Wound healing and fibrosis: current stem cell therapies

Abstract

Scarring is a result of the wound healing response and causes tissue dysfunction after injury. This process is readily evident in the skin, but also occurs internally across organ systems in the form of fibrosis. Stem cells are crucial to the innate tissue healing response and, as such, present a possible modality to therapeutically promote regenerative healing while minimizing scaring. In this review, the cellular basis of scaring and fibrosis is examined. Current stem cell therapies under exploration for skin wound healing and internal organ fibrosis are discussed. While most therapeutic approaches rely on the direct application of progenitor-type cells to injured tissue to promote healing, novel strategies to manipulate the scarring response are also presented. As our understanding of developmental and stem cell biology continues to increase, therapies to encourage regeneration of healthy functional tissue after damage secondary to injury or disease will continue to expand.

Tissue repair after injury or disease can be understood as a spectrum ranging from “overhealing,” as in hypertrophic scarring, keloids, and organ fibrosis, to functional tissue regeneration, as in fetal wound healing, liver regeneration, and amphibian limb regeneration. Normal adult tissue healing lies in between these two extremes with the formation of nonfunctional scar. Both chronic wounds and the consequences of overhealing, such as disfiguring scars and contractures, cause patient suffering, and are significant sources of health care spending. A multitude of therapies have been introduced to promote wound healing while minimizing scar formation and fibrosis, but the efficacy of commercially available therapies remains limited. Understanding the cellular basis of wound healing and tissue regeneration has received significant research attention in recent years.

Epidermal stem cells are critical to innate wound healing processes. The epidermis itself is composed of diverse cell types with multiple stem cells populations. Dedifferentiation can occur among differentiated cells in these populations to acquire stem cell characteristics during repair after injury.¹ Fibroblasts and myofibroblasts play a crucial role in the healing process^2,3 and harbor reprogramming capabilities.⁴ This flexibility of resident tissue cells is a crucial component of the wound healing process.⁵ As our knowledge regarding stem cell properties and capabilities has expanded, a variety of stem cells therapies have been explored to optimize tissue repair in the setting of injury or disease.

TYPES OF THERAPEUTIC STEM CELLS

Stem cells are defined by plasticity, which is the potential to differentiate into multiple tissue types, and the capacity for self-renewal. In the stem cell literature, a variety of “stem” and “progenitor” cell types have been described, each with specific capacity for self-renewal and multipotency. Stem cells in current therapeutic use represent a heterogeneous group, with diverse tissue sources and methods of isolation and culture. Some research protocols use mixtures of “progenitor-type” cells rather than pure extracts of homogenous cells. Pluripotent stem cells are divided into two broad categories: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs are isolated from human embryos,⁶ while iPSCs result from forced dedifferentiation of adult cells such as fibroblasts.⁷ Multipotent hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) can be derived from marrow.⁸ MSCs can also be isolated from the placenta, umbilical cord, peripheral blood, skin, and synovial fluid.^9,10 Adipose-derived stem cells (ASCs) are an attractive source of stem cells given the abundance and ease of harvesting adipose tissue^11,12 (Fig. 1). The current literature provides a multitude of studies suggesting therapeutic benefits of each of these stem cell types in a variety of disease states, but comprehensive comparisons to determine the most efficacious cellular compositions are sparse.^13,14 Significantly, several studies have demonstrated the therapeutic potential of acellular extracts composed of the secretome of stem cells.^15,16 These healing substrates contain paracrine factors and cytokines and appear to provide similar benefit to cell-based therapies.^10,17

Fig. 1.

Common sources for therapeutic stem cells. ESCs (blue) are isolated from human blastocysts. iPSCs (green) are obtained from dedifferentiation of adult cells like fibroblasts. The marrow is the home of HSCs (red) and an important source of MSCs (purple). MSCs can also be isolated from various other tissues such as the placenta, umbilical cord, and adipose tissue. Finally, ASCs (orange) are extracted from adult adipose tissue.

