Biomedical subjects

Amy Li

Publications and source records attributed to Amy Li.

8 recordsLinked to original sources

PubMed2006

Side population in adult murine epidermis exhibits phenotypic and functional characteristics of keratinocyte stem cells.

Based on functional studies in the bone marrow, it has been suggested that the ability to efflux Hoechst 33342 may represent a universal stem cell trait. In this phenotypic and functional characterization of the Hoechst side population (SP) in adult murine epidermis, we demonstrate that these cells are a rare subset of the keratinocyte stem cell-enriched alpha(6)(bri)CD71(dim) fraction comprising SSC(low)/K14(+)/CD34(-)/Oil red O(-)/c-kit(-)/CD45(-) keratinocytes. Epidermal SPs have the smallest cell and nuclear size but exhibit the highest nuclear-to-cytoplasmic ratio of any fraction examined, consistent with a primitive cell type. Although SPs demonstrated poor cumulative in vitro proliferative output, they exhibited sustained epidermal tissue-regenerative activity in vivo compared with unfractionated and non-SP cells. Collectively, these results indicate that the epidermal SP contains the most potent keratinocyte stem cell population in skin epithelium.

Animals↗

PubMed2005

FACS enrichment of human keratinocyte stem cells.

Recent work from our laboratory has led to the development and validation of fluorescence-activated cell sorting (FACS)-based techniques to prospectively isolate viable keratinocyte stem cells from both human and murine skin. Here we describe a step-by-step method to apply our technique to isolate epidermal keratinocytes from skin tissue, process them for immunofluorescent staining for cell surface markers, and subject them to fluorescence-activated cell sorting to obtain the stem, transient amplifying, and early differentiating keratinocyte fractions. These viable cells can then be placed into culture for further analysis or directly into keratinocyte assays, such as organotypic cultures or in vivo transplantation. This method will be useful for the complete biological characterization of keratinocyte progenitors with respect to wound healing, carcinogenesis, and therapeutic manipulation.

Animals↗

PubMed2004

Keratinocyte stem cell assays: an evolving science.

Although the existence of epithelial stem cells in the skin has been known for some decades from cell kinetic studies performed in vivo, attempts to prospectively isolate these cells for further biological characterization have been made possible relatively recently facilitated by the availability of antibodies that detect cell surface markers on epidermal cells. Elegant gene marking studies in vivo have provided confirmation of the patterns of epithelial tissue replacement predicted by classical cell turnover studies. But, the identification of candidate epidermal stem cells ex vivo remains an area of great controversy, requiring the re-evaluation of current experimental approaches that rely of necessity on predicted epidermal stem cell behavior in culture. Here we review the diverse experimental approaches utilized to identify keratinocyte stem cells and their underlying assumptions. We conclude that hair follicles and interfollicular epidermis each have their own self-renewing stem cell populations, contributing to distinct regions of the epithelium during homeostasis, although this is perturbed during wound healing. The need for the development of more rigorous assays for stem cell activity is highlighted given our recent observations using current assays and the discovery of new surface markers that identify putative epidermal stem cells.

Animals↗

PubMed2004

Location and phenotype of human adult keratinocyte stem cells of the skin.

The location and identity of interfollicular epidermal stem cells of adult human skin remain undefined. Based on our previous work in both adult murine and neonatal human foreskin, we demonstrate that cell surface levels of the alpha6 integrin and the transferrin receptor (CD71) are valid markers for resolving a putative stem cell, transit amplifying and differentiating compartment in adult human skin by flow cytometry. Specifically, epidermal cells expressing high levels of alpha6 integrin and low levels of the transferrin receptor CD71 (phenotype alpha6 (bri)CD71(dim)) exhibit several stem cell characteristics, comprising a minor population (2%-5%) of the K14(bri) fraction, enriched for quiescent and small blast-like cells with high clonogenic capacity, lacking the differentiation marker K10. Conversely, the majority of K14(bri) K10(neg) epidermal cells express high levels of CD71 (phenotype alpha6 (bri)CD71(bri)), and represent the actively cycling fraction of keratinocytes displaying greater cell size due to an increase in cytoplasmic area, consistent with their being transient amplifying cells. The alpha6 (bri)CD71(bri) population exhibited intermediate clonogenic capacity. A third population of K14(dim) but K10 positive epidermal cells could be identified by their low levels of alpha6 integrin expression (i.e. alpha6 (dim) cells), representing the differentiation compartment; predictably, this subpopulation exhibited poor clonogenic efficiency. Flow cytometric analysis for the hair follicle bulge region (stem cell) marker K15 revealed preferential expression of this keratin in alpha6 (bri) cells (i.e., both stem and transient amplifying fractions), but not the alpha6 (dim) population. Given that K15 positive cells could only be detected in the deep rete ridges of adult skin in situ, we conclude that stem and transient amplifying cells reside in this location, while differentiating (K15 negative) cells are found in the shallow rete ridges.

Adult↗

PubMed2004

Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny.

