Measuring stem cell frequency in epidermis: a quantitative in vivo functional assay for long-term repopulating cells

Abstract

Epidermal stem cells play a central role in tissue homeostasis, wound repair, tumor initiation, and gene therapy. A major impediment to the purification and molecular characterization of epidermal stem cells is the lack of a quantitative assay for cells capable of long-term repopulation in vivo, such as exists for hematopoietic cells. The tremendous strides made in the characterization and purification of hematopoietic stem cells have been critically dependent on the availability of competitive transplantation assays, because these assays permit the accurate quantitation of long-term repopulating cells in vivo. We have developed an analogous functional assay for epidermal stem cells, and have measured the frequency of functional epidermal stem cells in interfollicular epidermis. These studies indicate that cells capable of long-term reconstitution of a squamous epithelium reside in the interfollicular epidermis. We find that the frequency of these long-term repopulating cells is 1 in 35,000 total epidermal cells, or in the order of 1 in 104 basal epidermal cells, similar to that of hematopoietic stem cells in the bone marrow, and much lower than previously estimated in epidermis. Furthermore, these studies establish a novel functional assay that can be used to validate immunophenotypic markers and enrichment strategies for epidermal stem cells, and to quantify epidermal stem cells in various keratinocyte populations. Thus further studies using this type of assay for epidermis should aid in the progress of cutaneous stem cell-targeted gene therapy, and in more basic studies of epidermal stem cell regulation and differentiation.

Fig. 1.

Reconstitution of murine epidermis on the fascia of SCID mice. (a) Donor cells are from interfollicular epidermis. Note epidermal–dermal split (asterisks), leaving hair follicles in the lower portion of the sample. After scraping, dermis shows a full complement of hair follicles (not shown). (Original magnification, ×40.) (b) Silicone chamber implanted on the fascia of a SCID mouse. (c and d) Grafted murine keratinocytes form an epidermis in 2 weeks. (c) Grafted keratinocytes have formed an epidermis in the chamber at 2 weeks. (Original magnification, × 10.) (d) A high-power view of c. Chamber epidermis shows normal morphology. (Original magnification, ×110.) E, epidermis; SC, stratum corneum; D, dermis; H, host tissue. (e and f) GFP-positive epidermis in a chamber. (e) Grafted GFP-positive keratinocytes have formed an epidermis (E) in the chamber at 2 weeks (fluorescence microscopy). The entire epidermis has positive fluorescence that is particularly bright in the stratum corneum. (f) Hematoxylin staining for detailed morphology (same section). Dotted line indicates dermal/epidermal junction. SC, stratum corneum; D, dermis; H, host tissue. (Original magnification, ×20.) (g and h) Chambers show no ingrowth of host epidermis. (g) There was no evidence of epidermal ingrowth when an empty chamber remained in place for 9 weeks. (Original magnification, ×10.) Normal murine epidermis (E) with hair follicles (HF) is seen on the left. The silicone chamber (location marked C) marks an abrupt end to normal epidermis. In the base of the chamber is the fascia (F) with no signs of viable epidermis. (h) Pancytokeratin–phycoerythrin immunofluorescence of host epidermis adjacent to the chamber, with absence of staining on the fascia inside the chamber. (Original magnification, ×20.)

Fig. 2.

Competitive repopulating units (CRUs) in grafted epidermis. (a and b) The same GFP-positive CRU surrounded by GFP-negative epidermis (whole mount preparation, viewed with an inverted fluorescent microscope) at 4 weeks. (a) The FITC/GFP channel. At such early time points (4 weeks), some individual GFP-positive cells can also be seen, whereas later (e.g., 9 weeks) they are not seen, presumably because they were differentiated non-SC originally added that are subsequently sloughed off. (b) The red channel as a control for autofluorescence, demonstrating the extent of the piece of tissue (dotted line). (Original magnification, ×2.5.) (c–h) Fluorescence microscopy images of CRUs in grafted epidermis, serially sectioned. (c–e) A single GFP-positive CRU consisting of thousands of GFP-positive cells, surrounded by GFP-negative epidermis (counterstained with propidium iodide). (c) The FITC/GFP channel. (d) The red channel. (e) The overlay of c and d. (f–h) Another such CRU. (Original magnification: c–e ×10; f–h, ×40.)