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Department of Anatomy and Cell Biology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USADepartment of Dermatology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USA
Department of Anatomy and Cell Biology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USADepartment of Dermatology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USA
Department of Anatomy and Cell Biology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USADepartment of Dermatology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USA
Department of Anatomy and Cell Biology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USADepartment of Dermatology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USA
Department of Anatomy and Cell Biology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USADepartment of Dermatology, Caver College of Medicine, The University of Iowa, Iowa City, Iowa, USA
Homeostasis of continuously renewing tissues, such as the epidermis, is maintained by somatic undifferentiated, self-renewing stem cells, which are thought to persist throughout life. Through a series of labeling experiments, we previously showed that stem cells from mouse skin did not divide often, but they did divide at a steady rate in vivo. Using our recently redefined sorting method, we isolated epidermal stem and transit amplifying (TA) cells from mouse skin. When injected into a developing blastocyst or into damaged tissues, the stem cells, but not the TA cells, could participate in the formation of new tissues. We hypothesize that all tissues contain reserved undifferentiated stem cells that are primed to react if needed. These reserve stem cells could restore the tissue in which they reside or they could be called upon to help restore another tissue that was severely damage.
LRC
label-retaining cell
TA
transit amplifying
Somatic Stem Cells of the Epidermis
Overall, it is generally agreed that homeostasis of the continuously renewing interfollicular epidermis is maintained by a small population of stem cells. Basically, a stem cell is considered to be an undifferentiated cell, which is both self-renewing and the source of all cells in that tissue. This is thought to be accomplished by a mechanism of differential cell division, where the undifferentiated stem cells divide to produce daughter cells that maintain the stem cell phenotype and daughter cells that are somehow changed in that they now can undergo only a finite number of cell divisions before they differentiate and leave the proliferative basal compartment. How this differential cell division is accomplished is not entirely understood, but in the epithelia of the small intestine, cells in the stem cell niche appear to segregate their DNA during cell division such that the stem cells retain the original DNA template while the transit amplifying (TA) daughter cells obtain the newly replicated DNA (
). Thus, any mutations that occurred during replication would be passed on to the TA daughter cells, leaving the stem cell DNA unperturbed. Whether this is also true for stem cells in skin epidermis has yet to be proven (for a review, see
Until recently, it was thought that somatic stem cells contributed cells only to their particular tissue. However, it is clear from several reports that many somatic stem cells, including epidermal stem cells, have the capacity to transdifferentiate into other cell types (e.g., see
). This bodes well for the use of epidermal stem cells to repair tissues. The skin is the largest organ of the body and the most easily accessible, making epidermal stem cell isolation less problematic for the patient, provided we can identify and isolate the appropriate cells. This latter has been under investigation for several decades.
Before the early 1980s, information available from radiation and structural studies of the skin (
) suggested that the continuously renewing epidermis should have a self-renewing stem cell population. However, no direct evidence existed. Heterogeneity of the epidermal proliferative compartment was first identified through a series of nuclear label-retention studies (
). Young growing mice were given several injections of tritiated thymidine over 2 days, then chased for up to 240 days. Samples of various epithelia were fixed, sectioned, and coated with liquid emulsion. The number of silver grains over each labeled cell was counted for each time point and for each tissue (for example of epidermis, see Figure 1). We showed that a few cells in the interfollicular epidermis retained a nuclear label for up to 240 days. We called these label-retaining cells (LRCs). The presence of LRCs with their slow loss of tritiated thymidine over time (a reduction of the number of grains per cell) proved that the epidermal proliferative population contained cells which cycled intermittently. Since that time LRCs have been identified in several tissues, and to date, this is an accepted method to identify epidermal stem cells (
Figure 1Percentage of LRCs at 15, 30, 60, 90, and 240 days after labeling. Cells were graphed and clustered according to the number of grains counted per nucleus. Note, the gradual decrease in the number of grains per nuclei with increasing time (purple to dark blue bar in each cluster). Also note that at each time point after labeling, a few cells were heavily labeled (>30 grains/nucleus).