METHODS OF THERAPEUTIC STEM CELL ADMINISTRATION

Described methods for stem cell administration are as diverse as the cellular types in play. Delivering “the seed and the soil” to injured tissue is an important paradigm in stem cell therapy. The “seed” represents stem cells and the “soil” is a supportive matrix in which cells are distributed.¹⁸ Since injured tissue is inflamed and potentially fibrosed, it is a hostile environment for stem cells and can prevent their function.^19–21 Delivery of seed and soil together can recapitulate a favorable environment and maximize stem cell therapeutic abilities.¹⁸

In the setting of dermal injury, skin is exposed and easily accessible for stem cell delivery. A gamut of materials have been developed for stem cell delivery to the skin, including gels, sprays, sponges, and silicone chambers.^22–26 Other delivery modalities include a sundew-inspired adhesive hydrogel²⁷ and an extracellular matrix patch.²⁸ Studies show improved efficacy when stem cells are applied in a supportive matrix, but comprehensive comparisons of delivery systems are lacking.

With regard to stem cell administration to internal organs, cells are commonly given peripherally via intravenous (IV) cannulation. However, the innate filtering systems present in the spleen, liver, and lungs and requisite crossing of the vascular endothelial barrier to reach target organs are hurdles for stem cells when given IV.²² As such, invasive delivery methods have also been developed for direct stem cell application to specific organs. Examples include intracoronary injection via cardiac catheterization and transendocardial injection for heart disease²¹ and intrahepatic, intraportal, and intraperitoneal injection for liver disease.²⁹

THE CELLULAR BASIS OF CUTANEOUS FIBROSIS

Skin scarring is the most common manifestation of cutaneous fibrosis and occurs after traumatic injury or surgical incision.¹⁹ Disorders of “overscarring” include hypertrophic scars and keloids.³⁰ Additional insults causing cutaneous fibrosis include radiation therapy,^2,31 autoimmune disease such as scleroderma,³² and neoplasia.^2,33 Skin scarring is a serious clinical problem that can cause physical disability and psychosocial dysfunction.^34–36

The wound healing process leading to scaring and fibrosis is typically divided into three broad phases: inflammation, proliferation, and remodeling¹⁹ (Fig. 2). Primarily, there is an influx of platelets (PLTs) to arrest bleeding at the site of injury. Local tissues and blood vessels then release cytokines, which attract neutrophils and macrophages to the area. These cells release additional cytokines and growth factors and clear debris from the site of injury. During the proliferation phase, new tissue forms with migration of keratinocytes over the wound. Neovascularization occurs, and fibroblasts deposit extracellular matrix.^19,37 During remodeling, Type III collagen is replaced by Type I collagen; nerve regeneration progresses and the overall vascularity of the scar tissue decreases. This mechanism functions to close tissue defects and restore the skin’s barrier function, but ultimately results in weaker, nonfunctional tissue devoid of skin appendages.^19,38 Ideally, wounds would close in a regenerative fashion, with restoration of functional tissue, skin appendages, and normal tensile strength. This form of skin regeneration is observed in the fetus, but is lost before the third trimester.³⁹

Fig. 2.

The wound healing response and role of EPFs. Phases of wound healing after injury, including hemostasis, inflammation, proliferation, and remodeling. EPFs are key mediators of cutaneous scarring after injury. When these cells are inhibited, a more regenerative phenotype results with decreased scarring.

The discovery that fetal skin heals without a scar is a fundamental observation driving our group’s exploration of the cellular basis of scarring. We identified a single lineage of dorsal fibroblasts in mouse skin responsible for the bulk of cutaneous scarring after wounding. These cells are identified by embryonic expression of the Engrailed-1 gene (Engrailed-1 lineage-positive fibroblasts [EPFs]). In adult skin, EPFs express CD26. The fibrotic behavior of these cells appears to be cell intrinsic, as noted with reciprocal transplantation studies in buccal mucosa. Oral mucosa is a permissive environment for regeneration, as noted by the dearth of oral scarring after mucosal injury. However, when EPFs are transplanted to buccal wounds, a scarring phenotype is produced. Ablation of EPFs utilizing transgenic mice models (or by inhibition of the cell surface marker CD26) results in visually and histologically decreased scar formation after wounding.² EPFs are thus responsible for scar formation on the dorsal skin of mice and can be manipulated to decrease scarring (Fig. 2). While most stem cell therapies involve administration of exogenous progenitor cells to improve outcomes, our work demonstrates an example of exploiting a specific regenerative cell type to achieve a desired phenotype.

