Results of the Phase 1 Open-Label Safety Study of Umbilical Cord Lining Mesenchymal Stromal/Stem Cells (Corlicyte®) to Heal Chronic Diabetic Foot Ulcers.

BACKGROUND: Mesenchymal stromal/stem cells (MSCs) play a critical role in wound healing. Corlicyte® is an MSC product derived from allogeneic umbilical cord tissue donated under an institutional review board-approved protocol and processed in accordance with section 501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act. This open-label phase 1 trial was performed under a United States Food and Drug Administration Investigational New Drug Application to establish the safety and tolerability of Corlicyte® in patients with diabetes and chronic diabetic foot ulcer (DFU). METHODS: Escalating doses were applied topically twice a week for up to 8 weeks after ulcer debridement, wound photography, and measurement. Subjects were followed for 4 weeks after the treatment phase. Adverse events were assessed at every visit. RESULTS: Nine subjects in 2 dosing cohorts completed the trial. No subjects experienced a serious adverse reaction to Corlicyte® or the development of anti-human leukocyte antigen (HLA) antibodies. Sixty percentage of subjects in the lower dose cohort experienced ulcer closure by Day 70 of follow-up, while the mean ulcer size was reduced by 54-67% in the other subjects. CONCLUSIONS: Topical administration of Corlicyte®, a novel biologic therapy consisting of allogeneic umbilical cord lining MSCs, appeared safe and tolerable and resulted in a significant decrease in ulcer area, demonstrating its potential as a therapy for healing of chronic DFU.

Epithelial and mesenchymal stem cells from the umbilical cord lining membrane.

Intense scientific research over the past two decades has yielded much knowledge about embryonic stem cells, mesenchymal stem cells from bone marrow, as well as epithelial stem cells from the skin and cornea. However, the billions of dollars spent in this research have not overcome the fundamental difficulties intrinsic to these stem cell strains related to ethics (embryonic stem cells), as well as to technical issues such as accessibility, ease of cell selection and cultivation, and expansion/mass production, while maintaining consistency of cell stemness (all of the stem cell strains already mentioned). Overcoming these technical hurdles has made stem cell technology expensive and any potential translational products unaffordable for most patients. Commercialization efforts have been rendered unfeasible by this high cost. Advanced biomedical research is on the rise in Asia, and new innovations have started to overcome these challenges. The Nobel Prize-winning Japanese development of iPSCs has effectively introduced a possible replacement for embryonic stem cells. For non-embryonic stem cells, cord lining stem cells (CLSCs) have overcome the preexisting difficulties inherent to mesenchymal stem cells from the bone marrow as well as epithelial stem cells from the skin and cornea, offering a realistic, practical, and affordable alternative for tissue repair and regeneration. This novel CLSC technology was developed in Singapore in 2004 and has 22 international patents granted to date, including those from the US and UK. CLSCs are derived from the umbilical cord outer lining membrane (usually regarded as medical waste) and is therefore free from ethical dilemmas related to its collection. The large quantity of umbilical cord lining membrane that can be collected translates to billions of stem cells that can be grown in primary stem cell culture and therefore very rapid and inexpensive cell cultivation and expansion for clinical translational therapies. Both mesenchymal and epithelial stem cells can be isolated from the umbilical cord lining membrane, usefully regenerating not only mesenchymal tissue, such as bone, cartilage, and cardiac and striated muscle, but also epithelial tissue, such as skin, cornea, and liver. Both mesenchymal and epithelial CLSCs are immune privileged and resist rejection. Clinically, CLSCs have proved effective in the treatment of difficult-to-heal human wounds, such as diabetic ulcers, recalcitrant chronic wounds, and even persistent epithelial defects of the cornea. Heart and liver regeneration has been shown to be successful in animal studies and await human trials. CLSCs have also been shown to be an effective feeder layer for cord blood hematopoietic stem cells and, more recently, has been recognized as an abundant and high-quality source of cells for iPSC production. Banking of CLSCs by cord blood banks in both private and public settings is now available in many countries, so that individuals may have their personal stores of CLSCs for future translational applications for both themselves and their families. Cord lining stem cells are strongly positioned to be the future of cell therapy and regenerative medicine.

