic spheres than the Wnt-positive/Notch-positive cells. Finally, CD133+ and CD117+ prostate cancer cells were only detected within the Wnt-positive and Notch-negative populations. These results indicate that prostate cancer TIC are enriched within prostate cancer cells that have activated Wnt signaling but lack activated Notch signaling and suggest that prostate cancer subsets of interest can be identified based on the presence of activated signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-266.

For gene therapy purposes, the skin is an attractive organ to target for systemic delivery of therapeutic proteins to treat systemic diseases, skin diseases, or skin cancer. To achieve long-term stable expression of a therapeutic gene in keratinocytes (KC), we have developed an approach using a bicistronic retroviral vector expressing the desired therapeutic gene linked to a selectable marker (multidrug resistant gene, MDR) that is then introduced into KC and fibroblasts (FB) to create genetically modified human skin equivalent (HSE). After grafting the HSE onto immunocompromised mice, topical colchicine treatment is used to select and enrich for genetically modified keratinocyte stem cells (KSC) that express MDR and are resistant to colchicine’s antimitotic effects. Both the apparatus for topical colchicine delivery and the colchicine doses have been optimized for application to human skin. This approach can be validated by systemic delivery of therapeutic factors such as erythropoietin and the antihypertensive atrial natriuretic peptide.

