Wound Healing: Stem Cells Repair and Restorations, Basic and Clinical Aspects

By Kursad Turksen

A comprehensive resource on the recent developments of stem cell use in wound healing

With contributions from experts in the field, Wound Healing offers a thorough review of the most recent findings on the use of stem cells to heal wounds. This important resource covers both the basic and translational aspects of the field. The contributors reveal the great progress that has been made in recent years and explore a wide range of topics from an overview of the stem cell process in wound repair to inflammation and cancer. They offer a better understanding of the identities of skin stem cells as well as the signals that govern their behavior that contributes to the development of improved therapies for scarring and poorly healing wounds.

Comprehensive in scope, this authoritative resource covers a wealth of topics such as: an overview of stem cell regeneration and repair, wound healing and cutaneous wound healing, the role of bone marrow derived stems cells, inflammation in wound repair, role and function of inflammation in wound repair, and much more. This vital resource:

• Provides a comprehensive overview of stem cell use in wound healing, including both the basic and translational aspects of the field
• Covers recent developments and emerging subtopics within the field
• Offers an invaluable resource to clinical and basic researchers who are interested in wound healing, stem cells, and regenerative medicine
• Contains contributions from leading experts in the field of wound healing and care

Wound Healing offers clinical researchers and academics a much-needed resource written by noted experts in the field that explores the role of stem cells in the repair and restoration of healing wounds. 

• Language: English
• Publisher: Wiley
• Release date: Nov 27, 2017
• ISBN: 9781119282501