The role of hepatocyte growth factor/c-Met system in keloid pathogenesis.

BACKGROUND: Keloid scar is a fibroproliferative disorder characterized by increased deposition of extracellular matrix components. Hepatocyte growth factor (HGF), also known as the "scatter factor," and its receptor, a product of the Met oncogene, play multiple roles in regulating cell behavior. However, the role of this system in pathogenic fibrosis is still unclear. Our aim was to investigate and to clarify the role of HGF and its receptor c-Met in pathogenesis of keloid scars. METHODS: This study investigated the expression profile of HGF and c-Met in keloid and normal skin tissue. In addition, the role of normal and keloid keratinocytes in modulating the expression of fibroblast HGF (epithelial-mesenchymal interactions) was examined using a two-chamber serum-free coculture model. The effect of serum stimulation on fibroblast expression of HGF and c-Met was also studied. RESULTS: Increased levels of HGF and c-Met were observed in tissue extracts obtained from keloid tissue. Increased levels of HGF and c-Met localization were observed in the basal epidermis and in the dermis of keloid tissue compared with normal skin. Serum stimulation seemed to upregulate the expression of both HGF and c-Met in fibroblasts. Finally, coculture of keloid keratinocytes with keloid fibroblasts upregulated levels of both HGF and c-Met in keratinocyte cell lysates and conditioned media obtained from fibroblast culture. CONCLUSIONS: These findings emphasize the importance of the HGF/c-Met system in keloid biology and pathogenesis and suggest a possible target for therapeutic intervention in the prevention and treatment of keloids.

Syndecan-2 and decorin: proteoglycans with a difference--implications in keloid pathogenesis.

BACKGROUND: Growth factors and cytokines involved in the wound healing process seem to be immobilized at the cell surface and extracellular matrix via binding with proteoglycans, making them important modulators of cell dynamics. Our aim was to investigate the expression of two proteoglycans, namely syndecan-2 and decorin, and to elucidate their role in the pathogenesis of an aberrant wound healing process leading to keloid scar. METHODS: Intrinsic expression of syndecan-2, fibroblast growth factor (FGF)-2, and decorin in keloid tissue was investigated using Western blotting and immunohistochemistry. Normal and keloid fibroblasts were treated with serum to see the effects of serum growth factors on the expression of syndecan-2 and decorin. The role of epithelial-mesenchymal interactions in modulating syndecan-2, FGF-2, and decorin expression was investigated using an established two-chamber serum-free coculture model. Finally, the antifibrotic effect of decorin was investigated by studying its effect on the expression of extracellular matrix components. RESULTS: Syndecan-2 and FGF-2 were upregulated in keloid tissue; decorin was downregulated. Normal and keloid fibroblasts treated with serum led to increase in syndecan-2 and decrease in decorin expression. Under coculture conditions, syndecan-2 was shed in the conditioned media. FGF-2 was also upregulated under coculture conditions and, when added to fibroblast monocultures, increased shedding of syndecan-2. Decorin levels were upregulated under coculture conditions only in normal cocultures. Decorin was also able to decrease extracellular matrix proteins, highlighting its importance as an antifibrotic agent. CONCLUSION: Syndecan-2 and FGF-2 are not only overexpressed in keloid tissues but may interact with each other resulting in the shedding of syndecan-2, which in turn might activate a whole cascade of events responsible for a keloidic phenotype. In addition, decorin had an antifibrotic effect and could well be used as a potential therapeutic agent for keloids.

Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts.

