The epidermis is prone to injury and epithelial cell loss; therefore, it is essential that it be able to maintain and repair itself throughout life from cells quiescent within the epidermal stem cell niche. Although the origin, location, and cellular characteristics of epidermal stem cells are begin- ning to become clearer, less is known about the genes that are critical for the formation and maintenance of epidermal stem cells as well as the signals that commit specific subpopulations within the epidermal lineages. Nevertheless, recent developments have made epidermal stem cells prime candidates for the development of various therapeutic modalities aimed at regen- eration of epidermal tissues. In this review, the current understanding of epidermal stem cell biology from embryonic and adult sources is summarized, as well as their potential for regen- erative medicine.
Throughout life, the skin acts as the interface between internal organs and the external envi- ronment, providing a physical barrier and first line of response to a variety of environmental insults including physical trauma, ultraviolet irradiation, chemical invasion, and microbial assault.1To maintain its physical integrity and function, the epidermis continuously renews itself from a pool of differentiating epidermal cells supplied by the epidermal stem cell niche.2 Advances in our understanding of the epidermal lineage have been substantial over the last 20 years, and although much remains to be learned, the field is positioned to devise ways to regener- ate skin in severe injury situations such as
observed in burn victims, patients with diabetic foot ulcers, as well as individuals with congeni- tal or acquired skin blistering diseases. In all such circumstances, the normal cycle of epider- mal differentiation is incapable of fulfilling the renewal demands of the epidermis, resulting in epidermal failure with all its life-threatening consequences; thus, the potential of skin regen- eration is enormous. To achieve this, however, will require a greater understanding of the self- renewal and directed differentiation of epider- mal stem cells as well as the signals and their transduction pathways that govern the prolifer- ation and differentiation of regeneration-com- petent cells. This review examines our current knowledge of epidermal stem cells and explores their potential use for epidermal regeneration and future clinical stem cell-based treatment regimens for epidermal defects.
Epidermis as a Developmental System
In mouse, the epidermis is derived from primi- tive ectoderm that gives rise to surface ectoderm during gastrulation at embryonic age E5–E6.3 After initial cell fate selection, surface ectoder- mal cells are selected to either the neuronal or epidermal fate.4 Pluripotent epidermal stem cells give rise to epidermal cells, which go through a complex series of morphogenic steps that give rise to the epidermis, hair follicles, and
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Skin Regeneration from Multipotent Adult and Embryonic Stem Cells
Kursad Turksen and Tammy-Claire Troy
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sebaceous glands where appropriate. From ges- tational days E8–E12, the putative epidermis is a single layer of proliferating cells (stratum ger- minativum), which is overlain by the periderm expressing characteristic markers of surface ectodermal cells [keratins 8 and 18 (K8/K18)].
At E12–E14, an intermediate layer of cells (stra- tum intermedium) develops from the stratum germinativum, and comes to separate it from the periderm. Differentiation to the mature epi- dermis begins at day E15, with epidermal strati- fication. At this stage, the stratum germinativum expresses K5/K14 (characteristic of basal cells), whereas the cells in the two layers of the stratum intermedium down-regulate the expression of K8/K18 and the expression of K1/K10 is induced in the suprabasal cells.5,6Differentiation of the mouse epidermis into mature structures is com- pleted by E17–E18 with the appearance of the granular and cornified layers of the epidermis6 giving rise to the insoluble cornified envelope and the completion of epidermal permeability barrier (EPB) formation.7–12 Similar steps and events in epidermal morphogenesis also take place (in their appropriate timeline) for other mammals.
