Myogenic potential of mesenchymal stem cells isolated from porcine adipose tissue.

Advances in stem cell biology and materials science have provided a basis for developing tissue engineering methods to repair muscle injury. Among stem cell populations with potential to aid muscle repair, adipose-derived mesenchymal stem cells (ASC) hold great promise. To evaluate the possibility of using porcine ASC for muscle regeneration studies, we co-cultured porcine ASC with murine C2C12 myoblasts. These experiments demonstrated that porcine ASC display significant myogenic potential. Co-culture of ASC expressing green fluorescent protein (GFP) with C2C12 cells resulted in GFP+ myotube formation, indicating fusion of ASC with myoblasts to form myotubes. The presence of porcine lamin A/C positive nuclei in myotubes and RTqPCR analysis of porcine myogenin and desmin expression confirmed that myotube nuclei derived from ASC contribute to muscle gene expression. Co-culturing GFP+ASC with porcine satellite cells demonstrated enhanced myogenic capability of ASC, as the percentage of labeled myotubes increased compared to mouse co-cultures. Enhancing myogenic potential of ASC through soluble factor treatment or expansion of ASC with innate myogenic capacity should allow for their therapeutic use to regenerate muscle tissue lost to disease or injury.

Proteomic analysis of fibroblastema formation in regenerating hind limbs of Xenopus laevis froglets and comparison to axolotl.

BACKGROUND: To gain insight into what differences might restrict the capacity for limb regeneration in Xenopus froglets, we used High Performance Liquid Chromatography (HPLC)/double mass spectrometry to characterize protein expression during fibroblastema formation in the amputated froglet hindlimb, and compared the results to those obtained previously for blastema formation in the axolotl limb. RESULTS: Comparison of the Xenopus fibroblastema and axolotl blastema revealed several similarities and significant differences in proteomic profiles. The most significant similarity was the strong parallel down regulation of muscle proteins and enzymes involved in carbohydrate metabolism. Regenerating Xenopus limbs differed significantly from axolotl regenerating limbs in several ways: deficiency in the inositol phosphate/diacylglycerol signaling pathway, down regulation of Wnt signaling, up regulation of extracellular matrix (ECM) proteins and proteins involved in chondrocyte differentiation, lack of expression of a key cell cycle protein, ecotropic viral integration site 5 (EVI5), that blocks mitosis in the axolotl, and the expression of several patterning proteins not seen in the axolotl that may dorsalize the fibroblastema. CONCLUSIONS: We have characterized global protein expression during fibroblastema formation after amputation of the Xenopus froglet hindlimb and identified several differences that lead to signaling deficiency, failure to retard mitosis, premature chondrocyte differentiation, and failure of dorsoventral axial asymmetry. These differences point to possible interventions to improve blastema formation and pattern formation in the froglet limb.

Muscle repair and regeneration: stem cells, scaffolds, and the contributions of skeletal muscle to amphibian limb regeneration.

Skeletal muscle possesses a robust innate capability for repair of tissue damage. Natural repair of muscle damage is a stepwise process that requires the coordinated activity of a number of cell types, including infiltrating macrophages, resident myogenic and non-myogenic stem cells, and connective tissue fibroblasts. Despite the proficiency of this intrinsic repair capability, severe injuries that result in significant loss of muscle tissue overwhelm the innate repair process and require intervention if muscle function is to be restored. Recent advances in stem cell biology, regenerative medicine, and materials science have led to attempts at developing tissue engineering-based methods for repairing severe muscle defects. Muscle tissue also plays a role in the ability of tailed amphibians to regenerate amputated limbs through epimorphic regeneration. Muscle contributes adult stem cells to the amphibian regeneration blastema, but it can also contribute blastemal cells through the dedifferentiation of multinucleate myofibers into mononuclear precursors. This fascinating plasticity and its contributions to limb regeneration have prompted researchers to investigate the potential for mammalian muscle to undergo dedifferentiation. Several works have shown that mammalian myotubes can be fragmented into mononuclear cells and induced to re-enter the cell cycle, but mature myofibers are resistant to fragmentation. However, recent works suggest that there may be a path to inducing fragmentation of mature myofibers into proliferative multipotent cells with the potential for use in muscle tissue engineering and regenerative therapies.

Employing the biology of successful fracture repair to heal critical size bone defects.