We previously reported an increase in signal transducer and activator of transcription 3 (Stat3) activation in keloid fibroblasts, which contributes to collagen production, cell proliferation, and migration. We further investigated the effect of epithelial-mesenchymal interaction on Stat3 in normal and keloid fibroblasts in noncoculture and coculture conditions. pY705 Stat3 was higher in keloid fibroblasts compared to normal fibroblasts in noncoculture. However, a more drastic decrease in pY705 Stat3 was observed in keloid fibroblasts compared to normal fibroblasts when cocultured with their respective keratinocytes over 5 days. To explore this paracrine effect, we examined the secretion of cytokines by cytokine arrays. Altered cytokine production was detected in keloid fibroblasts and keratinocytes, either in noncoculture or coculture conditions. IL-6, IL-8, monocyte chemoattractant protein-1, tissue inhibitor of metalloproteinases (TIMPs)-1, and TIMP-2 were major cytokines detected. Angiogenin, oncostatin M (OSM), vascular endothelial cell growth factor, IGF-binding protein-1, osteoprotegerin, and transforming growth factor-beta2 were present in keloid keratinocyte-fibroblast coculture, but absent in normal keratinocyte-fibroblast coculture. Only IL-6 and OSM stimulated strong pY705 Stat3 and cell proliferation in both normal and keloid fibroblasts. Other cytokines increased proliferation of keloid fibroblasts, but not normal fibroblasts, suggesting an altered state in keloid fibroblasts. Multiple cytokines likely contribute to keloid pathogenesis and a combinatorial neutralizing antibody/cytokine therapy may be effective in ameliorating keloid scars.

Innervation of the Face Studied Using Modifications to Sihler's Technique in a Primate Model.

BACKGROUND: There has been no reliable technique with which to display the innervation within whole-tissue specimens of the face. Such a technique preserves the architecture of the facial muscles and provides new data on intramuscular and sensory neural networks. Sihler's technique preferentially stains myelinated nerves within whole tissue, which is rendered transparent. On transillumination, entire neural networks can be studied in situ without the need for dissection and histologic examination. The purpose of this study was to apply Sihler's technique to study innervation patterns of the face, define end points, and improve specimen transillumination. METHODS: Eight macaque fascicularis monkeys were studied. The mimetic muscles of the face with intact facial nerve and sensory nerves were harvested as whole tissue composites. Sihler's technique was modified with formalin fixation before dissection to minimize autolysis of the myelin sheath. Prolonged immersion in glycerin improved tissue transparency. A replica of the skull was made with silicone and a light source embedded to restore three-dimensional configuration and provide transillumination. RESULTS: The facial nerve and sensory nerves were clearly seen up to their terminations in the transparent muscle and soft tissue. Observations were made with regard to the extramuscular and intramuscular innervation patterns of the facial nerve and sensory nerve patterns. CONCLUSIONS: Sihler's technique is a simple and reliable method with which to study the innervation of the face. This process may be applied to the human face to provide a much-needed roadmap to surgery, and the primate model may be developed for the study of facial reinnervation, facial reanimation, and dynamic facial transplantation.

Increased CCN2 transcription in keloid fibroblasts requires cooperativity between AP-1 and SMAD binding sites.

OBJECTIVE: We examined the transcriptional response to serum stimulation as an in vitro model of wound healing in keloid fibroblasts to identify molecular mechanisms leading to their aberrant growth. SUMMARY BACKGROUND DATA: Keloids are proliferative dermal growths representing a pathologic wound healing response. Although several groups have shown increased expression of profibrotic factors in keloids, there is little known about why they are expressed at higher levels than normal. METHODS: Fibroblasts derived from keloids and normal scar were subjected to serum stimulation as an in vitro model to mimic a component of the wound microenvironment to examine differential gene expression in keloid derived fibroblasts versus normal human fibroblasts. A promoter analysis was performed to identify specific enhancers involved in mediating the differential response of connective tissue growth factor (CTGF, CCN2). Point mutations in the enhancers were performed to confirm their role. Finally, we examined activation of transcription factors known to bind the targeted enhancers. RESULTS: Transcription of CCN2 after serum stimulation was significantly higher in keloid versus normal fibroblasts. Promoter analysis demonstrates the fragment from -625/-140 conferred increased serum responsiveness. Mutational analysis showed an AP-1 and SMAD binding site were both necessary for serum responsiveness. Preventing activation of either transcriptional complex will block CCN2 transcription. Additional experiments suggest that a single complex that includes components of the AP-1 and SMAD binding complexes is responsible for transactivation in response to serum. The key difference between keloid and normal fibroblasts appears to be the degree of activation of c-Jun. CONCLUSIONS: We suggest that altered responsiveness to cellular stress, based upon current data using serum stimulation and past data on response to mechanical strain, is a key defect leading to keloid formation.