Epidermis as a Self-Renewing Differentiation System
In response to discrete yet unidentified signals, throughout life, epidermal cells in the basal compartment become irreversibly committed to terminal differentiation and move upward away from the basal layer.13–16 This process can be very nicely followed by the expression profile of keratins (epidermal structural proteins) as well as other proteins known to be involved in termi- nal differentiation.12,17,18Upon leaving the basal layer, the expression of K5 and K14 is shut down whereas K1 and K10 expression is induced. As these cells progress to the spinous layer, they begin to express involucrin, a protein involved in the eventual formation of the insoluble corni- fied envelope. Granular cells, which directly fol- low the spinous cells, are characterized by electron-dense keratohyalin granules contain- ing filaggrin, a protein that facilitates the aggre- gation of keratin filaments.19 As the terminally differentiating cells transit from the granular layer to the cornified layer, cornified cells undergo a destruction of their organelles to
form the cornified envelope. Cornified envelope proteins include involucrin, loricrin, small proline rich proteins,20,21calcium binding S100 proteins, cystatin A (keratolinin), repetin, loricrin,22envo- plakin,23–25 sciellin,26 and late envelope pro- teins27 (reviewed in refs. 11, 19, 28). The cornified envelope is constructed through the sequential expression, processing, and deposi- tion of several other distinct proteins that are crosslinked by disulfide and Ne-(g-glutaminyl) lysine isodipeptide bonds, the formation of which is catalyzed by transglutaminase.29
Thus, the permeability barrier of the skin is formed in the last stage of epidermal differenti- ation consisting of corneocytes, an insoluble cornified envelope, and lipid-enriched intercel- lular domains.11,30–33The lipids for barrier func- tion are synthesized in the nucleated epidermal layers, stored in the lamellar bodies, and extruded into the intercellular space during the transition from the stratum granulosum to the stratum corneum forming a system of continu- ous membrane bilayers.8,34Using a qualitative, whole-mount assay for skin permeability, Hardman and colleagues10 demonstrated that barrier formation is indeed highly patterned during development. Morphologic and bio- chemical observations have been further cor- roborated recently by the transepidermal water loss assay as a powerful indicator of barrier function. Quantitative transepidermal water loss assays indicate that the EPB forms rapidly between days 19 and 21 of the 22-day rat gesta- tional period,35between days 17 and 19 of the 20-day mouse gestation period,10 and between weeks 30 and 33 of the 40-week human gesta- tional period.36 Generally, therefore, mam- malian EPB formation is completed just before birth as part of the preparation of the organism for terrestrial living. This barrier provides a first line of defense against bacterial infection, ther- moregulation, and dehydration, and the conse- quences of EPB dysfunction are generally deadly in the environment we occupy.
The histology of the mature epidermis as well as the overall changes in the expression pattern of structural molecules during development and adulthood have been described. However, the patterned expression of signaling molecules thought to be regulating cell fate selection16,37,38 as well as the epidermal and associated appendage lineages (i.e., hair and sebaceous glands) during embryologic development are slowly beginning to be identified from a series of
transgenic mouse studies. These include several transcription factors such as LEF-1/Tcf-3,39 p63,40 c-Myc,40–43 GATA-3,44 Foxn1 (nude),45,46 Hairless, RBP-J,47 and members of several sig- naling pathways including Wnts,48 Delta- Notch,49 sonic hedgehog,50 fibroblast growth factor (FGF),51 and bone morphogenetic pro- teins.52,53 However, very little is known of the early steps leading to the onset of cell lineage commitment, the allocation and identity of appropriate stem cells, or the control of the developmental program thereafter.
Epidermal Stem Cells and Their Potentiality
Continuous renewal of the epidermis and its appendages throughout life has predicted that there is a stem cell population with characteris- tics similar to those of other renewing sys- tems.54–57 Until recently, however, technical difficulties with culturing epidermal cells in vitro and the absence of markers to identify and isolate stem cells, as well as the lack of a bio- logical assay to demonstrate their stem cell-like characteristics, has plagued the epidermal stem cell field; hence, stem cells of the epidermis have not been explored with the same vigor of other lineages (such as hemopoietic cells). The first definitive study addressing the existence and location of putative stem cells in the epidermis was published in the 1980s, when Cotsarelis et al.58 demonstrated the existence of label- retaining,59 slowly proliferating cells concen- trated in the bulge region of the hair follicle, leading to the concept that these cells could con- stitute a stem cell pool in a specialized stem cell niche. The demonstration of the true potential of bulge region-derived epidermal stem cells and the ability to measure the frequency of puta- tive stem or more restricted precursor cells in vitro or in vivo have been hampered by the absence of appropriate assay systems and the lack of reliable/specific markers for epidermal stem cells and their immediate progeny.
