Chemotaxis-driven disease-site targeting of therapeutic adult stem cells in dystrophic epidermolysis bullosa.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB), a rare genodermatosis, is characterized by the formation of intra-epidermal blistering and the development of chronic nonhealing skin wounds. Recently, attempts have been made to develop cell-based therapies for this currently intractable disorder. The molecular mechanisms that govern directional migration of the adult stem cells, allowing their efficient and controlled homing to the skin affected with DEB, are poorly understood. The key mechanism that regulates recruitment of leukocytes and progenitor stem cells to distal anatomical tissues affected with disease is chemotaxis, which depends on the signaling molecules, chemokines, and acts primarily as part of the host defense and repair mechanism. METHODS: Comprehensive proteomic screening of chemokines in the blister fluids of DEB-affected mice was conducted to define the inflammatory and immune activities, thus providing potential to examine local biological mechanisms and define the protein signature within lesional skin as a potential marker of disease activity. Also, the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations. RESULTS: Assessment of blister fluid-derived chemokines showed a persistent presence of several chemotactic molecules, including CXCL1 + 2 and CXCL5. The majority of blister-originated chemotactic signals were associated with preferential recruitment of CD45(+)CXCR2(+) and CD11b(+)CXCR2(+) leukocytes. Systemic transplantation of an enriched CXCR2 population of mouse adipose-derived stem cells (mADSC) into DEB-affected mice demonstrated effective recruitment of cells to the blistering skin under the influence of blister-derived ligands and deposition of therapeutic type VII collagen. CONCLUSIONS: Collectively, these studies demonstrate that recruitment of mADSC into DEB skin is tightly controlled by disease-site chemotactic activities and suggest a potential mechanism for effective application of therapeutic stem cells for DEB.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

Human adipose-derived stem cell transplantation as a potential therapy for collagen VI-related congenital muscular dystrophy.

INTRODUCTION: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness within the first two years of life. Collagen VI-related muscle disorders have recently emerged as one of the most common types of CMD. COL6 CMD is caused by deficiency and/or dysfunction of extracellular matrix (ECM) protein collagen VI. Currently, there is no specific treatment for this disabling and life-threatening disease. The primary cellular targets for collagen VI CMD therapy are fibroblasts in muscle, tendon and skin, as opposed to muscle cells for other types of muscular dystrophies. However, recent advances in stem cell research have raised the possibility that use of adult stem cells may provide dramatic new therapies for treatment of COL6 CMD. METHODS: Here, we developed a procedure for isolation of human stem cells from the adipose layer of neonatal skin. The adipose-derived stem cells (ADSC) were examined for expression of ECM and related genes using gene expression array analysis. The therapeutic potential of ADSC was assessed after a single intramuscular transplantation in collagen VI-deficient mice. RESULTS: Analysis of primary cultures confirmed that established ADSC represent a morphologically homogenous population with phenotypic and functional features of adult mesenchymal stem cells. A comprehensive gene expression analysis showed that ADSC express a vast array of ECM genes. Importantly, it was observed that ADSC synthesize and secrete all three collagen VI chains, suggesting suitability of ADSC for COL6 CMD treatment. Furthermore, we have found that a single intramuscular transplantation of ADSC into Col6a1-/-Rag1-/- mice under physiological and cardiotoxin-induced injury/regeneration conditions results in efficient engraftment and migration of stem cells within the skeletal muscle. Importantly, we showed that ADSC can survive long-term and continuously secrete the therapeutic collagen VI protein missing in the mutant mice. CONCLUSIONS: Overall, our findings suggest that stem cell therapy can potentially provide a new avenue for the treatment of COL6 CMD and other muscular disorders and injuries.

Analysis of chemotactic molecules in bone marrow-derived mesenchymal stem cells and the skin: Ccl27-Ccr10 axis as a basis for targeting to cutaneous tissues.

BACKGROUND AIMS: Adult stem cells produce a plethora of extracellular matrix molecules and have a high potential as cell-based therapeutics for connective tissue disorders of the skin. However, the primary challenge of the stem cell-based approach is associated with the inefficient homing of systemically infused stem cells to the skin. METHODS: We examined chemotactic mechanisms that govern directional migration of mesenchymal stem cells (MSCs) into the skin by conducting a comprehensive expression analysis of chemotactic molecules in MSCs and defined cutaneous tissues from normal and hereditary epidermolysis bullosa (EB)-affected skin. RESULTS: Analysis of chemokine receptors in short-term and long-term MSC cultures showed tissue culture-dependent expression of several receptors. Assessment of epidermis-derived and dermis-derived chemokines showed that most chemotactic signals that originate from the skin preferentially recruit different sets of leukocytes rather than MSCs. Analysis of the chemotactic molecules derived from EB-affected non-blistered skin showed only minor changes in expression of selected chemokines and receptors. Nevertheless, the data allowed us to define the Ccl27-Ccr10 chemotactic axis as the most potent for the recruitment of MSCs to the skin. Our in vivo analysis demonstrated that uniform expression of Ccr10 on MSCs and alteration of Ccl27 level in the skin enhance extravasation of stem cells from circulation and facilitate their migration within cutaneous tissue. CONCLUSIONS: Collectively, our study provides a comprehensive analysis of chemotactic signals in normal and EB-affected skin and proof-of-concept data demonstrating that alteration of the chemotactic pathways can enhance skin homing of the therapeutic stem cells.