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Schwab SR, Pereira JP, Matloubian M et al. (2005) Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients. Science 309:1735–9 Shannon LA, McBurney TM, Wells MA et al. (2012) CCR7/CCL19 controls expression of EDG1 in T cells. J Biol Chem; (in press: Manuscript M111.310045; doi:10.1074/jbc.M111.310045).
Zaba LC, Krueger JG, Lowes MA (2009) Resident and ‘‘inflammatory’’ dendritic cells in human skin. J Invest Dermatol 129:302–8 Zhou X, Krueger JG, Kao MC et al. (2003) Novel mechanisms of T-cell and dendritic cell activation revealed by profiling of psoriasis on the 63,100-element oligonucleotide array. Physiol Genomics 13:69–78
See related article on pg 1707
Stem Cells in Tissue Repair and Regeneration Vincent Falanga1,2,3,4 The field of tissue repair and wound healing has blossomed in the past 30 years. We have gone from recombinant growth factors, to living tissue engineering constructs, to stem cells. The task now is to pursue true regeneration, thus achieving complete restoration of structure and function. Journal of Investigative Dermatology (2012) 132, 1538–1541. doi:10.1038/jid.2012.77
For most of this discussion, we will actually mean tissue repair, with its attendant compromise of achieving rapid wound closure at the expense of regeneration. Technically, the term ‘‘wound healing’’ implies complete restoration of tissue (i.e., regeneration) as the outcome. However, we believe that over time wound healing has also come to signify the overall processes taking place after injury, and more so from a biological standpoint. We should remain hopeful that true regeneration is possible in adult human tissues. Thus far, the evidence has not been encouraging, except for fetal wound healing. Even the liver, which many erroneously cite as an example of potential regeneration in adults, simply gets larger after lobe resection or injury; no true regeneration takes place. Figure 1 indicates the immense complexities that underlie chronic wounds. Regeneration
of the tissue shown in this figure would be extremely difficult, and would most likely first require complete removal of the non-healing and affected skin. Therefore, as we approach the topic of stem cells for wound healing/tissue repair, we must keep clinical realities in mind and what we can achieve with the methods and agents currently available. This is not meant to be a pessimistic view, but rather to stimulate progress toward the ultimate goal of achieving true regeneration. With a focus on wound healing and tissue repair, and particularly with the need represented by chronic non-healing human wounds, in the 1980s the excitement centered on growth factors (Falanga, 2005). However, in spite of the potential envisioned for recombinant growth factors, multiple clinical trials involving thousands of patients
1
Department of Dermatology and Skin Surgery, Roger Williams Medical Center, Providence, Rhode Island, USA; 2NIH Center of Biomedical Research Excellence, Roger Williams Medical Center, Providence, Rhode Island, USA; 3Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA and 4Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, USA Correspondence: Vincent Falanga, Department of Dermatology and Skin Surgery, Roger Williams Medical Center, Elmhurst Building, 50 Maude Street, Providence, Rhode Island 02908, USA. E-mail:
[email protected] or
[email protected]
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failed for the most part to show any, or more than modest, results. This failure surprised most investigators and pharmaceutical companies, because growth factors had substantial effects on many cell types in vitro (including fibroblasts, endothelial cells, and keratinocytes), and they had been shown to accelerate healing in experimental animal models. The problem was that laboratory animals do not suffer from chronic wounds, and even several innovative models could not reproduce the failure to heal that characterizes many human chronic wounds. Some investigators were not deterred. They then hypothesized that the growth factors could not penetrate the wound bed in sufficient concentrations, were easily broken down within the wound, or required complex methods of delivery, including the administration of several peptides in a specific sequence and at certain concentrations. We even hypothesized that growth factors were ‘‘trapped’’ or sequestered within the wound, for example, by albumin or a2-macroglobulin, the latter being a known scavenger molecule for platelet-derived growth factor (Falanga, 2005). This final hypothesis, similar to the others mentioned above, remains viable. Nevertheless, the problem with complex hypotheses is that, from a regulatory