CNS regeneration: a morphogen's tale

Tissue regeneration will soon become an avenue for repair of damaged or diseased tissues as stem cell niches have been found in almost every organ of the vertebrate body including the CNS. In addition, different animals display an array of regenerative capabilities that are currently being researched to dissect the molecular mechanisms involved. This review concentrates on the different ways in which CNS tissues such as brain, spinal cord and retina can regenerate or display neurogenic potential and how these abilities are modulated by morphogens.     

Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF’s blood vessel forming potential prior to clinical implantation.     

A breath of fresh air in lung regeneration

Enhancing the ability of the lungs to regenerate following injury could revolutionize the treatment of a wide range of different diseases. In this issue, Kumar et al. (2011) and Ding et al. (2011) dissect the cellular and molecular mechanisms of murine lung regeneration following injury and provide insights into the basic biology of the organ with implications for development of future therapeutic approaches.     

Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice

The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.     

Extracellular matrix: A dynamic microenvironment for stem cell niche

Background

Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue and able to regulate cell behavior. Stem cells have a key role in the maintenance and regeneration of tissues and they are located in a specific microenvironment, defined as niche.

Scope of review

We overview the progresses that have been made in elucidating stem cell niches and discuss the mechanisms by which ECM affects stem cell behavior. We also summarize the current tools and experimental models for studying ECM–stem cell interactions.

Major conclusions

ECM represents an essential player in stem cell niche, since it can directly or indirectly modulate the maintenance, proliferation, self-renewal and differentiation of stem cells. Several ECM molecules play regulatory functions for different types of stem cells, and based on its molecular composition the ECM can be deposited and finely tuned for providing the most appropriate niche for stem cells in the various tissues. Engineered biomaterials able to mimic the in vivo characteristics of stem cell niche provide suitable in vitro tools for dissecting the different roles exerted by the ECM and its molecular components on stem cell behavior.

General significance

ECM is a key component of stem cell niches and is involved in various aspects of stem cell behavior, thus having a major impact on tissue homeostasis and regeneration under physiological and pathological conditions. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Biomimetic approaches to protein and gene delivery for tissue regeneration

Novel therapeutic strategies that promote wound healing seek to mimic the response of the body to wounding, to regenerate rather than repair injured tissues. Many synthetic or natural biomaterials have been developed for this purpose and are used to deliver wound therapeutics in a controlled manner that prevents unwanted and potentially harmful side-effects. Here, we review the natural and synthetic biomaterials that have been developed for protein and gene delivery to enhance tissue regeneration. Particular emphasis is placed on novel biomimetic materials that respond to environmental stimuli or release their cargo according to cellular demand. Engineering biomaterials to release therapeutic agents in response to physiologic signals mimics the natural healing process and can promote faster tissue regeneration and reduce scarring in severe acute or chronic wounds.     

Electrospun scaffold tailored for tissue-specific extracellular matrix

The natural extracellular matrix (ECM) is a complex structure that is built to meet the specific requirements of the tissue and organ. Primarily consisting of nanometer diameter fibrils, ECM may contain other vital substances such as proteoglycans, glycosaminoglycan and various minerals. Current research in tissue engineering involves trying to replicate the ECM such that it provides the environment for tissue regeneration. Electrospinning is a versatile process that results in nanofibers by applying a high voltage to electrically charge a liquid. A variety of polymers and other substances have been incorporated into the artificial nanofibrous scaffold. Surface modification and cross-linking of the nanofibers are some ways to improve the biocompatibility and stability of the scaffold. Electrospun scaffolds with oriented nanofibers and other assemblies can be constructed by modifying the electrospinning setup. Using electrospinning, researchers are able to specifically tailor the electrospun scaffold to meet the requirements of the tissue that they seek to regenerate. In vitro and in vivo experiments demonstrate that electrospun scaffolds hold great potential for tissue engineering applications.     

Mind the gap: Cells respond to tissue damage by changing orientation of cell divisions

Nature presents plenty of examples of cellular behavior that determines the shape of an organ during development, such as epithelial polarity and cell division orientation. Little is known, however, about how organs regenerate or how cellular behavior affects regeneration. One of the most exciting aspects of regeneration biology is understanding how proliferation and patterning are coordinated, since it means that cells not only have to proliferate but also have to do so in an ordered manner so that organs are reconstructed proportionally. Drosophila wing imaginal discs and adult wings are models used in different approaches to investigate this issue; they have recently been used to reveal that, after localized cell death, neighboring cells change their cell division orientation toward the damaged zone. During this process, cell polarity and spindle orientation operate in coordination with cell proliferation to regenerate proper organ size and shape.     