Book preview

List of Contributors

Heidi Abrahamse PhD Laser Research Centre University of Johannesburg Johannesburg, South Africa Daniel D. Bikle MD, PhD VA Medical Center and University of California San Francisco, CA, USA Johanna M. Brandner PhD Department of Dermatology and Venerology University Hospital Hamburg-Eppendorf Hamburg, Germany Olivier Alexandre Branford PhD Queen Victoria Hospital East Grinstead, West Sussex, UK Joanna Bukowska PhD Institute of Animal Reproduction and Food Research, Polish Academy of Sciences Olsztyn, Poland Bruce A. Bunnell PhD Center for Stem Cell Research and Regenerative Medicine and Department of Pharmacology Tulane University School of Medicine New Orleans, LA, USA Melissa Crawford BSc (Hons) Department of Physiology and Pharmacology Children's Health Research Institute and Lawson Health Research Institute The University of Western Ontario London, Ontario, Canada Lina Dagnino PhD Department of Physiology and Pharmacology Children's Health Research Institute and Lawson Health Research Institute The University of Western Ontario London, Ontario, Canada Duncan Hieu M. Dam PhD Department of Dermatology Skin Disease Research Center (SDRC) Northwestern University Chicago, IL, USA Selami Demirci PhD National Heart, Lung, and Blood Institute (NHLBI) NIH, Bethesda MD, USA Luisa A. DiPietro DDS, PhD Center for Wound Healing and Tissue Regeneration University of Illinois at Chicago Chicago, IL, USA Ayşegül Doğan PhD National Cancer Institute (NCI) NIH, Frederick MD, USA Eduardo Escario MD Dermatology Service University General Hospital of Albacete Associate Professor, University of Castilla-La Mancha School of Medicine Spain Trivia Frazier PhD LaCell LLC, New Orleans, LA, USA and Center for Stem Cell Research & Regenerative Medicine and Department of Structural and Cellular Biology Tulane University School of Medicine New Orleans, LA, USA Barbara Gawronska-Kozak DSc, PhD Institute of Animal Reproduction and Food Research, Polish Academy of Sciences Olsztyn, Poland Shibnath Ghatak PhD Department of Regenerative Medicine and Cell Biology Medical University of South Carolina Charleston, SC, USA Jeffrey M. Gimble MD, PhD LaCell LLC, New Orleans LA, USA and Center for Stem Cell Research and Regenerative Medicine and Departments of Structural and Cellular Biology, Medicine, and Surgery Tulane University School of Medicine New Orleans, LA, USA Vincent C. Hascall PhD Department of Biomedical Engineering Cleveland Clinic Cleveland, OH, USA Nicolette Nadene Houreld DTech Laser Research Centre University of Johannesburg Johannesburg, South Africa Ander Izeta PhD Tissue Engineering Laboratory, Bioengineering Area Instituto Biodonostia, Hospital Universitario Donostia and Department of Biomedical Engineering, School of Engineering Tecnun-University of Navarra San Sebastián, Spain Sophia A. Jelsma BS Department of Dermatology Skin Disease Research Center (SDRC) Northwestern University Chicago, IL, USA Francisco Jimenez MD Mediteknia Dermatology and Hair Transplant Clinic Associate Professor University Fernando Pessoa Canarias, Gran Canaria, and Medical Pathology Group, ULPGC Canary Islands, Spain Jasreen Kular BSc, BMedSci(Hons), PhD Centre for Cancer Biology SA Pathology and University of South Australia Adelaide, South Australia, Australia Sathish Sundar Dhilip Kumar PhD Laser Research Centre University of Johannesburg Johannesburg, South Africa Feyzan Őzdal Kurt PhD Department of Biology Faculty of Sciences and Letters Manisa Celal Bayar University Manisa, Turkey Jeffery T. Kwock MD Department of Dermatology and Immunology Duke University Medical Center Durham, NC, USA Alexandra Laberge MD, Msc Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX Department of Surgery, Faculty of Medicine Université Laval and Centre de Recherche du CHU de Québec-Université Laval Québec, QC, Canada Michael T. Longaker MD, MBA Department of Surgery and Reconstructive Surgery The Stanford University Medical Center Stanford, CA, USA H. Peter Lorenz MD Department of Surgery and Reconstructive Surgery The Stanford University Medical Center Stanford, CA, USA Amanda S. MacLeod MD Department of Dermatology and Immunology, Duke University Medical Center Durham, NC, USA Roger R. Markwald PhD Department of Regenerative Medicine and Cell Biology Medical University of South Carolina Charleston, SC, USA Clement D. Marshall MD Department of Surgery and Reconstructive Surgery The Stanford University Medical Center Stanford, CA, USA María Luisa Martínez MD Dermatology Service Hospital General Universitario of Albacete Spain Suniti Misra PhD Department of Regenerative Medicine and Cell Biology Medical University of South Carolina Charleston, SC, USA Alessandra A. Moore MD Department of Surgery and Reconstructive Surgery The Stanford University Medical Center Stanford, CA, USA Ricardo Moreno Rodriguez PhD Department of Regenerative Medicine and Cell Biology Medical University of South Carolina Charleston, SC, USA Véronique J. Moulin PhD Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX Department of Surgery, Faculty of Medicine Université Laval and Centre de Recherche du CHU de Québec-Université Laval Québec, QC, Canada Paul E. O'Brien MD Hematology/Oncology Division Medical University of South Carolina Charleston, SC, USA Yuko Oda PhD VA Medical Center and University of California San Francisco, CA, USA Amy S. Paller MD Department of Dermatology, Skin Disease Research Center (SDRC) Northwestern University Chicago, IL, USA Hayley S. Ramshaw BSc (Hons), PhD Centre for Cancer Biology SA Pathology and University of South Australia Adelaide, South Australia, Australia and Adelaide Medical School Faculty of Health and Medical Sciences University of Adelaide Adelaide, South Australia, Australia Kerstin J. Rolfe PhD BCOM London, UK Fikrettin Şahin PhD Department of Genetics and BioEngineering Faculty of Engineering Yeditepe University Kayisdagi, Istanbul, Turkey Michael S. Samuel BSc (Hons), PhD Centre for Cancer Biology SA Pathology and University of South Australia Adelaide, South Australia, Australia and Adelaide Medical School Faculty of Health and Medical Sciences University of Adelaide Adelaide, South Australia, Australia Megan Schrementi PhD Department of Science and Health, DePaul University Chicago, IL, USA Amy Lin Strong MD PhD Center for Stem Cell Research and Regenerative Medicine and Departments of Structural and Cellular Biology Tulane University School of Medicine New Orleans, LA, USA Chia-ling Tu PhD VA Medical Center and University of California San Francisco, CA, USA Elgin Türköz Uluer MD Department of Histology and Embryology Faculty of Medicine Manisa Celal Bayar University Manisa, Turkey Hafize Seda Vatansever MD, PhD Department of Histology and Embryology Faculty of Medicine Manisa Celal Bayar University, Manisa, Turkey and Experimental Health Research Center of Health Sciences Near East University Mersin, Turkey Thomas Volksdorf PhD Department of Dermatology and Venerology University Hospital Hamburg-Eppendorf Hamburg, Germany Xiying Wu MD LaCell LLC New Orleans, LA, USA