Nevertheless, considerable data have been gen- erated documenting the high proliferative capacity/colony-forming capacity of cells iso- lated from carefully dissected regions along the hair follicle as well as within the heterogeneous population of basal layer epidermal cells.60 However, it is important to note that, given the
way these experiments were done, it was not possible to address the clonality of the colony- forming precursors. Indeed, the conditions that had been derived for mature keratinocyte growth61 fail to support growth of the isolated precursor cells at limiting dilution or single cell clonal levels. By the same token, the approaches were also not suitable for attempting to deter- mine lineage relationships and potentiality of putative multipotential epidermal stem cells and their more restricted progeny. Similarly, suit- able reconstitution-type biological assays were not available to test the potentiality of putative stem cells in vivo.
Identification of the hair follicle bulge as the likely location of stem cells has allowed identifi- cation of a number of potential stem cell mark- ers by region-specific immunohistochemical labeling62; these include β-integrin,63,64 K19,65,66 K15,67,68δ-1,49 p63,69CD34,70melanoma-associ- ated CSPG,67and Nestin.71None of these mark- ers is absolutely specific for putative stem cells nor is use of any one alone sufficient to allow isolation of homogeneous stem or precursor cells from the bulge; however, the markers pro- vide additional support for stem cells and a stem niche in the bulge region. Early attempts to enrich epidermal stem cells was based on the observation that the putative stem cells adhered strongly to basement membranes and exhibited high levels of β1-integrin (surface adhesion pro- teins).72,73 Thus, enriched β1-integrinbright cells exhibited high proliferative and colony-forming capacity in vitro.73Putative epidermal stem cells have also been reported to express not only high levels of β1 but also α6-integrins and low levels of CD71 (transferrin receptor).74 K19, a tailless keratin molecule, was also reported to be a puta- tive stem cell marker based on its restricted localization to the bulge region of the hair folli- cle.66Another keratin (K15), although associated with various epithelial cells,75may also be a good marker of bulge cells in adult epidermis.76,77
More recently, the hemopoietic cell marker CD34 has been said to be highly expressed by epidermal stem cells, based on the intense mem- brane staining on keratinocytes in the bulge region of the mouse hair follicle.70In these stud- ies, CD34 expression colocalized with both slowly cycling (label-retaining) cells and K15 expression. When cells were selected with fluo- rescence-activated cell sorting (FACS) with anti- bodies against CD34 and α6-integrin in combination, the positively selected cells were
found to be predominantly in G0/G1, character- istic of a quiescent or slowly cycling stem or pre- cursor population, in contrast to the CD34− cells which had well-defined G2/M and S phases.
Interestingly, the majority of the CD34+ cells (98%) were positive for K6, establishing this population as the basal keratinocytes of follicu- lar origin. Notably, when putative adult epider- mal stem cells were labeled with Hoechst 33342 and a side population (SP) was sorted by FACS, the cells within the SP did not exhibit the stem cell-like characteristics that SP cells have been reported to have in other adult stem cell iso- lates.78,79 This emphasizes the need for caution in attributing widely accepted stem cell charac- teristics to all stem cell populations.