Bone has the natural ability to remodel and repair. Fractures and small noncritical size bone defects undergo regenerative healing via coordinated concurrent development of skeletal and vascular elements in a soft cartilage callus environment. Within this environment bone regeneration recapitulates many of the same cellular and molecular mechanisms that form embryonic bone. Angiogenesis is intimately involved with embryonic bone formation and with both endochondral and intramembranous bone formation in differentiated bone. During bone regeneration osteogenic cells are first associated with vascular tissue in the adjacent periosteal space or the adjacent injured marrow cavity that houses endosteal blood vessels. Critical size bone defects cannot heal without the assistance of therapeutic aids or materials designed to encourage bone regeneration. We discuss the prospects for using synthetic hydrogels in a bioengineering approach to repair critical size bone defects. Hydrogel scaffolds can be designed and fabricated to potentially trigger the same bone morphogenetic cascade that heals bone fractures and noncritical size defects naturally. Lastly, we introduce adult Xenopus laevis hind limb as a novel small animal model system for bone regeneration research. Xenopus hind limbs have been used successfully to screen promising scaffolds designed to heal critical size bone defects.

Network based transcription factor analysis of regenerating axolotl limbs.

BACKGROUND: Studies on amphibian limb regeneration began in the early 1700's but we still do not completely understand the cellular and molecular events of this unique process. Understanding a complex biological process such as limb regeneration is more complicated than the knowledge of the individual genes or proteins involved. Here we followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs. RESULTS: We used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF) pathways and networks that regulate blastema formation in amputated axolotl limbs. The five most connected factors, c-Myc, SP1, HNF4A, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets). Network analysis showed that TGF-ß1 and fibronectin (FN) lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28 are also connected to c-Myc and SP1. CONCLUSIONS: Our study provides a systems biology approach to how different molecular entities inter-connect with each other during the formation of an accumulation blastema in regenerating axolotl limbs. This approach provides an in silico methodology to identify proteins that are not detected by experimental methods such as proteomics but are potentially important to blastema formation. We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration. Systems biology has the potential to map out numerous other pathways that are crucial to blastema formation in regeneration-competent limbs, to compare these to the pathways that characterize regeneration-deficient limbs and finally, to identify stem cell markers in regeneration.

Looking proximally and distally: 100 years of limb regeneration and beyond.

The experimental study of amphibian limb regeneration spans most of the 20th century and the first decade of the 21st century. We first review the major questions investigated over this time span: (1) the origin of regeneration blastema cells, the mechanism of tissue breakdown that liberates cells from their tissue organization to participate in blastema formation, (3) the mechanism of dedifferentiation of these cells, (4) how the blastema grows, (5) how the blastema is patterned to restore the missing limb structures, and (6) why adult anurans, birds and mammals do not have the regenerative powers of urodele salamanders. We then look forward in a perspective to discuss the many unanswered questions raised by investigations of the past century, what new approaches can be taken to answer them, and what the prospects are for translation of basic research on limb regeneration into clinical means to regenerate human appendages.

Xenopus laevis as a novel model to study long bone critical-size defect repair by growth factor-mediated regeneration.

We used the tarsus of an adult Xenopus laevis frog as an in vivo load-bearing model to study the regeneration of critical-size defects (CSD) in long bones. We found the CSD for this bone to be about 35% of the tarsus length. To promote regeneration, we implanted biocompatible 1,6 hexanediol diacrylate scaffolds soaked with bone morphogenetic proteins-4 and vascular endothelial growth factors. In contrast to studies that use scaffolds as templates for bone formation, we used scaffolds as a growth factor delivery vehicle to promote cartilage-to-bone regeneration. Defects in control frogs were filled with scaffolds lacking growth factors. The limbs were harvested at a series of time points ranging from 3 weeks to 6 months after implantation and evaluated using micro-computed tomography and histology. In frogs treated with growth factor-loaded scaffolds, we observed a cartilage-to-bone regeneration in the skeletal defect. Five out of eight defects were completely filled with cartilage by 6 weeks. Blood vessels had invaded the cartilage, and bone was beginning to form in ossifying centers. By 3 months, these processes were well advanced, and extensive ossification was observed in 6-month samples. In contrast, the defects in control frogs showed only formation of fibrous scar tissue. This study demonstrates the utility of a Xenopus model system for tissue engineering research and that the normal in vivo mechanism of endochondral bone development and fracture repair can be mimicked in the repair of CSD with scaffolds used as growth factor delivery mechanisms.

Proteomic analysis of blastema formation in regenerating axolotl limbs.

BACKGROUND: Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa) through the mid-tibia/fibula of axolotl hind limbs. RESULTS: We identified 309 unique proteins with significant fold change relative to controls (0 dpa), representing 10 biological process categories: (1) signaling, (2) Ca2+ binding and translocation, (3) transcription, (4) translation, (5) cytoskeleton, (6) extracellular matrix (ECM), (7) metabolism, (8) cell protection, (9) degradation, and (10) cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5), a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation. CONCLUSION: Our data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR). Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to neural and epidermal factors. Our findings indicate the general value of quantitative proteomic analysis in understanding the regeneration of complex structures.

Strategies to reduce variation in Xenopus regeneration studies.

In this study, we present strategies for experimental design that minimize variation in Xenopus hindlimb regeneration results. We have standardized our laboratory culture conditions for older stage Xenopus tadpoles. We have established a normal tadpole growth curve for our laboratory and characterized normal tadpole behaviors in an effort to eliminate abnormal tadpoles from our experiments. We have used large sample sizes and statistical analysis to establish normal regeneration performances for seven amputation planes in stages 55-57 tadpole hindlimbs. We have demonstrated that regeneration performance of abnormal tadpoles is significantly different than that of normal tadpoles. We have examined the kinetics of ossification in developing Xenopus hindlimbs, and have found that increasing ossification rates and rates of regeneration decline are inversely correlated.

Early regeneration genes: Building a molecular profile for shared expression in cornea-lens transdifferentiation and hindlimb regeneration in Xenopus laevis.

Recent studies in Xenopus laevis have begun to compare gene expression during regeneration with that of the original development of specific structures (e.g., the hindlimb and lens), while other studies have sought differences in gene expression between regeneration-competent and regeneration-incompetent stages. To determine whether there are any similarities between the regeneration of different structures, we have used a differential screen to seek shared early gene expression between hindlimb regeneration and cornea-lens transdifferentiation in the Xenopus tadpole. We have isolated 13 clones representing genes whose expression is up-regulated within the first few days of both regenerating processes and which are not demonstrably up-regulated in the context of basic wound healing. Furthermore, all of these genes also show prominent late embryonic expression. The expression patterns and putative identities of all 13 genes are presented, and a model is considered that allows us to characterize and profile important changes in gene expression, which might be shared among various regenerating and developmental systems.

Extending the table of stages of normal development of the axolotl: limb development.

The existing table of stages of the normal development of the axolotl (Ambystoma mexicanum) ends just after hatching. At this time, the forelimbs are small buds. In this study, we extend the staging series through completion of development of the forelimbs and hindlimbs.

Regeneration of the urodele limb: a review.

Urodele amphibians have been widely used for studies of limb regeneration. In this article, we review studies on blastema cell proliferation and propose a model of blastemal self-organization and patterning. The model is based on local cell interactions that intercalate positional identities within circumferential and proximodistal boundaries that outline the regenerate. The positional identities created by the intercalation process appear to be reflected in the molecular composition of the cell surface. Transcription factors and signaling molecules involved in patterning are discussed within the context of the boundary/intercalation model.

Urodele spinal cord regeneration and related processes.

Urodele amphibians, newts and salamanders, can regenerate lesioned spinal cord at any stage of the life cycle and are the only tetrapod vertebrates that regenerate spinal cord completely as adults. The ependymal cells play a key role in this process in both gap replacement and caudal regeneration. The ependymal response helps to produce a different response to neural injury compared with mammalian neural injury. The regenerating urodele cord produces new neurons as well as supporting axonal regrowth. It is not yet clear to what extent urodele spinal cord regeneration recapitulates embryonic anteroposterior and dorsoventral patterning gene expression to achieve functional reconstruction. The source of axial patterning signals in regeneration would be substantially different from those in developing tissue, perhaps with signals propagated from the stump tissue. Examination of the effects of fibroblast growth factor and epidermal growth factor on ependymal cells in vivo and in vitro suggest a connection with neural stem cell behavior as described in developing and mature mammalian central nervous system. This review coordinates the urodele regeneration literature with axial patterning, stem cell, and neural injury literature from other systems to describe our current understanding and assess the gaps in our knowledge about urodele spinal cord regeneration.