Chapter 1

Stem Cell Regeneration and Repair – Overview

Clement D. Marshall, Alessandra A. Moore, Michael T. Longaker and H. Peter Lorenz

Department of Surgery and Reconstructive Surgery, The Stanford University Medical Center, Stanford, CA, USA

Introduction

While at first glance the skin appears to be no more than a static unchanging surface, it is in fact a complex, dynamic organ that continuously replenishes its cellular and molecular content. In addition to this homeostatic maintenance, skin has evolved the remarkable ability to rapidly repair itself after injury. For our most distant ancestors, there was presumably evolutionary pressure to rapidly restore the barrier function of skin before infection could set in. The result of this evolutionary necessity is scar tissue, which serves as a protective barrier, but falls short in several ways compared with uninjured skin. In humans, the end point of healing all but the smallest injuries is the formation of scar [1]. For most people, scars are a cosmetic concern, but many patients are affected by major scars that result in debilitating contractures as well as disfigurement in aesthetically sensitive areas such as the face. Children with major scars in visible areas such as the face often suffer from long-term psychological stress and impaired self-esteem [2]. If excessive scar formation represents one end of the human wound healing spectrum, the other end consists of chronic and non-healing wounds. Often arising in patients with diabetes, peripheral arterial disease, impaired mobility, and other comorbidities, chronic wounds typically require months of intensive treatment and consume substantial healthcare resources [3].

Stem cells are a key cellular player in the repair of skin after injury and during normal homeostasis. Tremendous progress has been made in recent years toward delineating the role of stem cells in these processes. An improved understanding of the identities of skin stem cells as well as the signals that govern their behavior will hopefully allow for the development of improved therapies for scarring and poorly healing wounds. This chapter will begin with an overview of the events of normal wound healing and stem cell biology and will then review our current understanding of the role of stem cells in skin regeneration and repair.

Overview of Skin Wound Healing

The major function of skin is to provide a barrier that excludes noxious and infectious agents of the outside world while protecting underlying structures from trauma and preventing the loss of valuable body fluid. Wound repair appears to have evolved in a way that rapidly restores these functions while simultaneously preventing infection of the wound (Figure 1.1).

Diagram shows inflammation stage wound with labels for fibrin clot, oxygen, bacteria, platelet et cetera and wound during new tissue formation with labels for eschar, monocyte, macrophage, new blood vessel and granulation tissue.

Figure 1.1 There are three classic stages of wound repair: (a) inflammation, (b) new tissue formation, and (c) remodeling.

(a) Inflammation. This stage lasts until about 48 h after injury. Depicted is a skin wound at about 24–48 h after injury. The wound is characterized by a hypoxic (ischaemic) environment in which a fibrin clot has formed. Bacteria, neutrophils, and platelets are abundant in the wound. Normal skin appendages (such as hair follicles and sweat duct glands) are still present in the skin outside the wound. (b) New tissue formation. This stage occurs about 2–10 days after injury. Depicted is a skin wound at about 5–10 days after injury. An eschar (scab) has formed on the surface of the wound. Most cells from the previous stage of repair have migrated from the wound, and new blood vessels now populate the area. The migration of epithelial cells can be observed under the eschar. (c) Remodeling. This stage lasts for a year or longer. Depicted is a skin wound about 1–12 months after repair. Disorganized collagen has been laid down by fibroblasts that have migrated into the wound. The wound has contracted near its surface and the widest portion is now the deepest. The re-epithelialized wound is slightly higher than the surrounding surface and the healed region does not contain normal skin appendages. (Reproduced from Gurtner et al. [1], with permission from Nature Publishing Group.)

The first events after a skin injury has occurred relate to the restoration of hemostasis. A fibrin and platelet plug prevents ongoing bleeding from blood vessels. The fibrin matrix that composes the plug provides a scaffold for wound healing cells that will migrate in later. Activated platelets in the injury provide early chemical signals that activate other cells and potentiate further wound healing events [4].

The first phase of a true wound repair is known as the inflammatory phase. Immune cells such as macrophages, neutrophils, and lymphocytes enter the wound tissue and begin the process of removing bacteria, dead cells, and other debris [5]. Cytokines released during wounding and hemostasis are critical for the recruitment of these immune cells to the wound [4]. Immune cell influx is accompanied by a local inflammatory reaction characterized by increased blood flow and capillary leaking, causing the typical symptoms of redness, swelling, and increased warmth. In addition to cleaning the wound area and removing infectious agents, immune cells release a host of cytokines and other chemical mediators that encourage other cells to engage in healing behaviors [4].

Inflammation is followed by the proliferative phase of wound healing. This refers to the migration of cells into the wound, particularly fibroblasts and keratinocytes, that are responsible for building new tissue to reconstruct the wound. These cells are highly responsive to chemical mediators released by immune cells during the inflammatory phase [6]. New epidermis and dermis are constructed to replace the empty space left by the wound during this phase. In almost all wounds, the new skin is built in the form of scar [1]. Compared with normal skin, scar lacks hair follicles and sweat glands, is stiffer, and is often raised and hyperpigmented. The basement membrane of the epidermis in scar is flat and does not contain the rete pegs that normally project down into the dermis [7] (Figure 1.2). Large scars, particularly those located over a joint, often contract as a result of myofibroblast action. This contraction occurs in the remodeling phase of wound healing, during which scar extracellular matrix, including collagen, is extensively remodeled [4]. Contractures can be painful and cause severe physical impairment, particularly in patients with large burn scars [8].

Image described by caption.

Figure 1.2 Masson's trichrome staining of the interface between normal (left) and scarred (right) dorsal skin in the adult mouse.

Normal skin contains hair follicles and other dermal appendages. Scarred skin does not contain these appendages and the epidermis is flattened. Note that that scarred dermis is thicker than the normal dermis. Scale bar, 500 μm.

While stem cells are normally active at a low level in uninjured skin to maintain homeostasis, they are recruited during the proliferative phase of wound healing to provide large numbers of new cells to populate the healing wound [9].

Stem Cell Definition: History

Most cells have short life spans and are not capable of indefinite self-renewal. In the 1970s, the concept of the stem cell was developed to describe a special population of cells that divide in order to replenish a population of differentiated cells but do not themselves differentiate. Over time the definition of the stem cell has evolved [10]. Today, stem cells are generally considered to be undifferentiated cells that self-renew and that produce differentiated cells as progeny [11]. Within this broad definition there are many types of stem cells that differ based on their capacity for long- or short-term self-renewal and the number of different cell types that they produce [11].

The presence of stem cells has been verified in most tissues of the body, although certain tissues such as the pancreas may not contain stem cells [10, 12]. The precise manner in which a stem cell behaves and produces differentiated progeny differs depending on the tissue involved. Cell surface markers and genes expressed by stem cells also differ markedly between tissues, making identification and isolation of stem cells sources challenging. Many stem cells express regulatory genes that are switched off in their progeny, making the identification of the progeny cells in vivo more difficult. Furthermore, a stem cell's progeny may change its morphology, cell surface marker expression, or migrate to a new location.