Recently, two groups61,80independently, and with somewhat different genetic strategies, tracked and isolated a stem cell-like subpopula- tion of cells from the hair follicle bulge. Based on their assumption that bulge stem cells would uniquely be both slow-cycling and active for a keratinocyte-specific promoter, Fuchs’ group61 engineered transgenic mice to express histone H2B–green fluorescent protein (GFP) controlled by a tetracycline (tet)-responsive regulatory ele- ment and crossed them to mice harboring a K5 promoter–tet repressor–VP16 transgene. Four weeks of tet treatment of the double transgenic offspring with tet-controlled regulation restricted to skin epithelium selected for a low frequency (<1%) of slowly cycling bulge cells.
Cotsarelis’ group,80 however, used a K15 pro- moter fragment to target mouse bulge cells with an inducible Cre recombinase construct or with the gene encoding GFP to mark bulge cells for lineage analysis in vivo and isolation, respec- tively. Further characterization of either of the GFP-positive populations on the basis of a num- ber of phenotypic and functional properties in vitro and in vivo indicate that the cells meet many of the hallmark definitions of stem cells.
The authors of both studies recognized the need to address whether any of the isolated cells are multipotential and generate or regenerate hair follicles and other cutaneous lineages, a fea- ture attributed to bulge stem cells.81,82 This is key, given that there is evidence both for multi- potent cells as well as more restricted progenitor cells (i.e., cells that give rise to only the hair fol- licle or only the sebaceous gland). By following the fate of marked label-retaining cells in rela- tion to coexpression of a variety of proliferation- associated markers during the normal hair cycle
and in response to injury, the Fuchs group con- cluded that only a few bulge label-retaining cells initiate each new follicle, and that upon exit from the bulge their progeny rapidly proliferate, markedly change their biochemical and gene expression profiles, and regenerate all differen- tiated cutaneous cell types.61Using somewhat dif- ferent methodologies, including isolated bulge cell cotransplantation with dermal cells, the Cotsarelis group reached similar conclusions.
Notably, however, the observations suggest that, although multipotent, under normal circum- stances the bulge stem cells have a preference for generating hair follicles over the other cutaneous lineages. Both studies also reported observations suggesting that after exit from the bulge, the progeny of bulge cells retain significant prolifera- tive and multilineage differentiation capacity.
This is consistent with numerous previous indi- cations that primitive progenitors for the cuta- neous lineages, as for many other lineages, retain extensive proliferative capacity and multipoten- tiality, and these features alone are insufficient to rigorously define definitive stem cells.
It remains of interest to define the lineage relationships between the putative multipotent stem cells, multipotent progenitors, and/or more restricted unipotent progenitors for any one of the cutaneous lineages, especially when one considers these new data in the context of previous work that suggested the existence of multiple classes of stem cells in the cutaneous epithelium.83 Ideally, one might attempt to address these latter issues by additional in vitro studies in combination with transplantation assays at single cell and clonal levels. Both the Fuchs and Cotsarelis groups FACS-enriched their marked cells and provided solid evidence that the populations with highest GFP expres- sion were relatively quiescent when isolated but were capable of extensive proliferation in vitro;
i.e., the efficiency of large colony formation was higher in their enriched versus nonbulge, basal keratinocytes, or GFP-positive populations with lower fluorescence intensity. They also showed that the most intensely fluorescent-sorted frac- tion of cells coexpressed other markers associ- ated with bulge stem cells, including β1-integrin and CD34. However, clonal differentiation assays to address self-renewal, multilineage dif- ferentiation capacity of single putative stem cells within the FACS-sorted enriched populations, and the detailed lineage relationships men- tioned above have not yet been done.
