Two‐photon microscopy for intracutaneous imaging of stem cell activity in mice

Abstract

The adult skin is a typical example of a highly regenerative tissue. Terminally differentiated keratinocytes are shed from the external layers of the epidermis or extruded from the skin as part of the growing hair shaft on a daily basis. These are effectively replenished through the activity of skin-resident stem cells. Precise regulation of stem cell activity is critical for normal skin homoeostasis or wound healing and irregular stem cell proliferation or differentiation can lead to skin disease. The scarcity and dynamic nature of stem cells presents a major challenge for elucidating their mechanism of action. To address this, we have recently established a system for visualizing stem cell activity, in real time or long term, in the intact skin of live mice using two-photon microscopy. The purpose of this review was to provide essential information to researchers who wish to incorporate two-photon microscopy and live imaging into their experimental toolbox for studying aspects of skin and stem biology in the mouse model. We discuss fundamental principles of the method, instrumentation and basic experimental approaches to interrogate stem cell activity in the interfollicular epidermis and hair follicle. | INTRODUCTIONThe replenishment of lost cells to maintain homoeostasis and the repair of the skin after wounding rely on the activity of resident stem cells. [1][2][3] Due to their unique properties of self-renewal and multipotency, stem cells can be indispensible for normal skin function or extremely detrimental when they deviate from their standard activity.[ [4][5][6][7][8] One of the major impediments on studying adult stem cells is that they usually constitute a relatively small fraction of the total population.While significant progress has been made in this front, with the identification of genes that are preferentially expressed in restricted populations that display stem cell properties in the skin, questions remain regarding the level of heterogeneity within these populations. Another significant challenge is that stem cell activity is by definition a highly dynamic process. While the microscopic analysis of frozen-or paraffinembedded skin sections-processed by conventional histology and immunohistochemistry-has been the workhorse of dermatological research, this method is not always sufficient to overcome the unique challenges of studying stem cell activity in vivo. Live imaging modalities such as two-photon microscopy-especially when combined with powerful mouse genetic models-can provide critical insight into the mechanisms that govern stem cell activity, by enabling the visualization of stem cells within their native environment in the intact living skin. [8] In contrast to other tissues, the skin being the most external organ in the adult body offers direct access for observation and requires minimal, non-invasive preparation for imaging. [9] This review discusses recent methodological advances in the use of two-photon microscopy for intracutaneous imaging of stem cell activity.

Divergent proliferation patterns of distinct human hair follicle epithelial progenitor niches in situ and their differential responsiveness to prostaglandin D2

“…identifying a ‘true’ stem cell population (which may not concretely exist, given context specific plasticity). We instead posit that, at least in the bulge, eHFSCs represent a heterogeneous and dynamic pool (Figs S1 and S2 ), likely varying greatly in three dimensions 7 , 49 , 50 . This cell pool can be recognised histologically by a band of nuclei within the basal layer with clearly limited Ki-67 expression/EdU uptake in the bulge region (Fig S2 ).…”

Functional anatomy of the hair follicle: The Secondary Hair Germ

“…Given the semi-quantitative nature of immunohistochemistry, making the list of reliable SHG protein markers is a challenging task. 4 Also of interest, while P-cad is crucial for HF development in humans, it does not appear to be essential for this process in mice, [36,37] suggesting that P-cad may play different roles in mouse and human HFs.…”

“…Here is where a reductionistic approach that seeks “ to understand the whole through the parts” (ie “functional anatomy,” if we talk about a biological structure) can be exceptionally efficient: through analysis of cell kinetics and morphological changes in distinct HF anatomical parts, this approach would help to understand the basic principles of HF functioning in its entirety. And modern state‐of‐the‐art methodologies, like lineage tracing or in vivo imaging, are providing effective tools for this endeavour.…”

P75 neurotrophin receptor activates epidermal stem cell differentiation via ERK, JNK and AKT pathways

“…The wound area of the seven groups on 3, 7, 14 day was compared with which of their own on 0 day by a metric ruler measure. (Wound area rate of (3, 7, 14) D) % = (the wound area of (3,7,14) D)/(the wound area of 0 D). Use a metric ruler to measure the area of the wound.…”

“…The dysfunction of skin homeostasis usually result in dire consequences, involving acute, chronic inflammation or infected wounds [3,4]. And skin wound healing is an intrinsic self-saving chain reaction, which is crucial to facilitate the replacement of damaged or lost tissues.…”