Jonathan C. Vogel

Characterization and isolation of stem cell–enriched human hair follicle bulge cells

The human hair follicle bulge is an important niche for keratinocyte stem cells (KSCs). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell-surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSCs. In this study, we determined the distribution of label-retaining cells to define the human anagen bulge. Using navigated laser capture microdissection, bulge cells and outer root sheath cells from other follicle regions were obtained and analyzed with cDNA microarrays. Gene transcripts encoding inhibitors of WNT and activin/bone morphogenic protein signaling were overrepresented in the bulge, while genes responsible for cell proliferation were underrepresented, consistent with the existence of quiescent noncycling KSCs in anagen follicles. Positive markers for bulge cells included CD200, PHLDA1, follistatin, and frizzled homolog 1, while CD24, CD34, CD71, and CD146 were preferentially expressed by non-bulge keratinocytes. Importantly, CD200+ cells (CD200hiCD24loCD34loCD71loCD146lo) obtained from hair follicle suspensions demonstrated high colony-forming efficiency in clonogenic assays, indicating successful enrichment of living human bulge stem cells. The stem cell behavior of enriched bulge cells and their utility for gene therapy and hair regeneration will need to be assessed in in vivo assays.

Expression of naked DNA in human, pig, and mouse skin.

The insertion and expression of genes in the epidermis may have a variety of therapeutic uses, including the treatment of skin diseases. Here we show that when both human skin organ cultures and human skin grafts on immunocompromised mice are injected with naked DNA, the DNA is taken-up and genes are expressed in the epidermis in a manner similar to both pig skin injected in vivo and injected pig skin organ cultures. In contrast, DNA injected into mouse skin is expressed not just in the epidermis, but also in the dermis and underlying fat and muscle tissue, and is expressed at lower levels. These findings suggest that genes can be expressed in human skin, after injection of naked DNA, and indicate that pig skin is an appropriate model for the study of DNA uptake and gene expression in human skin. The organ cultures of human and pig skin may be useful in understanding how naked DNA is internalized and expressed after in vivo injections. Additionally, skin obtained from patients with skin disease may be studied as skin grafts and organ cultures to help optimize genetic approaches for the treatment of skin diseases prior to clinical trials, by determining if the injected gene can provide a therapeutic benefit.

Bacterial DNA and CpG-containing oligodeoxynucleotides activate cutaneous dendritic cells and induce IL-12 production : Implications for the augmentation of Th1 responses

Background: Unmethylated CpG sequences in bacterial DNA act as adjuvants selectively inducing Th1 predominant immune responses during genetic vaccination or when used in conjunction with protein Ag. The precise mechanism of this adjuvant effect is unknown. Because dendritic cells (DC) are thought to be crucially involved in T cell priming and Th1/Th2 education during vaccination via skin, we characterized the effects of bacterial DNA and CpG–containing oligodeoxynucleotides (CpG ODN) on cutaneous DC. Methods and Results: Stimulation with CpG ODN 1826 (6 μg/ml) induced activation of immature Langerhans cell (LC)–like DC as determined by an increased expression of MHC class II and costimulatory molecules, loss of E–cadherin–mediated adhesion and increased ability to stimulate allogeneic T cells. Composition–matched control ODN 1911 lacking CpG sequences at equal concentrations was without effect. In comparison to LPS and ODN 1911, CpG ODN 1826 selectively stimulated DC to release large amounts of IL–12 (p40) and little IL–6 or TNF–α within 18 h and detectable levels of IL–12 p70 within 72 h. Stimulation with Escherichia coli DNA, but not calf thymus DNA, similarly induced DC maturation and IL–12 p40 production. Injection of CpG ODN into murine dermis induced enhanced expression of MHC class II and CD86 by LC in the overlying epidermis and intracytoplasmic IL–12 p40 accumulation in a subpopulation of activated LC. Conclusion: Bacterial DNA and CpG ODN stimulate DC in vitro and in vivo and may preferentially elicit Th1–predominant immune responses because they can activate and mobilize DC, inducing them to produce IL–12.

Nonviral Skin Gene Therapy

Nonviral skin gene therapy is an effective method to directly deliver and transiently express genes in the skin. Several different nonviral delivery methods have been successfully used and are analyzed here for their efficiency and efficacy in achieving specific therapeutic applications. For one important and frequently used application of nonviral skin gene therapy, genetic immunization, the types of resulting immune responses (Th1 versus Th2) will depend on which delivery method is used. In addition, we discuss the contributions of DNA as an immunostimulatory adjuvant in genetic immunization and how activation of skin dendritic cells and induction of IL-12 expression are mechanistically important in this process. Nonviral skin gene therapy has also been successfully used to enhance tumor regression in animal models, frequently by inducing a specific immune response against the tumor. In the future, nonviral skin gene therapy may be successfully used for the treatment of additional skin diseases if genes can be selectively delivered and expressed in specific skin cells, and if increased level and duration of gene expression can be achieved.