). The first was a method for isolating hematopoietic stem cells, which worked on the principle that stem cells had more ABCG2 cell surface pumps. This allowed them to easily exclude the vital Hoechst 33342 red/blue dye. Hematopoietic cells sorted by this method proved to be highly enriched for the long-term in vivo repopulative stem cells (
). The combination of both methods allowed us to use a flow cytometer to sort basal keratinocytes into several populations with different recapitulation and proliferative characteristics (
). One population that we called TA cells showed rapid in vitro proliferation, but inability to maintain an epithelium, and no long-term expression of a recombinant gene. Another population that we call stem cells was ∼0.1% of the total basal cell population, expressed keratin 14 (K14), formed large clones in culture, exhibited much slower proliferation but much greater growth potential in vitro than TA cells, reformed and maintained an epidermis in organotypic culture, and expressed a recombinant gene for a long period of time (
). Additionally, we showed that both neonatal and older adult epidermal stem cells, but not the TA cells, could produce cell lineages from all three germ layers in the developing mouse (
). Hematopoietic early progenitor cells have also been identified as side population cells by their ability to exclude Hoechst 33342 dye, and thus located on the low side of the fluorescence in the scatter plots from the flow cytometer (
). These hematopoietic cells express the three basic stem cell characteristics of self-renewal, unlimited proliferative capacity, and the ability to differentiate into various mature circulating blood cells (lymphocytes, macrophages, myelomonocytes, granulocytes, erythroid, etc) (
). In the past decade, it has been shown that pluripotentiality is not limited to hematopoietic stem cells. For example, stem cells located in intestinal crypts provide all four cell types of the small intestine (paneth, enteroendocrine, goblet, and intestinal epithelial cells) (
). In addition, stem cells can transdifferentiate into multiple tissues. For example, skeletal muscle cells have the capacity to differentiate into hematopoietic cells (
). Thus, given the appropriate environmental stimulus, somatic stem cells may have the plasticity to form a variety of tissues.
The plasticity of epithelial cells isolated from whisker and hair follicles have been extensively studied. It has been suggested that cells from the bulge of the hair follicle produce all the epithelial cells in the outer root sheath, inner root sheath, and matrix, as well as cells in the interfollicular epidermis, and cells in the sebaceous glands. Whether this is a typical homeostatic mechanism is not clear. We do know that when the skin is damaged, cells from the hair follicle migrate into the interfollicular epidermis and into the sebaceous gland (
). Recently, it was shown that a single-cell cultured from either the bulge or the matrix of the rat whisker follicle could contribute all the cell lineages of the hair follicle and the sebaceous gland if grafted to the basement membrane zone in neonatal mouse skin (
). However, the grafting technique in itself creates a wound, and thus may not truly represent homeostasis. Furthermore, with an ever-increasing array of molecular markers staining different epithelial cell population, it is becoming apparent that follicular and interfollicular stem cells may be distinct (
Both embryonic and somatic stem cells have been classified by their potential to contribute to other cell types or tissues as totipotent (able to give rise to all embryonic and extraembryonic cell types), pluripotent (able to give rise to all cell types of the embryo proper), multipotent (able to give rise to a subset cell lineages, usually in a tissue) (for a review, see
). Somatic stem cells have also been classified as plastic (able to change their phenotype or to transdifferentiate into another cell type). However, the mechanism of plasticity is poorly understood. It may be that transdifferentiation is a phenomenon of one type of cell fusing with another type of cell as suggested for the hematopoietic and neural stem cells (
). In our hands, neonatal epidermal stem cells did not appear to fuse with embryonic stem cells when grown together in culture. If fusion is not what is causing plasticity of somatic stem cells, then three other possibilities exist: (1) somatic stem cells might be disbursed throughout other tissues; (2) genetic reprogramming might take place when a somatic stem cell is placed in a new environment; or (3) pluripotent cells left undifferentiated from initial development might persist and be distributed throughout all tissues. The latter possibility would require a shift in the paradigm of stem cells. Furthermore, it appears clear that an inductive influence may be required, such as that found in a developmental environment or a damaged tissue (
Potential of Epidermal Stem Cells for Tissue Replacement
Follicular cells that were positive for CD34, but negative for keratin 15 were shown to form several types of cells in vitro, including neurons, glia, keratinocytes, smooth muscle cells, and melanocytes (
). These data suggested that such cells could be used to regenerate tissues after wounding. When CD34-positive, keratin 15-negative, nestin-positive cells were grafted into a gap in a severed sciatic nerve, the cells transdifferentiated primarily into Schwann cells. Importantly, they appeared to completely restore the function of the severed nerve, for example, the rats could walk again (
). Thus, even though transdifferentiation may not be a typical homeostatic function of epithelial stem cells, these findings suggest that using them for tissue regeneration is viable.