CURRENT STEM CELL THERAPY FOR WOUND HEALING

Stem cell therapy has been explored extensively in wound healing.^40,41 Traumatic wounds, most notably those as a result of burns, are of particular interest to clinicians as these wounds are plagued by slow healing, infection, pain, and hypertrophic scarring.⁴² Application of stem cells to burn wounds has shown to stimulate healing and decrease inflammation and fibrosis in both animal and human models.⁴³ Improved formation of granulation tissue and neovascularization contributes to this positive therapeutic effect.^43–47 Similar findings in radiation burns in animal models and humans are noted.^48–51 Subsequent investigations have focused on improved delivery of stem cells using biomimetic scaffolds as discussed. Wounds treated with tissue-engineered matrices seeded with stem cells demonstrate increased vascularization and keratinization with less contraction compared to stem cell application without a supportive matrix.⁵² Addition of growth factors and supportive cell types to enhance stem cell function has further improved healing in wounds and burns. Tested additives include human beta defensin 2 and 3, vascular endothelial growth factor, hepatocyte growth factor, and PLT-derived growth factor.^53–55 Finally, surgical fat grafting with ASCs has also been studied, with evidence suggesting enhanced angiogenesis and decreased inflammation, yielding decreased burn scar size in both murine models and humans.⁵⁶

Stem cells are thought to improve healing by acting on resident cells to reduce inflammation⁵⁷ and stimulate extracellular matrix production.⁵⁸ Some studies have examined stem cell therapy in diabetic and acute wounds, with encouraging results,^20,40,59,60 although other results have been less encouraging.⁶¹ MSCs may also increase inflammation by stimulating cytokines that promote both granulation and angiogenesis, which may lead to hypertrophic scarring.^62–64 Taken together, these results emphasize the need for further high-quality research into the role for stem cells therapies in skin wounds.

THE ORIGIN AND PROBLEM OF ORGAN FIBROSIS

Just as fibrotic scarring secondary after skin wounds can pose significant health challenges, injury or disease can activate the wound healing cascade and fibrotic response in internal organs. Again, this response can be understood as a spectrum of scarring and regeneration (Fig. 3). Organ fibrosis leads to dysfunction and even organ failure, as in ischemic heart failure and cirrhosis. This occurs secondary to replacement of functional parenchyma with scar tissue, which is nonfunctional and deforming. In blood vessels, this can manifest as postsurgical stenosis causing decreased blood flow through the affected vessel. After bowel anastomosis, fibrotic scar can likewise lead to stenosis of the bowel lumen and consequential obstruction. Other examples include pulmonary fibrosis, intraabdominal or pleural adhesions, kidney fibrosis, tumor stroma formation and fibrosis after radiation therapy, and spinal cord injury.

Fig. 3.

The spectrum of scarring and regeneration. Tissue repair is carried out on a spectrum from overhealing, as in hypertrophic scars, keloids, and cirrhosis, to regeneration, as in fetal wound healing, amphibian limb regeneration, and mucosal repair.

Building on our findings that the Engrailed fibroblast lineage is the cellular mediator of scar formation, we examined the contribution of these cells to fibrosis including stroma proliferation associated with neoplasia and radiation-induced fibrosis. We found that EPFs contribute to melanoma tumor stroma in the skin; when EPFs were ablated, tumor burden is significantly decreased secondary to decreased connective tissue deposition.² Similarly, we confirmed that EPFs contribute to dermal fibrosis after radiation therapy.

Given the wide variety of tissues in which fibrosis can be found, a common pathway for the formation of fibrosis encompassing different organ systems is proposed. Disease and injury states cause inflammation, which can be acute or chronic in duration. Similar to the wound healing cascade, with inflammation, vessels become leaky, releasing cytokines and recruiting further inflammatory cells. Transforming growth factor-β is found to be up regulated in the fibrotic response.⁶⁵ Fibroblasts deposit collagen fibers and myofibroblasts contribute to tissue contraction, which can further exacerbate the organ failure process secondary to deformity and decreased vascularity. The fibrotic tissue is poorly or nonfunctional and, as such, the affected organ can no longer perform its normal functions.

CURRENT STEM CELL THERAPY FOR ORGAN FIBROSIS

Stem cell therapies have been explored for the treatment of organ fibrosis. Novel methods for application continue to be developed and both preclinical and clinical trials are ongoing. To follow, we present an overview of cell-based therapies for fibrosis.

Lungs: chronic fibrosis and acute respiratory distress syndrome

Pulmonary fibrosis can result from chronic lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis, and idiopathic pulmonary fibrosis. “Spheroid lung cells,” defined as a heterogeneous population of pulmonary progenitor and stem cells, have been isolated from human subjects using minimally invasive techniques. When administered in a murine idiopathic pulmonary fibrosis model, improvements in lung function as well as decreased inflammation and fibrosis were noted. Human trials harnessing this technology are forthcoming.⁶⁶

Stem cell therapies have been studied for acute respiratory distress syndrome (ARDS). This disease is caused by an overly robust pulmonary inflammatory response to infection or trauma and leads to respiratory failure. Mortality from ARDS ranges from 20% to 40%.⁶⁷ Studies utilizing MSCs for ARDS have shown good therapeutic tolerance,⁶⁸ as well as improved recovery from ventilator-associated pulmonary injury,⁶⁹ decreased pulmonary edema, and increased presence of peripheral regulatory T cells.⁷⁰ Given that cell engraftment of MSCs appears to be very low, therapeutic efficacy is believed to result from anti-inflammatory cytokines or paracrine signals produced by MSCs.^71,72 Another proposed mechanism is that MSCs enact mitochondrial transfer to local alveolar epithelium, which facilitates tissue repair and promotes bacterial clearance by macrophages.⁷²

Cardiovascular disease: ischemic cardiomyopathy and vascular stenosis

Ischemic cardiovascular disease is the leading cause of death for adults worldwide.⁷³ Resident cardiac stems cells are present in developed myocardium, but they possess limited regenerative capacity after injury.^74,75 Multiple types of stem cell therapies have been investigated to promote cardiac muscle recovery including marrow-derived cells, skeletal myoblasts, ESCs, endogenous cardiac stem cells and MSCs.²¹ Marrow-derived progenitor cells have been shown to home to the site of injury in transplantation studies⁷⁶ and transdifferentiate into cardiomyocytes when transplanted into infarcted mouse heart.⁷⁷ Endothelial progenitors derived from the marrow have also been studied in cardiac regeneration, but do not appear to differentiate into cardiac tissue. However, these cells do facilitate cardiac neovascularization and thus delivery of key nutrients to the area of injury, in addition to providing supportive paracrine signaling.^78,79 Clinical trials of cell therapy in cardiac disease show small but potentially significant results in ischemic and nonischemic cardiomyopathy.⁸⁰ Overall, these trials show low rates of adverse events and variable improvements in clinical endpoints such as mortality, ejection fraction, 6-minute walk test, arrhythmias, and infarct size.^80–84 Therapeutic efficacy is again suspected to be due to paracrine and growth factors from stem cells.^17,80

Peripheral vascular stenosis can also form secondary to fibrosis, most commonly at surgical anastomotic sites after either elective operations for vascular disease or surgical vascular repairs after traumatic injury. Reduced vessel diameter depresses circulatory flow, resulting in tissue ischemia and thrombosis. Allogenic MSC injection has been explored as a therapeutic solution to postsurgical vascular stenosis. In a rat model, increased cell proliferation and vascular endothelial growth factor levels and decreased inflammatory cytokines were observed after MSC injection, which may indicate an increased rate of repair.⁸⁵ MSCs have also been shown to limit hypertension in the setting of renal artery stenosis in a rat model.⁸⁶