Sodium hydroxide based non-detergent decellularizing solution for rat lung

Lung transplantation is the last option for the treatment of end stage chronic lung disorders. Because the shortage of donor lung organs represents the main hurdle, lung regeneration has been considered to overcome this hurdle. Recellularization of decellularized organ scaffold is a promising option for organ regeneration. Although detergents are ordinarily used for decellularization, other approaches are possible. Here we used high alkaline (pH12) sodium hydroxide (NaOH)-PBS solution without detergents for lung decellularization and compared the efficacy on DNA elimination and ECM preservation with detergent based decellularization solutions CHAPS and SDS. Immunohistochemical image analysis showed that cell components were removed by NaOH solution as well as other detergents. A Collagen and GAG assay showed that the collagen reduction of the NaOH group was comparable to that of the CHAPS and SDS groups. However, DNA reduction was more significant in the NaOH group than in other groups (p < 0.0001). The recellularization of HUVEC revealed cell attachment was not inferior to that of the SDS group. Ex vivo functional analysis showed 100% oxygen ventilation increased oxygen partial pressure as artificial hemoglobin vesicle-PBS solution passed through regenerated lungs in the SDS or NaOH group. It was concluded that the NaOH-PBS based decellularization solution was comparable to ordinal decellularizaton solutions and competitive in cost effectiveness and residues in the decellularized scaffold negligible, thus providing another potential option to detergent for future clinical usage.     

Matrix metalloproteinases in liver injury,repair and fibrosis

The liver is a large highly vascularized organ with a central function in metabolic homeostasis, detoxification, and immunity. Due to its roles, the liver is frequently exposed to various insults which can cause cell death and hepatic dysfunction. Alternatively, the liver has a remarkable ability to self-repair and regenerate after injury. Liver injury and regeneration have both been linked to complex extracellular matrix (ECM) related pathways. While normal degradation of ECM components is an important feature of tissue repair and remodeling, irregular ECM turnover contributes to a variety of liver diseases. Matrix metalloproteinases (MMPs) are the main enzymes implicated in ECM degradation. MMPs not only remodel the ECM, but also regulate immune responses. In this review, we highlight some of the MMP-attributed roles in acute and chronic liver injury and emphasize the need for further experimentation to better understand their functions during hepatic physiological conditions and disease progression.     

Periodontal tissue regeneration by combined implantation of adipose tissue-derived stem cells and platelet-rich plasma in a canine model

Background aimsOne goal of periodontal therapy is to regenerate periodontal tissues. Stem cells, growth factors and scaffolds and biomaterials are vital for the restoration of the architecture and function of complex tissues. Adipose tissue-derived stem cells (ASCs) are an ideal population of stem cells for practical regenerative medicine. In addition, platelet-rich plasma (PRP) can be useful for its ability to stimulate tissue regeneration. PRP contains various growth factors and may be useful as a cell carrier in stem cell therapies. The purpose of this study was to determine whether a mixture of ASCs and PRP promoted periodontal tissue regeneration in a canine model.MethodsAutologous ASCs and PRP were implanted into areas with periodontal tissue defects. Periodontal tissue defects that received PRP alone or non-implantation were also examined. Histologic, immunohistologic and x-ray studies were performed 1 or 2 months after implantation. The amount of newly formed bone and the scale of newly formed cementum in the region of the periodontal tissue defect were analyzed on tissue sections.ResultsThe areas of newly formed bone and cementum were greater 2 months after implantation of ASCs and PRP than at 1 month after implantation, and the radiopacity in the region of the periodontal tissue defect increased markedly by 2 months after implantation. The ASCs and PRP group exhibited periodontal tissue with the correct architecture, including alveolar bone, cementum-like structures and periodontal ligament-like structures, by 2 months after implantation.ConclusionsThese findings suggest that a combination of autologous ASCs and PRP promotes periodontal tissue regeneration that develops the appropriate architecture for this complex tissue.     

Generation of monoclonal antibodies directed against organ-specific endothelial cell surface determinants.

Organ-specific determinants expressed on the luminal surface of vascular endothelia are often unstable when cells are removed from their normal tissue environment and grown in culture. Unspecific endothelial cells of large vessel origin [e.g., bovine aorta (BAEC)] can be modulated to express and preserve such determinants when they are grown on the extracellular matrix of the desired organ. Lung matrix-modulated BAEC were used here to generate MAb against lung-specific vascular endothelia. Immunization was accomplished with outside-out membrane vesicles obtained by incubating BAEC monolayers grown on lung matrix with a low-strength paraformaldehyde solution. In four of the six fusions performed, this active immunization was preceded by passive immunization with mouse antiserum directed against membrane vesicles from BAEC grown on plastic. Among the growing hybrids, 7.6% secreted MAb that bound efficiently to both BAEC grown on lung-derived matrix and BAEC grown on plastic, while 3.5% (50) secreted MAb that bound primarily to BAEC grown on lung matrix. The fusion data show that only a passive/active immunization protocol yielded MAb directed against lung-specific endothelia. For example, MAb 6D3 and 5F5 selectively recognized endothelia from small- and medium-sized venules of bovine lungs, but failed to react with endothelial cells in other organs and tissues.     