To examine the transdifferentiation potential of epidermal stem cells under stress, we created full-thickness skin wounds (6 mm punch biopsies) in the backs of young adult C57BL/6 mice. After 2 days, we injected green fluorescence protein (GFP)+ epidermal stem cells just beneath the wound bed. The GFP+ epidermal stem cells came from the back skin of GFP transgenic neonatal mice. Controls received TA cells or buffer only. The wounds that received the neonatal epidermal stem cells appeared to heal faster in that by 4 days after injection, the gross appearance of the wounds appeared healed, whereas both sets of control wounds did not appear healed until 7 days after injection. At 21 days, histological analysis indicated that all the wounds were fully healed. In wounds that received epidermal stem cells, we found GFP+ cells in the underlying dermal tissues, including cells with an adipocyte morphology within the fat layer (Figure 2). These findings suggest that under the stress of wounding, mouse epidermal stem cells have the ability to incorporate into the healing dermal tissues.
Figure 2Wounds receiving neonatal epidermal stem cells retain GFP+ cells. Full-thickness skin wounds were created in the back skin of C57BL/6 non-transgenic mice. After 2 days, epidermal stem cells were isolated from the back skin of neonatal GFP transgenic mice, and injected beneath the wound beds. Shown here are adjacent sections through the middle of the healed wound bed 21 days after GFP+ epidermal stem cells were injected. (a) Hematoxylin- and eosin-stained section. (b) Unstained adjacent section showing GFP fluorescence. Circle surrounds a cluster of GFP+ cells of varying types. Arrows point to single GFP+ cell.
Epidermal stem cells also show potential for non-skin tissue replacement therapy. When GFP+ epidermal stem cells were injected into the blood stream of lethally irradiated mice, they helped the mice survive and appeared to help repair their damaged bone marrow (Figures 3 and 4). Recipient mice were irradiated with 1,100 cGy whole-body γ-radiation from a Cesium-137 source (broken into two doses of 600 and 500 cGy, 3 h apart). After 24 h, 1 × 105 GFP+ epidermal stem cells were injected. For radioprotection, each mouse received 1 × 105 total bone marrow cells from non-GFP-expressing mice. Control mice received TA epidermal cells, bone marrow cells only, or buffer. We monitored the mice for 8 months. All control mice receiving 2 × 105 bone marrow cells lived for the entire 8 months; those who received 1 × 105 bone marrow cells died at 2 months. All mice receiving epidermal stem cells lived the entire 8 months (Figure 3). Mice receiving 1 × 105 TA cells did not fare as well. By 4 months after irradiation, 60% (12 of 20 mice) were dead, and by 8 months after injection, 80% were dead (Figure 3). At 8 months, surviving mice that received TA cells had no GFP+ cells in cell smears made from total bone marrow isolates. In the bone marrow cell smears from mice that received epidermal stem cells, only ∼1 in 1,000,000 were GFP+. Although this is a very low percentage of engraftment, some GFP+ cells in the circulating blood were also positive for a cocktail of lineage markers (Figure 4), indicating that at least a few epidermal stem cells contributed to repopulating the hematopoietic tissue. These data suggest that epidermal stem cells aid in the overall recovery of the irradiated mice.