A lineage can be defined as a particular population of cells and all subsequent cells descended from them, regardless of location or phenotype. The concept of lineage tracing was developed in order to follow cell offspring as they migrate to new locations and change surface marker and gene expression profiles [13] (Figure 1.3). Lineage tracing comprises multiple techniques that involve physically or genetically marking cells or populations of cells with a reporter in such a way that all of their progeny retain the reporter. This allows cells of a certain lineage to be detected even if they change location, gene expression, or surface marker profile [22].

Image described by caption.

Figure 1.3 Different approaches to lineage tracing.

(a) Direct observation, as pioneered by Whitman and colleagues (exemplified by a plate from Conklin, 1905) [14]. (b) Schematic showing agar chips with vital dyes applied on to the surface of an early stage amphibian embryo (top). These dyes label regions within later stage embryos (bottom) (based on Vogt, 1929; adapted from Gilbert, 2000 [15, 16]). (c) Use of soluble carbocyanine dyes to fate map chick neural crest. (Reproduced from Serbedzija et al., 1989, with permission from The Company of Biologists Ltd [17].) (d) Whole-mount of mouse epidermis showing DNA label-retaining stem cells in the hair follicle bulge. (Reproduced from Braun et al., 2003 [18].) Red: keratin 14; green: BrdU. Scale bar: 100 μm. (e) LacZ retroviral vector introduced into rat retinal cells (upper panel) and subsequently tracked in the reconstituted retina. (Reproduced from Price et al., 1987, with permission from C. Cepko [19].) (f) Schematic showing Spemann and Mangold's organizer experiment, which was performed by grafting tissues between amphibian embryos (adapted from Grove, 2008 [20]). (g and h) Adult mouse chimeras from GFP-positive and -negative mice. (g) Whole-mount of lung (reproduced from Giangreco et al., 2009 [21]). (h) Histology of skin tumor (reproduced from Arwert et al., 2010 [22]). The GFP-positive region in (h) is brown. (Reproduced with permission from Kretzschmar and Watt [13].)

In perhaps the most powerful and adaptable method for lineage tracing, a gene expressing the Cre recombinase enzyme is inserted into the genome under the control of a specific gene promoter that defines a lineage (i.e., the gene of interest). A reporter gene is inserted separately. When the gene of interest is expressed, the Cre recombinase permanently activates the reporter gene by altering the reporter gene's genetic sequence in that cell. In this way, the reporter remains activated in the cell and in all of its offspring, but not in cells that never expressed the gene of interest [24]. Further refinements of this technique allow the activation of the reporter gene to be inducible. In this way, a researcher can choose a specific time point to begin the lineage tracing. Lineage tracing with inducible methods has allowed for more precise characterization of the behavior of cells at different time points in the developing embryo, which in turn has revolutionized the field of developmental biology [25].

Stem Cells in Skin Homeostasis and Repair

Lineage tracing using Cre recombinase has been critical in defining the identities and roles of various stem cell populations of the skin. These experiments are generally carried out in mice and, as a result, much of our knowledge of skin stem cell biology may not be totally applicable to other species. In general, the stem cells of a particular structure within the skin are mainly responsible for maintaining the cell population of that niche. Often, though, stem cells based in a given location give rise to progeny that migrate to other locations and participate in the regeneration of distant tissues.

The skin contains several structures that each contain their own unique compartment of stem cells. These include the interfollicular dermis, hair follicles, sebaceous glands, eccrine sweat glands, and dermal papillae. Another group of stem cells that participate in skin regeneration and repair are circulating mesenchymal stem cells.

Interfollicular Epidermis

The interfollicular epidermis (IFE) is the region of epidermis located between hair follicles. In the uninjured state, stem cells residing in the basal layer of the IFE divide at a steady rate in order to provide new keratinocytes to populate the epidermis [9]. These cells are characterized by expression of the gene Lrig1 [26]. Loss of Lrig1 in these cells results in hyperproliferation of epidermal cells, suggesting that it has a role in preventing excessive growth. Lrig1+ cells also contribute to the growth of sebaceous glands and hair follicles [26].