As previously noted, hair follicle and epider- mal lineages pose significant problems in this regard. To date, no appropriate conditions have been reported by which primary epidermal cells can be maintained at single cell density in cul- ture, and, based on data reported in recent stud- ies, it seems likely that hair follicle stem cells may be similarly intransigent and require very different conditions than mature epidermal cells. Nevertheless, some hints may come from the gene expression profiling that was done in these studies. Studies from both the Fuchs and Cotsarelis groups include extensive transcrip- tional profiling of the isolated enriched bulge stem cell populations versus non-stem cell pop- ulations as well as, excitingly, other stem cell populations [i.e., hematopoietic, neural, and embryonic stem (ES) cells]. Generally, tran- scriptional profiling clearly supported other evi- dence presented that the hair follicle stem cells are quiescent or slowly cycling cells and are dis- tinctly different from the more rapidly prolifer- ating and differentiating progeny in expression of, for example, multiple growth factors and related signaling molecules including FGFs, transforming growth factor (TGF)-β, Wnts, and a variety of other molecular classes (extracellu- lar matrix molecules, transcription factors, etc).
In this regard, however, although there was con- siderable overlap in the stem cell transcriptional profiles reported in the two studies, there were some notable differences that may reflect varia- tions in the homogeneity of the different popu- lations assessed and/or the fact that the follicles compared in the studies were at different stages of the hair cycle. Interestingly, three genes were identified as common to hair follicle, neural, hemopoietic, and ES cells (namely, Eps8, Col18a1, and Pkd2); although it is premature to suggest that these comprise a definitive stem cell signature, the overlap in expression profiles as the transcriptomes of additional purified stem cell populations are compared will be interest- ing (e.g., ref. 84).
Stem Cell Niche and Regulation of Stem Cell Fate
Most mammalian continuously renewing tissues are maintained by stem cells located within stem cell niches2,85; the bulge area fits these criteria in the epidermis. For example, Blanpain et al.86
showed that within the bulge there are two dis- tinct populations, one of which maintains basal lamina contact and temporally precedes the other, which is suprabasal and arises only after the start of the first postnatal hair cycle. This spatial distinction endows them with discrete transcriptional programs; surprisingly, how- ever, both populations are growth inhibited in the niche, yet can self-renew in vitro and make epidermis and hair when grafted. These findings suggest that the niche microenvironment imposes intrinsic “stemness” features without restricting the establishment of epithelial polar- ity and changes in gene expression.
The abrupt change in proliferative and bio- chemical properties when cells exit the bulge as reported in the aforementioned studies from the Fuchs group is consistent with the notion of a distinct stem cell niche in the bulge.2,61,86What are the signals that may help to define this niche and what keeps bulge stem cells in a low cycling state but activates them to leave the niche? As just summarized, several different families of growth factors are differentially expressed in bulge cells enriched on the basis of the markers used. One particularly interesting family of can- didate molecules is the Wnt family, which has been demonstrated to be involved in stem cell renewal.87Defining the mechanism of action of Wnt signaling in epidermal stem cell mainte- nance in the context of the surrounding microenvironment and determining how this signal may integrate with other niche-derived signals represents the next challenge in epider- mal stem cell biology. Regulating these signaling pathways might pave the way for the regenera- tion of damaged epithelia by stimulating stem cell function and inducing rapid expansion of progenitor cells in tissue regeneration.
Skin and Regenerative Medicine
One successful approach for the treatment of epidermal dysfunction is cellular transplanta- tion, built on the techniques pioneered by Green and colleagues88,89 more than two decades ago for culturing committed epidermal cells. In this approach, bioengineered skin substitutes cre- ated by culturing living cell sheets of human epi- dermal keratinocytes together with dermal fibroblasts90–93have been used successfully, for example, to promote the healing of chronic skin
ulcers and to cover full-thickness burns.
Although this process allows the replacement of nonfunctional or lost epidermal cells and scar tissue with fully functional epidermal cells, resolving the immediate problem of skin failure (namely, barrier function),94its utility has been rather limited.92 Such problems as: i) lack of long-term integration, ii) incomplete healing/
generation of scar tissue, iii) lack of regenerative potential, iv) inadequate appendage contribu- tion, and v) overt rejection, have plagued the approach. Problems are further exacerbated by infection as well as the development of hyper- proliferative disorders as a consequence of the immunosuppressive treatments used for modu- lating rejection. It is also not yet entirely clear what the cellular composition of the grafts is, but likely they comprise heterogeneous and not well-controlled mixtures of already committed mature epidermal cells as well as some progeni- tors and very few putative stem cells. This may contribute to many of the problems seen, including the lack of long-term regenerative potential, because it seems unlikely that the lim- ited stem cells present would be able to home and reconstitute a stem cell in the disrupted or missing microenvironment of injured areas.
