o assessment of epidermal stem cell function and frequency has been well recognized as an important goal [1
]. We have developed and used a long term repopulating assay to test for sustained tissue regeneration and maintenance in vivo
. This may be considered the most rigorous definition of an epidermal stem cell. For this assay dissociated keratinocytes regenerate a differentiated epidermis on top of dermal fibroblasts seeded on the subcutaneous fascia of immunodeficient mice. GFP negative keratinocytes serve to ensure the production of an intact differentiated epidermis despite variations in the numbers of GFP positive cells in the test population. For the test population, a range of dilutions of GFP positive keratinocytes is used. Mixtures of test keratinocytes (GFP positive) are seeded into chambers along with a constant number of GFP negative keratinocytes, and the presence of GFP positive epidermal repopulating units is assessed 2 to 30 weeks after epidermal regeneration (). At each assessment the epidermis is scored as positive or negative, for the presence or absence of a cluster of GFP positive cells. By seeding a range of doses of GFP positive keratinocytes in this repopulating assay and waiting until all transit amplifying cells and their progeny have differentiated and been lost from the epidermis, limiting dilution analysis allows the frequency of cells with long-term repopulating ability in a given population to be quantified [3
Figure 1 Limiting dilution analysis for the determination of short term (TA) and long term (stem) repopulating cell frequency. (A) Diagram of the 6 mm diameter silicone chamber implanted onto the dorsal fascia of a NODSCID mouse into which the keratinocyte and (more ...)
is presented as an example of how this type of assay can be used to compare the frequency of stem cells in various populations of keratinocytes. As seen in , keratinocytes that rapidly adhere to collagen were implanted at a range of doses. At the highest dose of 240,000 keratinocytes, all chambers were positive for GFP positive repopulating units at all time points. At the lowest doses, of 1,900 and 7,500 keratinocytes, no chambers had GFP positive repopulating units at any time point. At intermediate doses, (e.g. 60,000 keratinocytes), it can be seen that, while at early time points (3 weeks) 9 of 10 chambers contained GFP positive repopulating units, at later time points (e.g., 9 weeks) only 5 of the 10 chambers remained positive. At sequential time points the repopulating frequency decreases until only long-term repopulating cells remain and the repopulating unit frequency remains constant thereafter. Using Poisson distribution statistics the calculated frequency of repopulating units corresponds to the derived cell dose at which 37% of the tests yield a negative response (L-Calc software v1.1, www.stemcell.com
). For the study in , we relied on previous work that found that the stem cell frequency of total unsorted cells at 9 weeks was 1 in 30,000 keratinocytes [3
]. As can be seen for the dose of 30,000 total unsorted keratinocytes at 9 weeks (), 4 out of 10 chambers are negative, consistent with Poisson statistic predictions. For each population of cells to be tested (for example, rapidly adherent cells, not-rapidly-adherent cells, unsorted cells) the doses to be used experimentally will depend on the expected long term repopulating unit frequency.
Frequency of repopulating units in rapidly adherent, not rapidly adherent, and unsorted murine keratinocytes over time.1
Phenotypic analysis of hematopoietic stem cells in in vivo
transplantation assays has allowed separation of long-term repopulating cells from cells detected in colony forming assays [4
]. These types of studies have defined a hierarchy of hematopoietic stem cell phenotypes (see [12
] ). Primitive progenitors that represent the closest stem cell descendents which can be prospectively isolated from the true stem cell by flow cytometry, are still multipotent, yet already have a decline in self-renewal capacity, underscoring long-term repopulating ability as the sine qua non of a stem cell [13
]. These progenitors produce distinct highly proliferative colonies, which can differentiate into specific lineages, but unlike a true stem cell are unable to repopulate all hematopoietic lineages for the life of the animal. Thus in vivo
transplantation assays have long been the gold standard for the study of hematopoietic stem cells, and after almost 20 years remain so [15
One important question regarding functional assays for stem cells is what duration of repopulation distinguishes the true epidermal stem cell from a short-term repopulating cell. In the epidermis, the short-term repopulating cells are termed transit amplifying cells. Cell cycle duration has been estimated to be 4 to 5 days and transit amplifying cells go through approximately 3 divisions [16
], before terminally differentiating. Our initial studies showed that there is no further decline in repopulating cell frequency after 7 weeks, indicating that at this point transit amplifying cells and their progeny have differentiated and been lost from the epidermis, and we are assaying the true long term repopulating epidermal stem cell [3
]. Thus in all subsequent epidermal stem cell studies we have selected an endpoint of 9 weeks or later to ensure the study of stem cells rather than short term repopulating transit amplifying cells.
As noted in similar hematopoietic transplantation assays [18
], estimates of stem cell frequency are most certainly underestimates since the detection efficiency of the assay procedure is not known, but is almost certain to be less than 1. In recognition of this we term the GFP positive clusters of cells repopulation units rather than epidermal stem cell units. This does not undermine the value of comparing the relative frequency of progenitor cells in different cell populations. While this assay estimates that 1 in 10,000 basal cells is a truly primitive epidermal stem cell, similar to stem cell frequencies in other tissues [11
], previous work on the epidermis showed that 1 in 10 basal cells was a colony forming stem cell (for review, see [20
]). However, more recently it has been shown that colony forming cells do not all represent stem cells [1
]. These findings lead us to believe that stem cell frequency is significantly less than previously thought [20
]. Using the in vivo
transplantation assay described here multiple studies have reported that the frequency of epidermal stem cells in young and in neonatal murine epidermis is approximately 1 in 10,000 basal cells [3
] strengthening the argument that the frequency of epidermal stem cells is similar to that of other somatic stem cell populations.
In this chapter we describe a method to determine the frequency of short and long term repopulating epidermal progenitors in vivo using limiting dilution analysis. First, primary keratinocytes are isolated from GFP positive and GFP negative neonatal murine epidermis. Then fibroblasts are isolated from the GFP negative skin. Next, a range of doses of GFP positive test keratinocytes are prepared and left on ice, while silicone chambers are implanted onto the fascia of NODSCID mice. Fibroblasts and then keratinocytes are seeded into the chambers. The regenerated epidermis is imaged over time and analyzed for presence or absence of GFP positive repopulating units. Analysis of the positive and negative results is performed using Poisson statistics for limiting dilution analysis, and allows a quantitative analysis of the repopulating unit frequency. The strengths of this assay are the long term functional nature of the repopulation carried out in vivo, which allows for the distinction between true long term repopulating stem cells and short term repopulating (transit amplifying) cells, and the ability to quantify the number of long term repopulating epidermal stem cells.