Characterization of the Growth of Chlamydia trachomatis in In Vitro-Generated Stratified Epithelium.

Chlamydia infection targets the mucosal epithelium, where squamous and columnar epithelia can be found. Research on Chlamydia-epithelia interaction has predominantly focused on columnar epithelia, with very little known on how Chlamydia interacts with the squamous epithelium. The stratification and differentiation processes found in the squamous epithelium might influence chlamydial growth and infection dissemination. For this reason, three-dimensional (3D) organotypic stratified squamous epithelial cultures were adapted to mimic the stratified squamous epithelium and chlamydial infection was characterized. Chlamydia trachomatis infection in monolayers and 3D cultures were monitored by immunofluorescence and transmission electron microscopy to evaluate inclusion growth and chlamydial interconversion between elementary and reticulate body. We observed that the stratified epithelium varied in susceptibility to C. trachomatis serovars L2 and D infection. The undifferentiated basal cells were susceptible to infection by both serovars, while the terminally differentiated upper layers were resistant. The differentiating suprabasal cells exhibited different susceptibilities to serovars L2 and D, with the latter unable to establish a successful infection in this layer. Mature elementary body-containing inclusions were much more prevalent in these permissive basal layers, while the uppermost differentiated layers consistently harbored very few reticulate bodies with no elementary bodies, indicative of severely limited bacterial replication and development. For serovar D, the differentiation state of the host cell was a determining factor, as calcium-induced differentiation of cells in a monolayer negatively affected growth of this serovar, in contrast to serovar L2. The apparent completion of the developmental cycle in the basal layers of the 3D cultures correlated with the greater degree of dissemination within and the level of disruption of the stratified epithelium. Our studies indicate that the squamous epithelium is a suboptimal environment for growth, and thus potentially contributing to the protection of the lower genital tract from infection. The relatively more fastidious serovar D exhibited more limited growth than the faster-growing and more invasive L2 strain. However, if given access to the more hospitable basal cell layer, both strains were able to produce mature inclusions, replicate, and complete their developmental cycle.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia.

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

Cutaneous cancer stem cells: beta-catenin strikes again.

Cancer stem cells (CSCs) are a subpopulation of tumor cells that retain properties of tissue-specific stem cells, including the ability to self-renew. In a recent article in Nature, Malanchi et al. (2008) identified a population of CD34(+) cells in epidermal tumors that require beta-catenin signaling to maintain a CSC phenotype.

Characterization of bipotential epidermal progenitors derived from human sebaceous gland: contrasting roles of c-Myc and beta-catenin.

The current belief is that the epidermal sebaceous gland (SG) is maintained by unipotent stem cells that are replenished by multipotent stem cells in the hair follicle (HF) bulge. However, sebocytes can be induced by c-Myc (Myc) activation in interfollicular epidermis (IFE), suggesting the existence of bipotential stem cells. We found that every SZ95 immortalized human sebocyte that underwent clonal growth in culture generated progeny that differentiated into both sebocytes and cells expressing involucrin and cornifin, markers of IFE and HF inner root sheath differentiation. The ability to generate involucrin positive cells was also observed in a new human sebocyte line, Seb-E6E7. SZ95 xenografts differentiated into SG and IFE but not HF. SZ95 cells that expressed involucrin had reduced Myc levels; however, this did not correlate with increased expression of the Myc repressor Blimp1, and Blimp1 expression did not distinguish cells undergoing SG, IFE, or HF differentiation in vivo. Overexpression of Myc stimulated sebocyte differentiation, whereas overexpression of beta-catenin stimulated involucrin and cornifin expression. In transgenic mice simultaneous activation of Myc and beta-catenin revealed mutual antagonism: Myc blocked ectopic HF formation and beta-catenin reduced SG differentiation. Overexpression of the Myc target gene Indian hedgehog did not promote sebocyte differentiation in culture and cyclopamine treatment, while reducing proliferation, did not block Myc induced sebocyte differentiation in vivo. Our studies provide evidence for a bipotential epidermal stem cell population in an in vitro model of human epidermal lineage selection and highlight the importance of Myc as a regulator of sebocyte differentiation.

Bone marrow cells engraft within the epidermis and proliferate in vivo with no evidence of cell fusion.

In adults, bone marrow-derived cells (BMDC) can contribute to the structure of various non-haematopoietic tissues, including skin. However, the physiological importance of these cells is unclear. This study establishes that bone marrow-derived epidermal cells are proliferative and, moreover, demonstrates for the first time that BMDC can localize to a known stem cell niche: the CD34-positive bulge region of mouse hair follicles. In addition, engraftment of bone marrow cells into the epidermis is significantly increased in wounded skin, bone marrow-derived keratinocytes can form colonies in the regenerating epidermis in vivo, and the colony-forming capacity of these cells can be recapitulated in vitro. In some tissues this apparent plasticity is attributed to differentiation, and in others to cell fusion. Evidence is also provided that bone marrow cells form epidermal keratinocytes without undergoing cell fusion. These data suggest a functional role for bone marrow cells in epidermal regeneration, entering known epidermal stem cell niches without heterokaryon formation.

Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis.

Mammalian epidermis is maintained by stem cells that have the ability to self-renew and generate daughter cells that differentiate along the lineages of the hair follicles, interfollicular epidermis and sebaceous gland. As stem cells divide infrequently in adult mouse epidermis, they can be visualised as DNA label-retaining cells (LRC). With whole-mount labelling, we can examine large areas of interfollicular epidermis and many hair follicles simultaneously, enabling us to evaluate stem cell markers and examine the effects of different stimuli on the LRC population. LRC are not confined to the hair follicle, but also lie in sebaceous glands and interfollicular epidermis. LRC reside throughout the permanent region of the hair follicle, where they express keratin 15 and lie in a region of high alpha6beta4 integrin expression. LRC are not significantly depleted by successive hair growth cycles. They can, nevertheless, be stimulated to divide by treatment with phorbol ester, resulting in near complete loss of LRC within 12 days. Activation of Myc stimulates epidermal proliferation without depleting LRC and induces differentiation of sebocytes within the interfollicular epidermis. Expression of N-terminally truncated Lef1 to block beta-catenin signalling induces transdifferentiation of hair follicles into interfollicular epidermis and sebocytes and causes loss of LRC primarily through proliferation. We conclude that LRC are more sensitive to some proliferative stimuli than others and that changes in lineage can occur with or without recruitment of LRC into cycle.