Given our recent discovery that it is possible to separate human epidermal stem cells of the skin from their more committed progeny (i.e., transit-amplifying cells and early differentiating cells) using FACS techniques, we sought to determine the comparative tissue regeneration ability of these keratinocyte progenitors. We demonstrate that the ability to regenerate a fully stratified epidermis with appropriate spatial and temporal expression of differentiation markers in a short-term in vitro organotypic culture system is an intrinsic characteristic of both epidermal stem and transit-amplifying cells, although the stem cell fraction is most capable of achieving homeostasis. Early differentiating keratinocytes exhibited limited short-term tissue regeneration under specific experimental conditions in this assay, although significant improvement was obtained by manipulating microenvironmental factors, that is, coculture with minimally passaged dermal cells or exogenous supply of the ECM protein laminin-10/11. Importantly, transplantation of all classes of keratinocyte progenitors into an in vivo setting demonstrated that tissue regeneration can be elicited from stem, transit-amplifying, and early differentiating keratinocytes for up to 10 weeks. These data illustrate that significant proliferative and tissue-regenerative capacity resides not only in keratinocyte stem cells as expected, but also in their more committed progeny, including early differentiating cells.

Cell Differentiation↗

PubMed2003

Enhanced immunogenicity to Mycobacterium tuberculosis by vaccination with an alphavirus plasmid replicon expressing antigen 85A.

The immunogenicity of a plasmid DNA vaccine incorporating Sindbis virus RNA replicase functions (pSINCP) and expressing antigen 85A (Ag85A) from Mycobacterium tuberculosis was compared with a conventional plasmid DNA vector encoding Ag85A. pSINCP-85A was highly immunogenic in mice and gave enhanced long-term protection against M. tuberculosis compared with the conventional vector.

Acyltransferases↗

PubMed2002

DNA vaccine combinations expressing either tissue plasminogen activator signal sequence fusion proteins or ubiquitin-conjugated antigens induce sustained protective immunity in a mouse model of pulmonary tuberculosis.

DNA vaccination has emerged as a powerful approach in the search for a more efficacious vaccine against tuberculosis. In this study, we evaluated the effectiveness of immunizing with combinations of 10 different tuberculosis DNA vaccines that expressed mycobacterial proteins fused at the N terminus to eukaryotic intracellular targeting sequences. In one vaccine combination, the genes were fused to the tissue plasminogen activator signal sequence (TPA), while in a second combination the same 10 genes were expressed as ubiquitin (Ub)-conjugated proteins. In ex vivo studies in which the secretion of gamma interferon was measured, cellular immune responses were detected in mice vaccinated with either the TPA DNA vaccine combination or the Ub DNA vaccine combination at 7 and 14 days following a low-dose Mycobacterium tuberculosis challenge. Moreover, mice vaccinated with the TPA combination, the Ub combination, and Mycobacterium bovis BCG were able to limit the growth of tubercle bacilli in the lung and spleen after a virulent tuberculous aerosol challenge. Histopathological analyses also showed that mice immunized with the DNA vaccine combinations had substantially improved postinfection lung pathology relative to the naïve controls. Finally, in three different long-term experiments, the survival periods following aerogenic challenge were extended as much as sevenfold for vaccinated mice compared to naïve controls. Interestingly, in all three experiments, no significant differences were detected in the mean times to death for mice immunized with the TPA combination or the Ub combination relative to the BCG controls. In conclusion, these studies demonstrate the effectiveness of immunization with DNA vaccine combinations against tuberculosis and suggest that further testing of these plasmid cocktails is warranted.

Adjuvants, Immunologic↗

PubMed2002

DNA immunization in a mouse model of latent tuberculosis: effect of DNA vaccination on reactivation of disease and on reinfection with a secondary challenge.

Individuals who are latently infected with Mycobacterium tuberculosis can develop active disease via either endogenous reactivation of the latent bacilli or exogenous reinfection with a second mycobacterial strain. In this study, we investigated whether immunization with a tuberculosis DNA vaccine cocktail that induces significant protective responses in mice could prevent reactivation of disease in a murine latent-tuberculosis model. In addition, we assessed whether DNA vaccination could retard the growth of a secondary aerogenic infection with M. tuberculosis (exogenous reinfection) in latently infected mice. In the reactivation studies, administration of the DNA vaccine combination did not prevent recrudescence of the latent infection after injection of dexamethasone. Moreover, for the reinfection experiments, only a modest decrease in the growth of a secondary M. tuberculosis challenge in DNA-vaccinated animals, compared to controls, was observed 14 and 28 days after the reinfection of previously exposed mice. Interestingly, although proliferation of the secondary challenge was reduced significantly in a nonvaccinated chronic-infection group relative to the naïve controls, the number of bacilli still increased by 500-fold 1 month after the secondary challenge in mice with active tuberculosis. These results indicate that novel immunotherapeutic approaches will be required to prevent reactivation of infection or reinfection of individuals with latent tuberculosis.

Animals↗