Thus, much pressure remains to develop alter- native stem cell-based epidermal transplant approaches.
Sources and Potential Usefulness of Stem Cells in Regenerative Medicine
Based on developments in stem cell biology over the last few years, there exist three principal sources of stem cells capable of generating organ-specific cell types: i) ES cells, ii) adult stem cells isolated from the target organ, and iii) adult stem cells from other organs.95–98ES cells of the mammalian blastocyst give rise to all the tissue lineages that begin to emerge at gastrula- tion and they can be propagated in vitro without loss of pluripotency. Many adult tissues also contain adult stem cells, which serve as self- renewing stem cells whose normal fate is to regenerate tissue-specific cells, in response to either physiological cell turnover or damage inflicted by injury or disease. In some cases, adult stem cells thought to be tissue-specific may possess plasticity or developmental potency far greater than their normal lineage-
restricted fate (e.g., bone marrow-derived stem cells). The growth potential and pluripotency of ES cells and the developmental plasticity of adult stem cells make them potentially useful for replacing tissues (via transplantation or con- struction of bioartificial tissues) that either do not regenerate naturally or are damaged beyond their natural capability for regeneration. In addition to these two ways of replacing tissue, a third strategy of regenerative medicine is to stimulate regeneration in vivo from resident stem cells. Thus, the regeneration of epidermal cells can, theoretically, involve ES cells, adult epidermal stem cells, or adult stem cells (includ- ing from bone marrow).
ES Cells
The ability to generate differentiated epidermal progeny from a continuously growing stem cell population in vitro would provide a unique sys- tem for the study of stem cell/very early progen- itor potential.99 It would also make possible a comprehensive analysis of the underlying molecular mechanisms for the onset of embry- onic epidermal commitment and differentia- tion. In fact, similar in vitro approaches have yielded invaluable information on the mecha- nism of differentiation of other cell types. In the past, several attempts have been made to con- duct similar studies on epidermal differentia- tion100,101(for review, see ref. 102). However, the difficulties encountered in stably maintaining the putative stem cell population in culture lim- ited the usefulness of these systems. Indeed, until recently, even the maintenance of mature epithelial cells in culture has been problem- atic103(for review, see ref. 104).
ES cells have been shown to provide excellent model systems in which to study lineage com- mitment and progression in vitro. ES cells were initially isolated from mice and more recently from monkeys and humans105–107 and can be maintained in culture under appropriate condi- tions in an undifferentiated state, theoretically indefinitely. A switch in culture conditions to induce embryoid body formation (aggregation) leads to spontaneous differentiation; depending on the conditions and factors to which they are exposed, cells representative of all three germ layers can occur. The use of mouse ES cell cul- ture models and more recently human-derived
ES cells has revealed invaluable information concerning the commitment of stem cells to var- ious lineages,108including neurons,109cardiomy- ocytes,110,111adipocytes112as well as osteoclast,113 epidermal,114and hair follicle115cells. Compared with certain other lineages, the use of ES cells for studies of the epidermal lineage has been rela- tively slow to advance. However, studies from three independent groups13,18,116–118 have indi- cated that mouse ES cells have the potential to be directed robustly along the epidermal line- age. The protocols for derivation of epidermal cells from murine ES cells were based on expos- ing ES cells to in vivo-like conditions, including extracellular matrix13 and bone morphogenetic protein-4 signaling,119 conditions known to be required for ectodermal lineage commitment.