Figure 3Survival graph of irradiated mice receiving epidermal stem (EpiSC) or epidermal transit amplifying cells (EpiTA). Note that all the mice receiving epidermal stem cells lived, whereas most of the mice receiving epidermal TA cells died, suggesting that epidermal stem cells participated in hematopoietic repopulation.
Figure 4GFP+ Lin+ cells present in peripheral blood of irradiated mice 8 months after receiving GFP+ epidermal stem cells. C57BL/6 mice were lethally irradiated, then injected with GFP-expressing epidermal stem cells, and the bone marrow examined. Panels show two pictures of the same area. (a) Smear stained with an antibody cocktail of hematopoietic lineage markers for macrophages/monocytes (CD11b, Mac-1), T-lymphocytes (CD5, CD3, CD4, CD8), B-lymphocytes (CD45R,B220), granulocytes (Gr-1), and erythrocytes (TER119), and then alkaline phosphatase-labeled secondary antibody. (b) Smear stained with an antibody to GFP, and then horseradish peroxidase-labeled secondary antibody. Arrows point to GFP+ cells that are also hematopoietic lineage marker positive.
Potential to Improve Targeting of Epidermal Stem Cells to Non-Skin Tissues
To improve engraftment of epidermal stem cells to non-skin tissue, it appears that we will have to alter the cells before injection. One way to approach this is to change the cell's surface receptors. This could be carried out by transfecting or transducing the cells with the appropriate gene. However, the efficiency of most transfection methods is only 10–20% in epidermal keratinocytes, thus requiring either injection of many more cells or selection of only the cells that have been transfected. Furthermore, if a viral vector is used to transduce the gene, then the added risk of transferring viral proteins might preclude these cells for future use in humans. We have taken a different approach, and allowed the epidermal stem cells to alter their own cell surface receptors in response to a change in their environment. We isolated and cultured mouse epidermal stem cells and epidermal TA cells. Both cell types were grown in low calcium medium with 10% chelexed serum, or in keratinocyte serum-free medium with bovine pituitary extract in place of the serum, or in defined keratinocyte serum-free medium, which is a defined medium. To each cell type grown in each medium, we added 100 ng/ml of IL-3, IL-6, stem cell factor, or stromal-derived factor-1α, or a combination of 100 ng/ml IL-6 plus 100 ng/ml stromal-derived factor-1α. These factors have been shown to influence hematopoietic progenitor cells to enter specific lineage pathways. After 3 days in culture, cells were double-stained with antibodies to Sca-1 and K14. Sca-1 is found on hematopoietic progenitors cells that have been in contact with bone marrow stromal cells, and K14 is an intermediate filament, which is expressed by basal keratinocytes. Cells were scored as expressing Sca-1, K14, or both markers (Figure 5). Five separate experiments were performed, and 200 cells counted in each experiment. TA cells did not express Sca-1. Epidermal stem cells responded by expressed Sca-1 in all treatment groups, with cells exposed to stromal-derived factor-1α showing the most complete change. Approximately, 97% of cells expressed Sca-1+ and no longer expressing K14 (Figure 5). These data indicate that under certain in vitro growth conditions, epidermal stem cells can specifically respond to cytokines and alter their cell surface expression to that of a hematopoietic cell surface marker. It is interesting that the expression of Sca-1 on the cell's surface appears to be incompatible with the intermediate filament K14, which was always seen in partial collapse in co-expressing cells. Surprisingly, the type of medium in which the cells were grown appeared to make no difference in the overall results. We had expected that the presence of serum in the medium might drastically upregulate the Sca-1 expression. However, the control cultures grown in serum, but without the addition of the cytokines, showed continual expression of K14 and no upregulation of Sca-1. Whether changing the epidermal expression of Sca-1 in vitro before injecting the cells results in an increased bone marrow engraftment of these cells remains to be determined.