The gene Lgr6 is a marker of primitive epidermal stem cells that in the prenatal state establish all lineages of the skin, including cells of the hair follicle, sebaceous gland, and interfollicular dermis. In postnatal life, Lgr6-expressing cells reside above the hair follicle bulge and regenerate the sebaceous gland and the IFE [27].

While the IFE can receive contributions of cells from other structures such as the hair follicle, the IFE is also capable of repairing and renewing itself in the absence of these other cells [9].

Hair Follicle

The cellular content of the hair follicle is exceptionally well studied and serves as a model for stem cell biology as a whole. The hair follicle is a complex structure with several distinct regions that contain unique populations of hair follicle stem cells (HFSCs) (Figure 1.4). While the HFSCs can regenerate the follicle itself, an important unresolved question is to what extent HFSCs have a meaningful role in regeneration of the skin following injury.

Image described by caption.

Figure 1.4 Heterogeneity of epidermal and hair follicle stem cells.

Skin epithelia feature distinct stem cell populations both in epidermis and, most prominently, in hair follicles. Epithelial stem cells in different microanatomical locations have different lineage potentials. Stem cells in the follicular infundibulum and interfollicular epidermis physiologically are restricted to epidermal fate. Interfollicular epidermal stem cells can be identified as slow-cycling in label retention studies, but distinct markers remain elusive. The isthmus and junctional zone of hair follicles harbor several distinct epithelial cell populations. Most prominent among them are Lrig1+ (yellow), Gli1+, and Lgr6+ stem cells (green), all of which physiologically maintain the isthmus and contribute to sebaceous gland, infundibulum and in some instances to interfollicular epidermis. Blimp1 identifies unipotent sebaceous gland progenitors (orange). The bulge stem cells (blue) normally contribute to all hair follicle lineages and can be identified based on the expression of Krt15, CD200, Lgr5, CD34, Sox9, Lhx2, Tcf3 and Nfatc1. The secondary germ of telogen hair follicles (purple) contains committed hair follicle-fated progenitors that express CD200, Gli1 and Lgr5. (Reproduced from Plikus et al. [9], with permission from Elsevier.)

In the developing embryo, the hair follicle is initially formed by separate populations of HFSCs expressing Lhx2 and Sox9, respectively [28]. The Lhx2+ cells appear to contribute transiently to hair follicle development while Sox9+ cells persist for longer. The developed hair follicle normally cycles through three stages of growth: catagen (regression), telogen (resting), and anagen (growth), a process that also involves varying contributions from different HFSC populations [28].

Stem cells residing in the bulge region of the hair follicle were the first HFSCs to be discovered and are characterized by expression of the genes Krt15, Lgr5, and Gli1, among others [9, 29]. Initially it was thought that these cells could contribute to skin regeneration, since they were found in the epidermis after a scratch injury [30]. However, subsequent sophisticated analyses revealed that while bulge cells transiently contribute progeny to the healing epidermis, these cells eventually disappear and in the long-term bulge stem cells are only capable of regenerating the hair follicle itself [9].

The junctional zone of the hair follicle is above the bulge and adjacent to the sebaceous gland. It contains a complex population of stem cells that are generally defined by expression Lrig1 but express other markers differently and have different roles in regeneration [9, 26]. Those expressing Lgr6 in prenatal life contribute to the formation of the hair follicle, sebaceous gland, and interfollicular dermis. Postnatally, Lgr6+ cells contribute to repair of IFE and hair follicles. Given that these cells are capable of forming several skin structures, it has been proposed that they are the most primitive skin stem cell [27].