Proliferating keratinocytes in culture have the ability to undergo terminal differentiation as revealed by the expression of differentiation markers important for the generation, matura- tion, and maintenance of differentiated epider- mal cells (i.e., keratinocytes and hair follicle cells), ultimately forming an epidermis-like tis- sue. An obvious issue is whether very early multi- or bipotential progenitors can be identi- fied and expanded. In a recent study, the exis- tence of bipotential progenitors expressing K17 has been reported.118,120 ES-derived epidermal progenitor cells cultured at high density have also been induced to a hair follicle-like pheno- type accompanied by the up-regulation of struc- tural hair-cell proteins (keratins and terminal differentiation markers, suggesting that these cultures may indeed contain bipotential or puta- tive epidermal stem cells. However, conditions to generate the sebaceous gland lineage remain to be established.
The ability of stimulated ES cells to produce a stratified epidermal tissue was assayed in an organotypic culture model in which cells were cultured on a cell-free inert filter substrata at the air–liquid interface.119 Histologic and indirect immunofluorescent staining revealed an artifi- cial skin quite similar to that of mice, in which both epidermal and dermal compartments were formed. In addition, the spatial distribution of differentiation markers was consistent with that seen in vivo with: i) basal cells expressing K14, ii) a basement membrane containing collagen IV and VII, as well as laminin-1, β-integrin, α6 β4, fibronectin, and nidogen,119and iii) K1-express- ing cells in the suprabasal layer.119 In all cases reported to date, K14-positive basal epidermal-
like cells developed. Importantly, the derived cells function as bone fide early epidermal cells, following lineage progression and exhibiting appropriate markers in a temporal manner; in addition, a subpopulation of these cells retain progenitor characteristics, with the potential to generate epidermal cells in vitro. These studies support the notion that ES cell-derived epider- mal stem and/or progenitor cells can be gener- ated and can respond to cues in their local environment to undergo terminal differentia- tion, faithfully mimicking the developmental program observed in vivo. Although the mouse epidermis is far different than injured or dis- eased mammalian epidermis, nevertheless, these results are the first successful approach using ES cell-derived epidermal cells to generate epidermis and follow the differentiation pro- gram in vitro.
These initial studies convincingly demon- strate the potential of ES cells to commit and dif- ferentiate along the epidermal lineage in vitro;
however, there remains a considerable amount of work to do to define the conditions necessary to support the generation and expansion of a homogeneous population of ES, progenitor, or even relatively mature cells in vitro. This is cru- cial because any remaining undifferentiated ES cells injected into an organism have a high capacity for teratoma formation.121 There are also other challenges to consider; for example, because the dynamics of the in vivo setting are unknown, transplantation of predifferentiated cells (such as epidermal and hair follicle lineage cells) into the hair follicle could act to destabi- lize the differentiation milieu and lead to defects. Thus, among the technical challenges is the need to improve protocols by which ES cells can be induced to differentiate in a much more directed manner to specific lineages and devel- opmental stages. Developmental studies indi- cate that several factors alone and/or in combination may be involved in differentiation along the epidermal lineage. These include epi- dermal growth factor, FGF-2, TGF-β1, platelet- derived growth factor, retinoic acid, vitamin C, as well as a number transcription factors (i.e., KLF-4 and GATA-4).44,122–124The one-step cocul- ture protocol recently developed by our labora- tory should aid in identifying conditions that support a more efficient and highly enriched culture of ES cell-derived epidermal progenitor cells with controlled progression along the epider- mal lineage. The use of growth factors individually
in a sequential manner or in combination may provide the conditions needed to enrich and expand epidermal stem and progenitor cells in vitro.