Figure 5Percentage of cells expressing Sca-1, K14, or both after exposure to various cytokines in culture. Cells were cultured for 3 days in defined keratinocyte serum-free medium plus 100 ng/ml of stem cell factor, IL-3, IL-6, stromal-derived factor-α, or 100 ng/ml each of IL6+stromal-derived factor-1α, and then fixed and double-labeled with antibodies to K14 and Sca-1. The percentage of cells stained with only K14 (green bars), only Sca-1 (red bars), or both (yellow bars) antibodies were calculated. Experiments were performed five times and 200 cells were counted in each experiment. Means and standard deviations were determined for each group.
In conclusion, we have shown that stem cells can be highly enriched from the epidermis of the skin, and that given the appropriate stimulus, these cells appear to be induced to transdifferentiate into cell types in tissues other than the epidermis of the skin. How this takes place is not clear. It may be that all tissues contain a small population of uncommitted stem cells. Thus, we are not seeing true transdifferentiation. Instead, we are inducing the uncommitted stem cells population. We speculate that these cells remain sequestered as reserved stem cells that sit in each tissue primed to react if needed. These reserved stem cells could restore the tissue in which they reside or they could be called upon to migrate and help restore another tissue under severe damage. It has been shown that in amphibians pluripotent epi-like stem cells in the adult can form all types of cells and can express typical embryonic markers (
). Such cells were not lineage committed in that 1% of these cells could form any somatic tissue at any given time. They did not follow the typical lineage pathway established in the embryo during organogenesis. Tissue regeneration in the amphibian is owing to the activation of the reserved population of quiescent uncommitted stem cells to proliferate and commit to a specific tissue lineage (
). If this is true, then the mechanism underlying the plasticity (fusion, or transdifferentiation) of the stem cells is immaterial. For example, somatic neural stem cells have a very broad developmental capacity and the ability to contribute to the formation of a variety of organs in both mouse and chick embryos (
). These mouse cells were cultured as neurospheres, then injected as adherent neurospheres into chick amniotic cavities or dissociated and injected directly into mouse blastocysts. Both chick and mouse embryos showed a mosaic pattern of cells derived from the neural stem cells, indicating that the cells could commit to an altered lineage pathway in development. More interestingly, these cells appear to be able to form a committed lineage in a different organism, suggesting that they may be more uncommitted than previously thought. Our data suggest that epidermal stem cells may also have this ability. We found that somatic epidermal stem cells could contribute to a variety of tissues during the development of a mouse and that they persisted into adulthood (
), but we have not tested whether they are interacting with the zebrafish cells. Hematopoietic stem cells have been shown to incorporate and function in a variety of murine tissues (
). Furthermore, these uncommitted cells appear to exist in all tissues and persist into adult tissues. Long-term persistence of grafted male cells in females have been reported (
). Although lifelong persistence of single uncommitted stem cell has not been proved, it is an attractive hypothesis and leads us to wonder whether stem cells change with increasing age or whether they remain as uncommitted, undifferentiated cells in all tissues – just waiting to be called to act.
Conflict of Interest
The authors state no conflict of interest.
ACKNOWLEDGMENTS
We thank the current members of the Bickenbach Laboratory for their helpful discussions, and the members of The University of Iowa Flow Cytometry Core for their excellent technical help. We also thank Dr Luchuan Liang, Jason Marley, and Dr Yubin Kang for their help, and Dr Ian Mackenzie for his support and intelligent arguments. This work was supported by the National Institutes of Health (NIAMS, NIA) and a focused giving award from Johnson & Johnson.
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Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves.
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