The role of the activin system in keloid pathogenesis.

Keloid scars represent a pathological response to cutaneous injury under the regulation of many growth factors. Activin-A, a dimeric protein and a member of the transforming growth factor-beta superfamily, has been shown to regulate various aspects of cell growth and differentiation in the repair of the skin mesenchyme and the epidermis. Thus our aim was to study the role of activin and its antagonist, follistatin, in keloid pathogenesis. Increased mRNA expression for activin was observed in keloid scar tissue by performing RNase protection assay. Immunohistochemistry showed increased localization of both activin-A and follistatin in the basal layer of epidermis of keloid tissue compared with normal tissue. ELISA demonstrated a 29-fold increase in concentration of activin-A and an approximately 5-fold increase in follistatin in conditioned media in keloid fibroblasts compared with normal fibroblasts. Although keloid keratinocytes produced 25% more follistatin than normal keratinocytes, the amounts of activin-A, in contrast, was approximately 77% lower. Proliferation of fibroblasts was stimulated when treated with exogenous activin-A (46% increase in keloids fibroblasts) or following co-culture with hbetaAHaCaT cells (66% increase). Activin-A upregulated key extracellular matrix components, namely collagen, fibronectin, and alpha-smooth muscle actin, in normal and keloid fibroblasts. Co-treatment of follistatin with activin-A blocked the stimulatory effects of activin on extracellular matrix components. These findings emphasize the importance of the activin system in keloid biology and pathogenesis and suggest a possible therapeutic potential of follistatin in the prevention and treatment of keloids.

Differential transcriptional responses of keloid and normal keratinocytes to serum stimulation.

BACKGROUND: Keloids are benign tumors that occur only in response to injury, for which there is no effective treatment. We demonstrated previously that keloid keratinocytes (KKs) promote fibroblast proliferation more than normal keratinocytes (NKs) and that transforming growth factor (TGF)-beta is a component of that signal. We used the transcriptional response to serum stimulation to examine how TGF-beta expression is stimulated in KKs. MATERIALS AND METHODS: Quiescent KKs and NKs were stimulated using serum; harvested using RNA at 0, 1, 6, 12, and 24 h; and analyzed using quantitative real-time polymerase chain reaction. TGF-beta activity in the conditioned medium was measured with an MLEC/PAI-luciferase assay. Inhibition of ERK1/2, p38 kinase, and JNK pathways was performed with PD98059, SB203580, and SP600125, respectively. RESULTS: Increased transcription of TGF-beta2 occurs within 1 h of serum stimulation in KKs but not in NKs. In contrast, TGF-beta3 transcription was suppressed in KKs compared with NKs. No significant differences were observed in the transcriptional response of TGF-beta1. Increased TGF-beta2 mRNA correlated with increased TGF-beta biological activity in the conditioned medium. Inhibition of the ERK, p38 kinase or JNK signal transduction pathways blocked the transcriptional up-regulation of TGF-beta2, TbetaR1, and TbetaR2 in KKs. CONCLUSIONS: KKs produce more TGF-beta2 mRNA than NKs in response to serum stimulation, resulting in increased TGF-beta activity in conditioned medium. Combining these results with our previous data lead us to propose a model of keloid formation characterized by an exaggerated response to cellular stress and abnormal epithelial-mesenchymal signaling promoting keloid formation.

Increased transcriptional response to mechanical strain in keloid fibroblasts due to increased focal adhesion complex formation.