keratinocyte stem cells Keratinocyte stem cells (KSC) maintain epidermal structure, and are an attractive target of regenerative medicine in human skin and hair disorders. In human and mouse hair follicles, KSC are localized in a specific portion of hair follicles called the “bulge” (Stenn and Cotsarelis, 2005Stenn K.S. Cotsarelis G. Bioengineering the hair follicle: fringe benefits of stem cell technology.Curr Opin Biotechnol. 2005; 16: 493-497Crossref PubMed Scopus (89) Google Scholar; Fuchs, 2007Fuchs E. Scratching the surface of skin development.Nature. 2007; 445: 834-842Crossref PubMed Scopus (582) Google Scholar). Previously, CD200 expression has been described in the outer root sheath of murine hair follicles (Rosenblum et al., 2004Rosenblum M.D. Olasz E.B. Yancey K.B. Woodliff J.E. Lazarova Z. Gerber K.A. et al.Expression of CD200 on epithelial cells of the murine hair follicle: a role in tissue-specific immune tolerance?.J Invest Dermatol. 2004; 123: 880-887Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). We recently found that CD200 is preferentially expressed by human KSC in the bulge, and used anti-CD200 mAb to successfully purify KSC from human scalp (Ohyama et al., 2006Ohyama M. Terunuma A. Tock C.L. Radonovich M.F. Pise-Masison C.A. Hopping S.B. et al.Characterization and isolation of stem cell-enriched human hair follicle bulge cells.J Clin Invest. 2006; 116: 249-260Crossref PubMed Scopus (490) Google Scholar). In murine models, evidence exists that CD200 may suppress immune responses to protect keratinocytes in hair follicles from destruction by inflammation through the interaction with the CD200 receptor on immunologically active cells (Gorczynski et al., 1999Gorczynski R.M. Cattral M.S. Chen Z. Hu J. Lei J. Min W.P. et al.An immunoadhesin incorporating the molecule OX-2 is a potent immunosuppressant that prolongs allo- and xenograft survival.J Immunol. 1999; 163: 1654-1660PubMed Google Scholar; Rosenblum et al., 2004Rosenblum M.D. Olasz E.B. Yancey K.B. Woodliff J.E. Lazarova Z. Gerber K.A. et al.Expression of CD200 on epithelial cells of the murine hair follicle: a role in tissue-specific immune tolerance?.J Invest Dermatol. 2004; 123: 880-887Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Rosenblum et al., 2005Rosenblum M.D. Woodliff J.E. Madsen N.A. McOlash L.J. Keller M.R. Truitt R.L. Characterization of CD 200-receptor expression in the murine epidermis.J Invest Dermatol. 2005; 125: 1130-1138Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, Rosenblum et al., 2006Rosenblum M.D. Yancey K.B. Olasz E.B. Truitt R.L. CD200, a “no danger” signal for hair follicles.J Dermatol Sci. 2006; 41: 165-174Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar; Cherwinski et al., 2005Cherwinski H.M. Murphy C.A. Joyce B.L. Bigler M.E. Song Y.S. Zurawski S.M. et al.The CD200 receptor is a novel and potent regulator of murine and human mast cell function.J Immunol. 2005; 174: 1348-1356Crossref PubMed Scopus (85) Google Scholar; Yu et al., 2005Yu K. Chen Z. Wang S. Gorczynski R. Decreased alloreactivity using donor cells from mice expressing a CD200 transgene under control of a tetracycline-inducible promoter.Transplantation. 2005; 80: 394-401Crossref PubMed Scopus (19) Google Scholar). We wanted to determine if CD200 is present in human keratinocytes from neonatal foreskins, which lack hair follicles, and to determine if CD200 could identify keratinocytes with KSC behavior. If so, foreskins would allow us to easily harvest a large number of CD200(+) KSC by enzymatic treatments and gentle mechanical dissociation. Epidermal cells were prepared from human neonatal foreskin specimens, which were collected with informed consent of parents or guardians, and institutional approval in adherence to the Declaration of Helsinki Principles. Flow cytometry can distinguish three cellular populations in human skin epidermis: CD45(+) lymphocytes and Langerhans cells (1%), c-kit(+) melanocytes (5%), and the remaining CD45(−) c-kit(−) cells (94%), mostly keratinocytes (Terunuma et al., 2003Terunuma A. Jackson K.L. Kapoor V. Telford W.G. Vogel J.C. Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells.J Invest Dermatol. 2003; 121: 1095-1103Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). A fraction of melanocytes was moderately positive for CD200 (Figure 1a), whereas CD45(+) cells did not express CD200 (Figure 1b). A small population (3.8±0.8%; mean±SEM; n=19) of CD45(−) c-kit(−) cells expressed higher levels of CD200 (Figure 1a and b). CD200(+) CD45(−) c-kit(−) cells were positive for keratin 5 and negative for keratin 10 (Figure 1c), suggesting that they are basal keratinocytes. Forward scatter characteristics indicated that CD200(+) keratinocytes are not necessarily small (Figure 1d). When human epidermal cells are cultured, CD200(+) cells disappeared after 2 weeks (Figure S1), consistent with the data on cultured keratinocytes by others (Rosenblum et al., 2004Rosenblum M.D. Olasz E.B. Yancey K.B. Woodliff J.E. Lazarova Z. Gerber K.A. et al.Expression of CD200 on epithelial cells of the murine hair follicle: a role in tissue-specific immune tolerance?.J Invest Dermatol. 2004; 123: 880-887Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Relative to the total keratinocyte population, CD200(+) keratinocytes were enriched for α6 integrin-bright CD71-dim cells (Figure 1e), which are known to be enriched for KSC (Li et al., 1998Li A. Simmons P.J. Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.Proc Natl Acad Sci USA. 1998; 95: 3902-3907Crossref PubMed Scopus (412) Google Scholar; Tani et al., 2000Tani H. Morris R.J. Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype.Proc Natl Acad Sci USA. 2000; 97: 10960-10965Crossref PubMed Scopus (334) Google Scholar). Download .pdf (1.23 MB) Help with pdf files Supplementary Figure S1CD200(+) keratinocytes disappear from primary culture in serum-free medium. Primary human keratinocytes from neonatal foreskin tissue were cultured in keratinocyte serum-free medium supplemented with recombinant EGF and bovine pituitary extract (Invitrogen, Carlsbad, CA). On days 0, 5, 8 and 14, cells were harvested and analyzed for their CD200 expression by flow cytometry However, as not all α6-bright CD71-dim cells are KSC, we first assessed the stem cell ability of CD200(+) keratinocytes using in vitro colony formation assays. As expected, α6-bright CD71-dim keratinocytes showed higher colony-forming efficiency than the total keratinocyte control (Figure 2a and b). In contrast, CD200(+) keratinocytes performed no better than the total keratinocyte control, indicating that the CD200(+) keratinocytes in foreskins, unlike their counterparts in hair follicles, do not behave as KSC in vitro. The long-term repopulation potential was tested in an in vivo competitive repopulation assay, in which KSC are tracked through multiple rounds of tissue turnover using an intrinsic marker, HLA-A2 (Terunuma et al., 2007Terunuma A. Kapoor V. Yee C. Telford W.G. Udey M.C. Vogel J.C. Stem cell activity of human side population and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay.Stem Cells. 2007; 25: 664-669PubMed Google Scholar). Keratinocytes were collected from HLA-A2(+) donors, mixed with HLA-A2(−) cultured keratinocytes, and used to make human skin equivalents, which were grafted and maintained on the backs of nude mice for 4–5 months, during which the epidermis turns over at least four times. After multiple turnovers, KSC will survive and expand, whereas non-stem cells will be lost. Therefore, the percentage of HLA-A2(+) keratinocytes in this competitive repopulation assay (Figure 2c) correlates with the number of HLA-A2(+) KSC in the original preparation. In two experiments (Figure 2d), CD200(+) keratinocytes lacked a competitive advantage over either the total control or α6-bright CD71-dim keratinocytes, indicating that the CD200(+) population is not enriched for KSC. Thus, results of in vitro and in vivo assays consistently demonstrated that CD200(+) keratinocytes in neonatal foreskin, unlike their counterparts in hair follicles, do not behave as KSC. Although CD200(+) keratinocytes in hair follicles behave as KSC in colony formation assays (Ohyama et al., 2006Ohyama M. Terunuma A. Tock C.L. Radonovich M.F. Pise-Masison C.A. Hopping S.B. et al.Characterization and isolation of stem cell-enriched human hair follicle bulge cells.J Clin Invest. 2006; 116: 249-260Crossref PubMed Scopus (490) Google Scholar), CD200(+) keratinocytes from foreskins lacking hair follicles behave differently in colony formation assays and do not demonstrate KSC behavior (this study). CD200(+) KSC in hair follicles contribute to the repair of wounded non-follicular epidermis but are not responsible for its homeostasis, which is independently maintained by interfollicular KSC (Ito et al., 2005Ito M. Liu Y. Yang Z. Nguyen J. Liang F. Morris R.J. et al.Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.Nat Med. 2005; 11: 1351-1354Crossref PubMed Scopus (929) Google Scholar; Kaur, 2006Kaur P. Interfollicular epidermal stem cells: identification, challenges, potential.J Invest Dermatol. 2006; 126: 1450-1458Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar; Watt et al., 2006Watt F.M. Lo Celso C. Silva-Vargas V. Epidermal stem cells: an update.Curr Opin Genet Dev. 2006; 16: 518-524Crossref PubMed Scopus (160) Google Scholar). Consistent with the notion that KSC in hair follicles and interfollicular epidermis are distinct, CD200 allowed the identification of KSC in human hair follicles but not in neonatal foreskin. As shown in current and previous studies (Terunuma et al., 2007Terunuma A. Kapoor V. Yee C. Telford W.G. Udey M.C. Vogel J.C. Stem cell activity of human side population and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay.Stem Cells. 2007; 25: 664-669PubMed Google Scholar), the combination of α6 integrin and CD71 allows the two- to four-fold enrichment for KSC from human foreskin tissues. This is consistent with the results observed in human adult breast skin (Webb et al., 2004Webb A. Li A. Kaur P. Location and phenotype of human adult keratinocyte stem cells of the skin.Differentiation. 2004; 72: 387-395Crossref PubMed Scopus (189) Google Scholar) and in mouse back skin, where 11% (53–62 in 500 cells) of the total keratinocytes and 19% (78–113 in 500 cells) of α6-bright CD71-dim keratinocytes were detected as label-retaining KSC (Tani et al., 2000Tani H. Morris R.J. Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype.Proc Natl Acad Sci USA. 2000; 97: 10960-10965Crossref PubMed Scopus (334) Google Scholar). To achieve further enrichment for KSC, identification of additional KSC markers is required. The discovery of KSC markers as well as the development and usage of in vivo stem cell assays mimicking human hair follicles and interfollicular epidermis, as shown here, will enhance the progress of regenerative medicine for skin and hair disorders. The authors state no conflict of interest. This research was supported by the Intramural Research Program of the NIH, NCI, CCR. Figure S1. CD200(+) keratinocytes disappear from primary culture in serum-free medium.