Liver injury and cirrhosis

Liver transplantation is the only definitive treatment for hepatic failure, but organ shortage and the risks associated with this major procedure as well as the need for life-long immunosuppression make stem cell therapy an attractive alternative to whole organ transplantation.⁸⁷ Three main cell types are utilized for liver stem cell therapy; these include hepatocytes derived from iPSCs and ESCs.^88,89 All of these cell types can be tailored to address specific components of the underlying disease state, whether targeting acute liver failure, inherited liver diseases, or cirrhosis from chronic destructive states such as viral hepatitis or alcohol consumption.⁹⁰ Granulocyte colony stimulating factor administration has also been explored to induce marrow-derived stem cell proliferation and peripheral circulation in patients with end-stage liver disease.⁹¹ Clinical trials investigating these cellular therapies in the treatment of acute and chronic liver disease show short-term improvements in hepatic function; however, long-term outcomes are not well characterized. Additionally, more data are needed regarding posttherapy risk of neoplasm, including hepatocellular carcinoma and teratoma.^29,92–96

An interesting approach to stem cell–based therapy for cirrhosis involves in vivo transformation of hepatic myofibroblasts into iHeps. Myofibroblasts are key mediators of liver fibrosis, but can be transformed into iHeps in animal models via viral vector transduction of appropriate transcription factors.^97,98 This highlights the dedifferentiation flexibility of many cell types that may not be classically considered stem cells.⁴ Bioengineered hepatocytes have also been explored as alternatives to whole organ transplantation. Functional hepatic tissue sheets have been constructed from both primary hepatocytes and ASCs cocultured with nonparenchymal hepatic cells. These are implanted subcutaneously, can engraft, and maintain hepatocyte-specific functions.^99,100

Intraabdominal fibrosis: anastomotic stricture and adhesions

As with postanastomotic vascular stenosis, anastomotic stricture after intestinal surgery can also occur as scar tissue and fibrosis forms at the site and can lead to obstruction. Inflammatory bowel disease (IBD) involves acute on chronic inflammation that results in fibrosis. As such, bowel resections for IBD are particularly high risk for anastomotic stricture. Ischemia can also increase the risk of stricture development in the bowel. Transplantation of ASCs and MSCs into the bowel wall in an ischemic colonic anastomosis rat model showed improved anastomotic healing with higher bowel bursting pressure, as well as decreased strictures, ulceration, and morbidity.¹⁰¹ Fistulas may also arise in the setting of IBD. When implanted into fistulous tracts with or without fibrin glue, adipose-derived MSCs can accelerate fistula closure.^102,103

Adhesions are a major problem after abdominal surgery in which fibrotic tissue forms between the visceral and parietal peritoneum. These can cause bowel obstructions, chronic pain, and infertility.¹⁰⁴ Similar tissue can form between the parietal and visceral pleura after lung surgery causing decreased pulmonary function. Adhesion prevention has been explored with administration of MSCs as well as skeletal muscle–derived stem cells.¹⁰⁵

Application of stem cells have been explored in many other injury settings, such as a kidney diseases including acute kidney injury, chronic kidney disease such as diabetic renal disease, and glomerulosclerosis, and in the setting of kidney transplant.¹⁰⁶ Stem cells have also been explored to manage spinal cord stenosis after surgery or injury.

CONCLUSION

Tissue repair and regeneration exists on a spectrum, from keloids, hypertrophic scarring, and fibrosis to functional tissue regeneration. The replacement of normal parenchyma with stiff, nonfunctional collagenous tissue can result in end-organ dysfunction. This process is most readily evident in cutaneous scarring. Stem cell therapies hold promise to promote regeneration of healthy, functional tissue and limit the overhealing, scarring response. As our knowledge of the developmental and cellular basis of fibrosis increases, key targets to reduce scarring and fibrosis continue to emerge. The Engrailed-1, CD26-positive lineage of cells and their role in cutaneous fibrosis is a key example. Prevailing regenerative paradigms such as fetal wound healing and amphibian limb regeneration will continue to provide invaluable insight into how to approach and manipulate fibrotic disease states.

ABBREVIATIONS:

ARDS

acute respiratory distress syndrome

ASC(s)

adipose-derived stem cell(s)

EPF(s)

Engrailed-1 lineage– positive fibroblast(s)

ESC(s)

embryonic stem cell(s)

IBD

Inflammatory bowel disease

iPSC(s)

induced pluripotent stem cell(s)

MSC(s)

mesenchymal stem cell(s)