Clinicians have observed that keloids preferentially form in body areas subject to increased skin tension. We hypothesized a difference exists in the transcriptional response of keloid fibroblasts to mechanical strain compared with normal fibroblasts. Normal and keloid fibroblasts were seeded in a device calibrated to deliver a known level of equibiaxial strain. We examined the transcriptional response of TGF-beta isoforms and collagen Ialpha, genes differentially expressed in keloids. Keloid fibroblasts produced more mRNA for TGF-beta1, TGF-beta2, and collagen Ialpha after mechanical strain compared to normals, and this was correlated with protein production. Inhibiting the major mechanical signal transduction pathway with the ERK inhibitor, U0126, blocked upregulation of gene expression. In addition, keloid fibroblasts formed more focal adhesion complexes as measured by immunofluorescence for focal adhesion kinase, integrin beta1, and vinculin. Finally, there is increased activation of focal adhesion kinase when we detected the phosphorylated form of focal adhesion kinase with immunofluorescence and immunoblotting. In summary, keloid fibroblasts have an exaggerated response to mechanical strain compared to normal fibroblasts leading to increased production of pro-fibrotic growth factors. This may be one molecular mechanism for the development of keloids.

Complex epithelial-mesenchymal interactions modulate transforming growth factor-beta expression in keloid-derived cells.

Keloids are proliferative dermal growths representing a pathologic wound healing response. We have previously demonstrated that coculture of fibroblasts derived from either keloid or normal skin have an elevated proliferation rate when cocultured with keloid-derived keratinocytes vs. normal keratinocytes. In these studies, we examined the contribution of transforming growth factor-beta (TGF-beta) to this phenomenon using a two-chamber coculture system. Fibroblast proliferation in coculture was slower with the addition of a pan-TGF-beta neutralizing antibody. Keloid keratinocytes in coculture expressed more TGF-beta1, -beta3, and TGF-beta receptor 1 than normal keratinocytes. Keloid fibroblasts cocultured with keloid keratinocytes expressed more mRNA for TGF-beta1, -beta2, TGF-beta receptor 1, and Smad2. Keloid fibroblasts also produced more type I collagen, connective tissue growth factor, and insulin-like growth factor-II/mannose-6-phosphate receptor when cocultured with keloid keratinocytes vs. normal keratinocytes. Levels of total and activated TGF-beta activity increased when fibroblasts were cocultured with keratinocytes, correlating with the changes in transcriptional activity of TGF-beta. In conclusion, we find a complex paracrine interaction regulates TGF-beta mRNA expression and activation between keratinocytes and fibroblasts. These data suggest that keloid pathogenesis may result from both an increased TGF-beta production and activation by the keloid keratinocyte, and elevated TGF-beta expression, utilization, and signaling in keloid fibroblasts.

From scarless fetal wounds to keloids: molecular studies in wound healing.

Surgical researchers were among the first to describe the different phases of wound healing and the events in tissue repair and regeneration that were taking place during each phase. The understanding of these events has been significantly enhanced in recent years by modern techniques in molecular and cellular biology. In this article, we discuss new findings in scarless fetal repair, angiogenesis in wound healing, and keloid pathogenesis. This serves to highlight the advances that have been made and also how much remains to be understood.

Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids.

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2-5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I-III, laminin beta2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I-III production and significantly decreased laminin beta2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.

Differences in collagen production between normal and keloid-derived fibroblasts in serum-media co-culture with keloid-derived keratinocytes.

Keloids are characterized by the deposition of excessive extracellular-matrix collagen by abnormal fibroblasts in response to cutaneous injury. Studies to date have largely concentrated on the role of the keloid fibroblast in the pathogenesis of this lesion. Recent studies have highlighted the important concept of epithelial-mesenchymal interactions in normal skin biology. Extrapolating this to keloids in two recent serum-free in vitro studies, we demonstrated increased growth and proliferation, as well as induction of keloid-like collagen secretory characteristics in normal fibroblasts co-cultured with keloid-derived keratinocytes. Most fibroblast culture work to date has been performed in nutrient and growth factor-rich serum media. To investigate how a serum co-culture system might influence epithelial-mesenchymal interactions, [3H] proline incorporation was examined in normal and keloid fibroblasts co-cultured in serum with keratinocytes derived either from normal skin or keloid tissue. Results showed increased [3H] proline incorporation when normal fibroblasts were co-cultured with keloid keratinocytes, which was significantly increased when keloid fibroblasts were co-cultured with keloid keratinocytes. Taken with previous results, this study demonstrates a good correlation between both serum and serum-free co-culture systems, and supports the significance of epithelial-mesenchymal interactions in keloid pathogenesis.