The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP-KCs) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow-derived side population (SP) hematopoietic stem cells, have been identified in human skin, but their potential to function as keratinocyte stem cells (KSCs) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of beta1 and alpha6 integrins and are distinct from SP-KCs, which express low levels of integrins, may be enriched for KSCs based on reported results of in vitro cell culture assays. When in vitro assays were used to measure total cell output of human SP-KCs and integrin-bright keratinocytes, we could not document their superior long-term proliferative activity versus unfractionated keratinocytes. To further assess the KSC characteristics in SP-KCs and integrin-bright keratinocytes, we used an in vivo competitive repopulation assay in which bioengineered human epidermis containing competing keratinocyte populations with different human major histocompatibility (MHC) class I antigens were grafted onto immunocompromised mice, and the intrinsic MHC class I antigens are used to quantify expansion of competing populations. In these in vivo studies, human SP-KCs showed little competitive expansion in vivo and were not enriched for KSCs. In contrast, keratinocytes expressing elevated levels of alpha6 integrin and low levels of CD71 (alpha6-bright/CD71-dim) expanded over 200-fold during the 33-week in vivo study. These results definitively demonstrate that human alpha6-bright/CD71-dim keratinocytes are enriched with KSCs, whereas SP-KCs are not.

Bullous pemphigoid antigen 2 (BPAG2) is targeted by autoantibodies in patients with bullous pemphigoid (BP), and absent in patients with one type of epidermolysis bullosa (OMIM #226650). A keratin 14 promoter construct was used to produce transgenic (Tg) mice appropriately expressing human BPAG2 (hBPAG2) in murine epidermal basement membrane (BM). Grafts of Tg skin placed on gender-matched, syngeneic wild type (Wt) or major histocompatibility complex I (MHC I)-/- mice elicited IgG that bound human epidermal BM and BPAG2. Production of such IgG in grafted mice was prompt (detectable within 16+/-2 days), robust (titer > or = 1,280), durable (present > or = 380 days), and correlated with the involution and loss of Tg skin grafts. MHC II-/- mice grafted with Tg skin did not develop anti-hBPAG2 IgG or graft loss indicating that MHC II:CD4+ T cell interactions were crucial for these responses. Tg skin grafts on Wt mice developed neutrophil-rich infiltrates, dermal edema, subepidermal blisters, and deposits of immunoreactants in epidermal BM. This model shows fidelity to alterations seen in patients with BP, has relevance to immune responses that may arise in patients with epidermolysis bullosa following BPAG2 gene replacement, and can be used to identify interventions that may block production of IgG against proteins in epidermal BM.

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin alpha2beta1(hi) and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 microg/ml insulin (DMEM+10% FBS+Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.