HOX decoy peptide enhances the ex vivo expansion of human umbilical cord blood CD34+ hematopoietic stem cells/hematopoietic progenitor cells.

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 β1 and α6 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 α6 integrin and low levels of CD71 (α6‐bright/CD71‐dim) expanded over 200‐fold during the 33‐week in vivo study. These results definitively demonstrate that human α6‐bright/CD71‐dim keratinocytes are enriched with KSCs, whereas SP‐KCs are not.

Immunization through dermal delivery of protein-encoding DNA: a role for migratory dendritic cells.

The early mechanisms by which DNA‐dependent immunization occurs remain poorly understood. We determined whether intradermal injection of a cytomegalovirus (CMV) promoter‐driven plasmid encoding hen egg lysozyme (pCMV  :  HEL) induced sensitization against the encoded protein, and whether cutaneous dendritic cells (DC) were involved in this sensitization. Both humoral and cellular responses to HEL were observed. DC that migrated from skin explant culture 3 days after injection of pCMV  :  HEL DNA contained mRNA encoding HEL. They induced a 3.5 –7‐fold increase in [ 3 H]thymidine incorporation by HEL protein‐primed CD4+ T cells compared to that induced by DC from mice injected with control plasmid. DC emigrating from skin explants recovered from pCMV  :  HEL injected mice also sensitized naive mice after adoptive transfer and induced the generation of CTL. Thus following DNA delivery within the dermis, DC can induce primary and secondary immune responses.

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

ABSTRACT For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.

Absence of donor-derived keratinocyte stem cells in skin tissues cultured from patients after mobilized peripheral blood hematopoietic stem cell transplantation

OBJECTIVE Recent studies suggest that primitive bone marrow-derived cells contribute to regeneration of many tissues, including muscle, endothelium, myocardium, neural tissues, liver, and skin. Conversely, primitive cells resident in muscle and other tissues have been reported to reconstitute hematopoiesis. We investigated the contribution of cells with a primitive hematopoietic phenotype to human epidermal skin formation in recipients of allogeneic mobilized peripheral blood hematopoietic stem cell (HSC) transplantation. PATIENTS AND METHODS Our study population included female patients who had received granulocyte colony-stimulating factor mobilized peripheral blood HSC transplants from male donors for a variety of benign and malignant hematologic disorders at least 6 months before study entry, with a history of skin graft-vs-host disease. Epidermal skin cells (keratinocytes) obtained from punch biopsies of the skin were cultured under conditions specific for growth and expansion of homogenous populations of keratinocytes from keratinocyte stem cells. After multiple passages, DNA was extracted from cultured cells and evaluated by two different polymerase chain reaction (PCR) method for detection of Y chromosome specific sequences. RESULTS Neither sensitive PCR-based technique revealed the presence of male donor-derived keratinocyte stem cells in keratinocytes cultured from skin biopsies of female allogeneic transplantation recipients. CONCLUSIONS We could not confirm the contribution of donor mobilized peripheral blood hematopoietic stem cells to keratinocyte stem cell populations after HSC transplantation. These results cannot explain the presence of donor-derived cells with keratinocyte phenotypic markers in tissue sections of HSC transplant recipients.

Selection of Keratinocytes Transduced with the Multidrug Resistance Gene in an in Vitro Skin Model Presents a Strategy for Enhancing Gene Expression in Vivo

In gene therapy studies, achieving prolonged, high-level gene expression in a significant percentage of cells has been difficult. One solution to enhance expression would be to select for cells expressing both the desired gene and a linked selectable marker gene in a bicistronic vector. As a potential target tissue, the skin is easily accessible for safe topical application of a selecting agent that could lead to significant gene expression in a high percentage of keratinocytes. To test the feasibility of such an approach, a skin raft culture model was developed. Human keratinocytes were transduced with the multidrug resistance (MDR) gene, which confers resistance to a variety of cytostatic and antimitotic compounds, such as colchicine. While growing on acellular dermis, transduced keratinocytes were treated with various doses of colchicine (10-50 ng/ml). Colchicine treatment increased the percentage of keratinocytes expressing MDR to almost 100% in raft cultures, Significantly, keratinocytes in colchicine-treated, MDR-transduced raft cultures were able to proliferate normally and form a stratified, differentiated epidermis. This model suggests that topical selection for MDR-expressing keratinocytes in vivo should be feasible without hampering the biologic integrity of skin. Thus, topical selection leading to enhanced expression of a desired gene, linked to a resistance gene, holds future promise for skin gene therapy.

Identification and characterization of tumor initiating cells in human primary cutaneous squamous cell carcinoma

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