Sebaceous Gland

The sebaceous gland is a separate structure that is intimately connected with the hair follicle as it secretes sebum on to the hair shaft. A population of unipotent stem cells defined by expression of Blimp1 is thought to control cellular contribution to the sebaceous gland [31]. Interestingly, ablation of Blimp1 does not cause loss of the sebaceous gland but rather hyperplasia, suggesting that Blimp1 serves an inhibitory role in sebaceous gland maintenance. Recently evidence has emerged that Blimp1+ cells of the sebaceous gland may in fact represent terminally differentiated cells rather than true stem cells [32]. While they are required for homeostatic maintenance of the sebaceous gland, they do not appear to produce progeny that replenish the cellular population of the gland.

Sweat Gland

The eccrine sweat gland is separate from the hair follicle and is responsible for secretion of sweat on to the skin surface. The nature of sweat gland cells remains less well understood than that of the hair follicle. Lu et al. demonstrated that the sweat gland itself and the duct that connects the gland to the skin contain separate populations of epithelial cells. Duct cells but not gland cells are able to contribute progeny to the injured epidermis. Following injury to the skin and to the gland, ductal cells regenerate the gland itself and also help to repopulate the epidermis near the gland [9, 33].

Circulating Stromal Cells

There is some evidence that bone marrow-derived mesenchymal stromal cells circulating in the blood may contribute to the repopulation of damaged skin. When the skin is injured in mice containing bone marrow from a pan-GFP donor, a small percentage of new keratinocytes express GFP, suggesting that circulating bone marrow cells may transform into skin cells [34, 35]. However, in the long run the number of these bone marrow-derived cells within the skin drops to nearly negligible numbers, raising the questions regarding the significance of their contribution [9].

Dermal Papilla

The dermal papilla (DP) is a region immediately below the hair follicle that contains a population of stem cells expressing CD133. Different subpopulations of these CD133+ DP cells are able to influence the type of hair follicle that develops above [36]. The presence of DP cells is critical for the formation of new hair follicles [36] and recent work using sophisticated culture techniques has shown that new hair follicles can be grown in vitro using DP cells [37]. This result raises the possibility of regenerating hair in patients affected by hair loss conditions such as alopecia.

Therapies for Wound Healing and Scarring that Target or Utilize Stem Cells

The availability of transgenic mouse models and lineage tracing has allowed a major expansion in our understanding of how skin stem cells produce cell populations that participate in regeneration and repair. The critical question, though, is how this knowledge can be harnessed to improve treatment for patients who suffer from chronic non-healing wounds, disfiguring facial scars, debilitating scar contractures, and other disorders of skin healing. Recently, there has been a concerning rise in the availability of questionable stem cell therapies offered to the public that are based on little to no evidence [38, 39]. Reassuringly, there are many legitimate and rigorously designed trials underway investigating the use of stem cells and other progenitor cells in a variety of human diseases. The majority of studies so far using human stem cells to improve wound healing have been performed in the setting of chronic non-healing wounds.

Bone Marrow Mononuclear Cells

Bone marrow mononuclear cells (BMMNCs) are cells of hematopoietic origin that can be obtained through bone marrow aspiration. They are capable of homing into areas of injury and participate in angiogenesis [40]. Several pilot studies in patients suffering from critical limb ischemia suggested that intravenous administration of bone marrow mononuclear cells could improve rates of spontaneous healing of chronic ulcers [36, 41–47]. Based on these results, the JUVENTAS trial randomized 160 patients with non-revascularizable limb ischemia to receive arterial injections of BMMNCs or placebo injections [48]. There was no significant difference between the groups in rates of amputations or quality of life measures. These results highlight the importance of performing high quality randomized, placebo-controlled trials in order to confirm the results of early pilot trials.

Peripheral Bone Marrow Mononuclear Cells

Peripheral bone marrow mononuclear cells (PBMNCs) are circulating hematopoietic cells defined by positivity for CD34 that have been shown in preclinical animal studies to improve regeneration of ischemic limbs [49]. In a human randomized trial, 28 patients with critical limb ischemia received injections of either CD34+ cells derived from their own peripheral blood or a control solution. There was a non-significant trend toward lower amputation rates in the treated patients [50]. In a recent small trial, three patients with sacral pressure ulcers received injections of CD34+ cells to one half of the ulcer while the other half was injected with normal saline. At the end of the study there was not a significant difference in healing between the two sides.

Adipose-Derived Stromal Cells

Adipose-derived stromal cells (ASCs) are a promising form of regenerative cell because they can readily be isolated from a patient's own fat tissue and are able to generate de novo bone and fat tissue in vivo and in vitro [51]. There is ample evidence in animal models that autologous and allogeneic ASCs can regenerate tissue following injury [52]. There is some evidence in a mouse model that factors released by ASCs may reduce hypertrophic scar formation [53]. At least two non-controlled trials with small numbers of patients showed improved wound healing following the administration of ASCs [54, 55]. In a phase II trial, ASCs along with fibrin glue accelerated closure of perianal fistulae [56], which may be thought of as a type of non-healing wound. However, a subsequent phase III trial showed no improvement with ASC administration [57].

Circulating Mesenchymal Stromal Cells

As discussed earlier, there is in vitro evidence that circulating non-hematopoietic mesenchymal stromal cells (MSCs) may transiently contribute to skin repair after injury [9]. MSCs would be a convenient therapeutic source because they can easily be isolated from a patient's own blood. The concept of using MSCs to improve wound healing has been tested in several trials in humans. Eight patients with chronic wounds were treated in a non-controlled fashion with topical application of autologous MSCs. The wounds subsequently healed, leading the authors to suggest that MSC application may accelerate wound closure [58]. In a randomized controlled trial in which patients with critical limb ischemia received injections of either allogeneic MSCs or control solution, no difference in outcomes was seen [59]. In a later randomized trial, 24 patients with non-healing ulcers were randomized to receive autologous cultured MSCs or control treatment [60]. Those treated with MSCs experienced greater symptom improvement and ulcer healing compared with the control group.

Conclusion

Major advances have been made in recent years toward understanding the role of stem cells in skin growth and repair. At the same time, excessive skin scarring and chronic wounds continue to pose significant problems for patients, clinicians, and the healthcare system. There is hope that advances in stem cell biology will lead to new therapies that utilize stem cells to promote the regeneration of functional skin tissue after injury. However, as is the case with many areas of investigation, exciting findings in animal models are not always borne out in humans. Additionally, early promising results in small, non-controlled human trials often do not persist when tested in large randomized trials. Given the public's frequent exposure to advertising for dubious stem cell therapies based on little or no scientific evidence, clinicians must critically evaluate new treatment options and educate patients about evidence-based medicine.

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Chapter 2

Cadherins as Central Modulators of Wound Repair

Melissa Crawford and Lina Dagnino

Department of Physiology and Pharmacology, Children's Health Research Institute and Lawson Health Research Institute, The University of Western Ontario, London, Ontario, Canada

Introduction

All humans will sustain skin wounds at some time in their lives. For this reason, and similar to other living organisms, they have developed efficient processes to repair injuries in skin and in other tissues. However, in some elderly individuals, or in those with morbidities such as diabetes, cutaneous wounds fail to heal properly and become chronic ulcers. In the United States, chronic wounds affect over 6.5 million people, with associated annual health care costs of over US$15 billion. Similarly, current estimates indicate that as much as 2% of the human population in developed countries will be affected by chronic ulcers during their lifetime [1].

In the injured skin, repair begins with the formation of a clot that temporarily re-establishes a permeability barrier [2]. Immune and inflammatory cells, including macrophages and neutrophils, are recruited from capillaries close to the wound. These cells secrete factors that activate adjacent dermal fibroblast and epidermal keratinocytes, and also serve as defense against pathogen invasion of the breached tissue. Activated keratinocytes subsequently undergo a complex phenotypic change, essential for efficient re-epithelialization. Specifically, they exit quiescence and become proliferative. They also undergo morphological changes associated with acquisition of migratory capacity. The latter requires alterations in the interactions between these cells and the underlying extracellular matrix substrates, which ultimately promote forward movement. Simultaneously, cell–cell adhesions are remodeled in a way that allows motility of keratinocytes