As discussed above, currently there are no markers that can be reliably used in the purifi- cation of a homogeneous population of epider- mal stem cells. However, one approach to enrich very early progenitor cells in the absence of appropriate markers is to isolate cells using cell- marker/separation techniques. The key to this process is to use a specific marker for the epi- dermal lineage that is ideally able to distinguish (for instance) hair follicle cells from other epi- dermal cells. One such method might involve the transfection of ES cells with a fusion gene of a K15 (an adult stem cell marker77) or a K17 (a bipotential early progenitor marker120) pro- moter linked to a cDNA encoding aminoglyco- side phosphotransferase and an antibiotic resistance gene. After differentiation, antibiotic selection of cells of the epidermal lineage is pos- sible, generating a relatively pure epidermal sample (approximately 99%). To date, this cell- marker/separation technique has been used suc- cessfully with an αMHC promoter in the muscle lineage95,125 as well as with the 2’3’-cyclic nucleotide 3’-phosphodiesterase (CNP) promoter in the oligodendrocyte progenitors.126Of course, the ultimate utility of these approaches for regen- erative medicine in a clinical setting will require the development of protocols for human ES cells, including the demonstration of the potential, fea- sibility, and success of human ES cell-derived epi- dermis formation in vivo.
Recent success in the generation of a number of established human ES cell lines has raised great promise for stem cell and regenerative biology106,127–129; however, their isolation and proposed studies thereafter generated a highly controversial social debate of legal and ethical implications. Despite controversy, some studies with human ES cells are underway, although protocols for maintaining undifferentiated cells and controlled differentiation have not been easily achieved. Initial conditions to generate and maintain human ES cells were based on the use of mouse fibroblasts as feeders,129a less than ideal protocol for clinical consideration. Some human ES cell lines have been shown to remain undifferentiated when grown in the presence of either human fetal fibroblasts, adult human epithelial cells, foreskin cells, or a matrigel/
laminin matrix in medium conditioned by
MEFs.130–132 Unfortunately, these protocols too will be problematic for regulatory agencies.
Adult Epidermal Stem Cells
As summarized, recent advances in identifying adult mouse epidermal stem cells in the hair fol- licle bulge have generated excitement on the possibility of this adult stem cell source. Of course, the race is on to isolate the human equivalent of these mouse cells. In this regard, one of the most interesting corollaries to the potential clinical use of adult hair follicle stem cells for hair growth is their additional potential as a cell source for other clinically important cel- lular therapies (e.g., epidermal and hair regener- ation in burn patients). Recent studies indicated that the bulge region, as predicted earlier, con- tains multipotential stem cells that have the potential to contribute to all three lineages of the epidermis. Protocols to isolate, expand, and direct differentiation of human adult epidermal stem cells remain to be devised,96 but the gene expression profiling done with isolated mouse cells gives some guidance as to potential mark- ers and factors of interest.
Challenges and Summary
There are a number of advantages and disad- vantages with the use of adult or ES cells for application in patients; however, for the thera- peutic administration of human ES cells to be an option, the rejection of cells by the immune sys- tem will have to be addressed. Although the immune response should be less intense than the response to xenotransplants, the major his- tocompatibility complex differences between human ES cells and a recipient will require immunosuppression (e.g., ref. 133), the extent of which must be determined for the transplanta- tion of cells into the epidermis. It is conceivable that stem cell-based therapy alone will not be the ultimate solution for the treatment of epi- dermal damage and loss of regeneration, and that therapies of the distant future might be a combination of stem cell transplantation, gene therapy, and drug treatment. Such a combina- tion of therapies will be customized to the par- ticular cellular ailment of the individual patient.
At this point, it is difficult to predict how stem cell-based therapy will be translated into clinical practice for epidermal tissues. Some of the limi- tations and potential issues regarding ES cell and adult stem cell plasticity have been dis- cussed; nevertheless, the promise is real and basic science advances continue to spur new skin cell biology that holds promise for novel skin cell therapeutics.
Acknowledgments
The authors thank Dr. Jane Aubin for many stimulating dis- cussions on stem cells over the years. Our work has been supported by a grant from the Canadian Institutes of Health Research (CIHR).
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