standpoint, it is difficult to move from the bench to the bedside. However, the field of wound healing, being very important, has kept moving forward. Tissue engineering and bioengineered skin constructs, first developed many years earlier, began to dominate the field in the 1990s. Indeed, the scientific and clinical rationale for certain constructs, especially those comprising living cells such as fibroblasts, keratinocytes, or both, was that a tissuelike product could overcome the deficiencies potentially uncovered using growth factors. In engineering terms, a living construct could act in a ‘‘smart’’ way, meaning that it would adapt to the wound microenvironment, release just enough peptide growth factors and in the right sequence, and possibly provide additional and much needed extracellular matrix proteins. Basically, this approach and hypothesis was a way to admit our own ignorance about the underpinning of failure to heal, and
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a
b
Some reasons for impaired healing Fibrosis, prolonged hypoxia Failure to reepithelialize Faulty angiogenesis and ECM Excessive, inappropriate MMPs Bacterial colonization/biofilms Phenotypic cellular changes
Impaired healing
Inflammation
Remodeling
Coagulation
Proliferation, migration Short-circuited processes of wound healing in chronic wounds
Figure 1. Difficulties underlying chronic wounds. (a) Panel a on the left shows a non-healing chronic wound attributable to a combination of venous and lymphatic disease. Both arrows point to the nonmigrating epidermis. The solid arrow shows the steep edges at the periphery of the wound, whereas the dashed arrow is directed at an island of fully epithelialized tissue within the wound bed, but with continued failure of its epidermis to migrate. (b) Panel b on the right shows a diagrammatic representation of the phases of wound healing which, in the case of chronic wounds, are not linear or predictable in their sequence. In addition, different areas of the wound are likely to be in a different phase of wound healing. Broken lines illustrate that this is a dynamic situation and not easily predictable. ECM, extracellular matrix; MMPs, matrix metalloproteinases.
therefore relying on cells and constructs to do the work. It should be noted that the cells in allogeneic constructs are not detectable after 4–6 weeks, further suggesting that the constructs act in a pharmacological mode, rather than replacement therapy (Phillips et al., 2002). There have been notable successes with the use of bioengineered skin, and indeed some are used in major burns and some are approved by the Food and Drug Administration (FDA) for use in chronic wounds (Falanga and Sabolinski, 1999; Marston et al., 2003). Important lessons were learned from these advanced therapies, which could benefit the future clinical use of stem cells in burns and chronic wounds. After regulatory approval of these advanced therapies, it was quickly noted that the favorable results obtained during clinical trials were not matched in clinical practice. This discrepancy was eventually remedied, but the finding points to an often-underestimated aspect of science and clinical practice. We generally adhere to the paradigm that we are already doing our best clinically and that new therapies will result from further ‘‘bench’’ studies of mechanisms of disease. In fact, in wound healing and other diseases, it is the introduction of a new therapy that, in turn, leads to
a better understanding of pathogenic mechanisms. In the case of chronic wounds, our knowledge of failure to heal proved to be quite unsatisfactory. As a result of the availability of new advanced therapies and because we desperately needed to have them work well clinically, among other findings, we discovered or realized the following: that resident wound fibroblasts did not respond well to the stimulatory actions of growth factors; that frequent debridement was also a way to remove these and other phenotypically altered cells; that the excessive wound exudate would break down bioengineered skin, associated with a shift in wound matrix metalloproteinases thereby impairing epithelialization; that wound bacterial colonization and the presence of biofilms were critical parameters; and that even the choice of wound dressings and how they were removed could minimize epidermal and dermal reinjury. We have previously reviewed these abnormalities and findings in chronic wounds (Falanga, 2005). Eventually, we developed the now widely adhered to concept of wound bed preparation (WBP), in which clinical and mechanistic aspects of impaired healing are corrected as much and as promptly as possible (Falanga, 2000; Panuncialman and Falanga, 2009).
It was not long before interest in stem cells would offer fresh hope in the field of wound healing. This interest now dominates our time and is obscuring, perhaps unwisely, some of the useful approaches achieved with growth factors and bioengineered constructs. Turning to human embryonic stem cells seemed attractive in principle, were one to overcome the ethical issues regarding their use (and which may also compromise the ‘‘quality’’ or reproducible behavior of cells kept long term in culture). After all, although not totipotent (i.e., able to also form placental tissues), embryonic stem cells are pluripotent and can give rise to all cells by using appropriate culture conditions. The ethical issues seemed to diminish overnight when in 2007 two separate publications described the development of induced pluripotent stem cells (iPS cells) from fibroblast cultures using a cocktail of genes. One investigative team used Oct3/4, Sox2, Klf4, and c-Myc by retroviral delivery (Takahashi et al., 2007), whereas the other team group used Oct4, Sox2, Nanog, and Lin28 by lentiviral vectors (Yu et al., 2007). The likelihood of producing a single iPS cell from fibroblast cultures was relatively low (requiring 5–10 thousand cells), but a major coup had taken place. Suddenly, human embryonic stem cells and their pluripotential capacity lost some of their attractiveness, because we were now able to develop pluripotent cells from adult skin fibroblasts. Over time, other methods (i.e., naked DNA, proteins, etc.) of delivering these and other critical reprogramming genes have been reported. Questions linger about whether iPS cells are truly embryonic-like, but nobody can deny the importance of taking a differentiated cell back to its possible origin. For wound healing and tissue repair, however, pluripotent stem cells remain a problem. Pluripotentiality may not be required to treat wounds safely, and the teratoma concern and regulatory and safety hurdles remain. The importance of the immunological rejection of these cells, recently identified as an unexpected finding, remains to be determined. However, alternative strategies are enjoying greater attention. Among these are bone marrow mobilization www.jidonline.org 1539
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Clinical Implications
It is appropriate to use the term ‘‘tissue repair’’ rather than ‘‘wound healing’’ when discussing clinical outcome, because ‘‘wound healing’’ refers to regeneration, which implies complete restoration of structure and function.
Multiple pathogenic abnormalities have roles in impaired healing, including phenotypic changes in resident wound cells, excessive exudate, and altered matrix metalloproteinases profiles, and the presence of biofilms and bacterial colonization.
Use of multipotent stem cells in chronic wounds and repair is easier to study, has better reproducibility, and is more compatible with stringent regulatory steps and requirements.
Wound bed preparation (WBP) is going to become an important aspect of treating difficult wounds with stem cells, just as it has been in the use of recombinant growth factors and bioengineered skin constructs.
and peripheral blood harvesting of stem cells with apheresis after the systemic administration of granulocyte colonystimulating factor, the use of stem/progenitor cells from umbilical cord blood, and culturing of multipotent (giving rise to one tissue type) stem cells from bone marrow, epidermis, hair, and (as reported by the current publication by Danner et al. this issue, 2012) sweat glands. From a scientific and practical standpoint (which ultimately has ramifications for safety and regulatory oversight), an advantage of multipotent stem cells is that we can more reliably depend on certain surface markers and appearance or behavior in culture to confirm that we are growing the same type of cells. For example, when our group first decided to use autologous cultured cells from bone marrow in wounds, we focused on mesenchymal stem cells. Although mesenchymal cell surface markers are not specific, the combination of such markers (i.e., lineage negative and CD34 , positivity for CD29, CD44, CD105, CD166), adherence to tissue culture plastic and cell culture appearance, and functional assays showing differentiation to bone, cartilage, or adipose tissue, provide a reproducible system (Falanga et al., 2007). We could have chosen a hematopoietic stem/progenitor cell type, which might have been more effective, but then we would not have known necessarily the attributes of that cell and we could not have ensured consistent and reproducible cultures in our good manu-
facturing practice facilities. The mesenchymal stem cells we have used in human wounds appear to differentiate along fibroblastic pathways and, in an animal model, do not seem to persist for more than 30 days, in spite of them being autologous. Nevertheless, they appear to be promising for use in difficult wounds and, as suggested by published reports, may decrease scarring—a major advantage in treating burns. We are intrigued by the report by Danner et al. 2012 that stem cells isolated from sweat glands exhibit features highly suggestive of endothelial cells. This raises the issue of whether stem cell transdifferentiation or plasticity, a process regarded by some as controversial, is playing a role. Some have suggested that plasticity is the result of co-purification with dormant pluripotent very small embryonic-like (VSEL) stem cells in various adult organs and tissues (Kucia et al., 2006). VSEL stem cells might be of interest in wound healing, provided that an adequate yield from apheresis could be achieved and that the potential for tumor formation, which is associated with embryonic stem cells, is not as great. The use of multipotent, rather than pluripotent, stem cells in wound healing appears to be beneficial and more practical at this point, given the regulatory and safety concerns. Regulatory requirements continue to increase, even as (or because) we are placing more emphasis on translational research. Therefore, investigators interested in
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applied approaches for stem cells must take into account whether the stem cells they choose to study will be deemed to be a relatively low risk in tissue repair. Moreover, lessons from tissue engineering constructs and from WBP have taught us that true replacement of cells may not be necessary for most wounds. There could be a ‘‘didactic paradigm’’ whereby certain subpopulations of multipotent stem cells may ‘‘teach’’ the wound microenvironment how to rectify the cellular and molecular phenotypic flaws that lead to impaired healing. It is also plausible that recruitment of circulating stem cells or neighboring tissuedifferentiated cells may take place with the topical administration of multipotent stem cells. The number of delivered stem cells seems critical. In our mesenchymal stem cell studies in humans, we found that at least one million autologous cultured stem cells per cm2 of wound surface were required to achieve a beneficial outcome. Progress continues in our ability to isolate stem cells, to characterize them, and to think of ways to deliver and use them in translational research. For now, multipotent stem cells are more useful for the acceleration of healing, particularly owing to their generally favorable risks/benefits ratio. Nevertheless, we do not reject the notion that pluripotent stem cells may ultimately find an extraordinary role in wound healing. When properly handled and controlled, such cells could be directed toward diverse differentiation pathways that might bring the field of tissue repair closer to regeneration, or true wound healing. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This work was supported by funds from the following National Institutes of Health Grants (V. Falanga as the Principal Investigator): NIH NIAMS R01 grant, Award #AR060342; NIH Center of Biomedical Research Excellence (COBRE) from NCRR Award #P20RR018757-10; and NIGMS Award (8 P20 GM103414-10), including the COBRE Administrative and Imaging Cores. It was also supported by the Wound Biotechnology Foundation.
REFERENCES Danner S, Kremer M, Petschnik AE et al. (2012) The use of human sweat gland-derived stem
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cells for enhancing vascularization during dermal regeneration. J Invest Dermatol 132:1707–16
cells identified in adult bone marrow. Leukemia 20:857–69
Falanga V (2000) Classifications for wound bed preparation and stimulation of chronic wounds. Wound Repair Regen 8:347–52
Marston WA, Hanft J, Norwood P et al. (2003) The efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers: results of a prospective randomized trial. Diabetes Care 261701–5
Falanga V (2005) Wound healing and its impairment in the diabetic foot. Lancet 366:1736–43 Falanga V, Iwamoto S, Chartier M et al. (2007) Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng 13:1299–312
Panuncialman J, Falanga V (2009) The science of wound bed preparation. Surg Clin North Am 89:611–26 Phillips TJ, Manzoor J, Rojas A et al. (2002) The longevity of a bilayered skin substitute after application to venous ulcers. Arch Dermatol 138:1079–81
Falanga V, Sabolinski M (1999) A bilayered living skin construct (APLIGRAF) accelerates complete closure of hard-to-heal venous ulcers. Wound Repair Regen 7:201–7
Takahashi K, Tanabe K, Ohnuki M et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–72
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Targeting the Palm: A Leap Forward Toward Treatment of Keratin Disorders Wera Roth1, Mechthild Hatzfeld2 and Thomas M. Magin1 Any rational therapy benefits from an understanding of basic biology and the simplicity of its strategy. Among keratinopathies, epidermolytic palmoplantar keratoderma stands out by virtue of hotspot mutations in the KRT9 gene, exclusively expressed in the palmoplantar epidermis. In this issue, Leslie Pedrioli et al. report on the successful application of KRT9-specific siRNAs in cultured cells and in a mouse model. The study beautifully illustrates the potency of a thorough experimental approach and the challenges that remain, especially in its delivery. Journal of Investigative Dermatology (2012) 132, 1541–1542. doi:10.1038/jid.2012.99
Efficacy, specificity, and potency of a drug represent the lynchpins of a successful therapy. In the case of genetic disorders, onset of disease and the cell type of origin mount additional hurdles to be overcome. Keratinopathies are caused mostly by dominant mutations in at least 23 of the 54 human keratin genes expressed as the ‘‘keratin pairs’’ that typify epithelial differentiation (Szeverenyi et al., 2008; http://www.interfil.org). Therefore, sites
of expression reveal the major site(s) of disease, despite the notion that most keratinocytes express 4–10 different keratin proteins. Further, there appears to be reasonable genotype–phenotype correlation, indicating that mutations severely compromising the cytoskeleton’s integrity cause more severe disease phenotypes than those that do not. Although pathomechanisms of the keratinopathies are more complex than originally thought
1
Division of Cell and Developmental Biology, Translational Centre for Regenerative Medicine and Institute of Biology, University of Leipzig, Leipzig, Germany and 2Institut fu¨r Molekulare Medizin, Martin-LutherUniversita¨t Halle-Wittenberg, Halle, Germany Correspondence: Thomas M. Magin, Division of Cell and Developmental Biology, Translational Centre for Regenerative Medicine and Institute of Biology, University of Leipzig, Philipp-Rosenthal-Strae 55, Leipzig D-04103, Germany. E-mail:
[email protected]
(Coulombe and Lee, 2012), one can reasonably argue that reducing the expression of the mutant allele in dominant keratin disorders should restore a more functional cytoskeleton from the intact allele, leading to greater tissue integrity. Proof of principle stemmed from mouse models in which the ratio of mutant and normal keratin alleles has been modified genetically (Cao et al., 2001; Hesse et al., 2007). Among keratinopathies, epidermolytic palmoplantar keratoderma (EPPK) is unique for several reasons: the expression of the culprit, KRT9, is restricted to the upper strata of the palmoplantar epidermis, forming a cytoskeleton together with at least six additional keratins. The majority of EPPK patients suffer from a missense mutation in one of the three hotspot codons, giving rise to a focal epidermolytic keratoderma (http://www.interfil.org). This setting invited Leslie Pedrioli et al. (this issue, 2012) to develop an siRNA-based therapy approach, testing both generic and mutation-specific siRNAs directed against KRT9. The team first scanned all possible 19-mer siRNAs for the repression of KRT9, using transiently expressed luciferase reporters to monitor specificity and efficacy of siRNAs. Next, siRNAs that efficiently inhibited the two prominent KRT9 missense mutations M157V and R163Q were identified using a similar strategy. The best siRNAs were able to repress a mutant KRT9 allele in the 50 pM range, without apparently affecting the expression of other keratins. Ultimately, siRNAs must be delivered in situ. Unfortunately, no mouse model for KRT9 is currently available. As a first step, Leslie Pedrioli et al. coinjected the most potent siRNA, siR163Q-13, together with a mutant KRT9-luciferase reporter carrying the same mutation, into mouse footpad epidermis. This delivery route had been previously approved in a phase Ib clinical trial for pachyonychia congenita (Leachman et al., 2010). To test for specificity, a wild-type KRT9-luciferase reporter was applied together with the above siRNA in another footpad. Despite the limitations imposed by the nature of the delivery, i.e., injection, the data suggest that the siRNA was more specific in repressing the mutant compared with the normal allele. www.jidonline.org 1541