Pro-angiogenic approach for skeletal muscle regeneration

The angiogenesis process is a phenomenon in which numerous molecules participate in the stimulation of the new vessels' formation from pre-existing vessels. Angiogenesis is a crucial step in tissue regeneration and recovery of organ and tissue function. Muscle diseases affect millions of people worldwide overcome the ability of skeletal muscle to self-repair. Pro-angiogenic therapies are key in skeletal muscle regeneration where both myogenesis and angiogenesis occur. These therapies have been based on mesenchymal stem cells (MSCs), exosomes, microRNAs (miRs) and delivery of biological factors. The use of different calls of biomaterials is another approach, including ceramics, composites, and polymers. Natural polymers are use due its bioactivity and biocompatibility in addition to its use as scaffolds and in drug delivery systems. One of these polymers is the natural rubber latex (NRL) which is biocompatible, bioactive, versatile, low-costing, and capable of promoting tissue regeneration and angiogenesis. In this review, the advances in the field of pro-angiogenic therapies are discussed.     

Regenerative biology and medicine

The replacement of damaged tissues and organs with tissue and organ transplants or bionic implants has serious drawbacks. There is now emerging a new approach to tissue and organ replacement, regenerative biology and medicine. Regenerative biology seeks to understand the cellular and molecular differences between regenerating and non-regenerating tissues. Regenerative medicine seeks to apply this understanding to restore tissue structure and function in damaged, non-regenerating tissues. Regeneration is accomplished by three mechanisms, each of which uses or produces a different kind of regeneration-competent cell. Compensatory hyperplasia is regeneration by the proliferation of cells which maintain all or most of their differentiated functions (e.g., liver). The urodele amphibians regenerate a variety of tissues by the dedifferentiation of mature cells to produce progenitor cells capable of division. Many tissues contain reserve stem or progenitor cells that are activated by injury to restore the tissue while simultaneously renewing themselves. All regeneration-competent cells have two features in common. First, they are not terminally differentiated and can re-enter the cell cycle in response to signals in the injury environment. Second, their activation is invariably accompanied by the dissolution of the extracellular matrix (ECM) surrounding the cells, suggesting that the ECM is an important regulator of their state of differentiation. Regenerative medicine uses three approaches. First is the transplantation of cells into the damaged area. Second is the construction of bioartificial tissues by seeding cells into a biodegradable scaffold where they produce a normal matrix. Third is the use of a biomaterial scaffold or drug delivery system to stimulate regeneration in vivo from regeneration-competent cells. There is substantial evidence that non-regenerating mammalian tissues harbor regeneration-competent cells that are forced into a pathway of scar tissue formation. Regeneration can be induced if the factors leading to scar formation are inhibited and the appropriate signaling environment is supplied. An overview of regenerative mechanisms, approaches of regenerative medicine, research directions, and research issues will be given.     

In vitro models of angiogenesis and vasculogenesis in fibrin gel

In vitro models of endothelial assembly into microvessels are useful for the study of angiogenesis and vasculogenesis. In addition, such models may be used to provide the microvasculature required to sustain engineered tissues. A large range of in vitro models of both angiogenesis and vasculogenesis have utilized fibrin gel as a scaffold. Although fibrin gel is conducive to endothelial assembly, its ultrastructure varies substantially based on the gel formulation and gelation conditions, making it challenging to compare between models. This work reviews existing models of endothelial assembly in fibrin gel and posits that differerences between models are partially caused by microstructural differences in fibrin gel.     

Numb Expression and Asymmetric versus Symmetric Cell Division in Distal Embryonic Lung Epithelium

Proper balance between self-renewal and differentiation of lung-specific progenitors is absolutely required for normal lung morphogenesis/regeneration. Therefore, understanding the behavior of lung epithelial stem/progenitor cells could identify innovative solutions for restoring normal lung morphogenesis and/or regeneration. The Notch inhibitor Numb is a key determinant of asymmetric or symmetric cell division and hence cell fate. Yet Numb proximal-distal expression pattern and symmetric versus asymmetric division are uncharacterized during lung epithelial development. Herein, the authors find that the cell fate determinant Numb is highly expressed and asymmetrically distributed at the apical side of distal epithelial progenitors and segregated to one daughter cell in most mitotic cells. Knocking down Numb in MLE15 epithelial cells significantly increased the number of cells expressing the progenitor cell markers Sox9/Id2. Furthermore, cadherin hole analysis revealed that most distal epithelial stem/progenitor cells in embryonic lungs divide asymmetrically; with their cleavage, planes are predicted to bypass the cadherin hole, resulting in asymmetric distribution of the cadherin hole to the daughter cells. These novel findings provide evidence for asymmetric cell division in distal epithelial stem/progenitor cells of embryonic lungs and a framework for future translationally oriented studies in this area.     

Imaging cardiac extracellular matrices: a blueprint for regeneration

Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated.