A regenerative approach to the pharmacological management of hard-to-heal wounds.

A wound is considered hard-to-heal when, despite the appropriate clinical analysis and intervention, the wound area reduces by less than a third at four weeks and complete healing fails to occur within 12 weeks. The most prevalent hard-to-heal wounds are associated with underlying metabolic diseases or vascular insufficiency and include arterial, venous, pressure and diabetic foot ulcers. Their common features include an abnormal immune response and extended inflammatory phase, a subdued proliferation phase due to cellular insufficiencies and finally an almost non-existent remodeling phase. Advances in wound care technology, tested in both pre-clinical models and clinical trials, have paved the way for improved treatment options, focused on regeneration. These interventions have been shown to limit the extent of ongoing inflammatory damage, decrease bacterial load, promote angiogenesis and deposition of granulation tissue, and stimulate keratinocyte migration thereby promoting re-epithelialization in these wounds. The current review discusses these hard-to-heal wounds in the context of their underlying pathology and potential of advanced treatment options, which if applied promptly as a standard of care, could reduce morbidity, promote quality of life, and alleviate the burden on a strained health system.

Co-culture of pro-inflammatory macrophages and myofibroblasts: Evaluating morphological phenotypes and screening the effects of signaling pathway inhibitors.

Skeletal muscle regeneration is a complex process influenced by non-myogenic macrophages and fibroblasts, which acquire different phenotypes in response to changes in the injury milieu or changes in experimental conditions. In vitro, serum stimulates the differentiation of fibroblasts into myofibroblasts, while lipopolysaccharide (LPS) stimulates the polarization of unstimulated (M0) macrophages to acquire an M1 pro-inflammatory phenotype. We characterized these phenotypes using morphology (with circularity as shape descriptor; perfect circularity = 1.0) and phenotype-specific markers. Myofibroblasts (high α-smooth muscle actin [SMA] expression) had high circularity (mean 0.60 ± 0.03). Their de-differentiation to fibroblasts (low α-SMA expression) significantly lessened circularity (0.47 ± 0.01 and 0.35 ± 0.02 in 2% or 0% serum culture media respectively (p < 0.05). Unstimulated (M0) macrophages (no CD86 expression) had high circularity (0.72 ± 0.02) which decreased when stimulated to M1 macrophages (CD86 expression) (LPS; 0.61 ± 0.02; p < 0.05). Utilizing these established conditions, we then co-cultured M1 macrophages with myofibroblasts or myoblasts. M1 macrophages significantly decreased relative myofibroblast numbers (from 223 ± 22% to 64 ± 7%), but not myoblast numbers. This pro-inflammatory co-culture model was used to rapidly screen the following four compounds for ability to prevent M1 macrophage-mediated decrease in myofibroblast numbers: L-NAME (inducible nitric oxide synthase inhibitor), SB203580 (p38 mitogen-activated protein kinase inhibitor), SP600125 (c-Jun N-terminal kinase inhibitor) and LY294002 (phosphoinositide 3-kinase [PI3K] inhibitor). We found that LY294002 rescued myofibroblasts and decreased macrophage numbers. Myofibroblast rescue did not occur with L-NAME, SB203580 or SP600125 incubation. In conclusion, these data suggest a PI3K-associated mechanism whereby myofibroblasts can be rescued, despite simulated pro-inflammatory conditions.

Ex vivo antioxidant preconditioning improves the survival rate of bone marrow stem cells in the presence of wound fluid.

The advancement of autologous mesenchymal stem cell (MSC) therapy for the treatment of non-healing diabetic wounds is hampered by endogenous MSC dysfunction and limited viability of cells post-transplantation into the pathological wound environment. The development of effective strategies to restore the functional capabilities of these impaired MSCs prior to transplantation may be a key to their ultimate success as wound repair mediators. The current study therefore investigated whether antioxidant preconditioning [7.5 mM N-acetylcysteine (NAC) + 0.6 mM ascorbic 2-phosphate (AAP)] could restore the growth rate, migration ability and viability of impaired MSCs and whether this restored state is maintained in the presence of diabetic wound fluid (DWF). Healthy control (source: wild type, C57BL/6J mice) (n = 12) and impaired/diabetic MSCs (source: obese prediabetic, B6.Cg-Lepob/J mice) (n = 12) were isolated from the bone marrow of mice. Treatment groups post-isolation were as follow: (a) No treatment (baseline phenotype): MSCs expanded in standard growth media (SGM) (±8 days) and only exposed to growth media. (b) DWF (baseline response): MSCs expanded in SGM (±8 days) followed by exposure to DWF (24 hours, 48 hours, 96 hours). (c) Antioxidant preconditioning (preconditioned phenotype): MSCs expanded in the presence of NAC/AAP (±8 days). (d) Antioxidant preconditioning + DWF (preconditioned response): MSCs expanded in the presence of NAC/AAP (±8 days) followed by exposure to DWF (24 hours, 48 hours, 96 hours). The results demonstrated that expansion of MSCs (both healthy control and impaired diabetic) in the presence of combined NAC/AAP treatment improved ex vivo MSC viability and protected MSCs in the presence of DWF. Despite improved viability, AAP/NAC could however not rescue the reduced proliferation and migration capacity of impaired diabetic MSCs. The protective effect of NAC/AAP preconditioning against the toxicity of DWF could however be a potential strategy to improve cell number post-transplantation.

Antioxidant Preconditioning Improves the Paracrine Responsiveness of Mouse Bone Marrow Mesenchymal Stem Cells to Diabetic Wound Fluid.

Mesenchymal stem cells (MSCs) are a promising therapeutic tool for the treatment of nonhealing diabetic wounds. The pathological nature of the niche microenvironment limits the use of autologous cell therapy in diabetic patients. Prolonged exposure of endogenous MSCs to a pathological microenvironment in vivo reduces their ability to respond to environmental cues. This study investigated the effectiveness of ex vivo antioxidant treatment [N-acetylcysteine (7.5 mM NAC) and Ascorbic acid 2-phosphate (0.6 mM AAP)] to restore the paracrine function of diabetic MSCs. Healthy control [bone marrow stem cells derived from wild-type mice (SCWT)] (source: wild-type C57BL/6J mice) (n = 12) and impaired/dysfunctional [bone marrow stem cells derived from ob/ob mice (SCob)] (source: obese diabetic, B6.Cg-Lepob/J mice) (n = 12) MSCs were isolated. Ex vivo treatment groups (SCWT vs. SCob) were as follows: (1) no treatment (baseline phenotype), (2) stimulated with diabetic wound fluid (DWF) (baseline response), (3) antioxidant preconditioning (preconditioned phenotype), and (4) antioxidant preconditioned with subsequent stimulation with DWF (preconditioned response). The paracrine responsiveness on both the molecular (mRNA expression of 80 cytokines and receptors, quantitative polymerase chain reaction microarray) and protein (23-plex bead-array Luminex assay) level was assessed. At baseline, 31 genes were overexpressed (> × 2-fold) and 39 genes were underexpressed (> × 2-fold) in SCob versus SCWT. In conditioned media, significant differences (P < 0.05) were detected at baseline for two proinflammatory cytokines [tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ)], four chemokines [keratinocyte chemoattractant (KC), granulocyte colony-stimulating factor (GCSF), Eotaxin, and macrophage chemoattractant protein (MCP1)], and one anti-inflammatory cytokine [interleukin 10 (IL10)]. Following stimulation with DWF, significant differences (P < 0.05) were detected in the secretion of two chemokines [granulocyte macrophage colony-stimulating factor (GMCSF) and Eotaxin], three proinflammatory cytokines (TNFα, IFNγ, and IL9), and four anti-inflammatory cytokines (IL10, IL4, IL13, and IL3). Antioxidant preconditioning significantly dampened the excessive TNFα response observed in SCob and improved the secretion of IL10. Taken together these data suggest that the combined ex vivo treatment of autologous stem cells with NAC and AAP could potentially be an effective strategy to restore the paracrine function of impaired diabetic MSCs before transplantation.

A triple co-culture method to investigate the effect of macrophages and fibroblasts on myoblast proliferation and migration.

The communication between nonmyogenic cells, such as macrophages and fibroblasts, and myoblasts is crucial for successful skeletal muscle repair. In vitro co-culture methods can be used to increase our understanding of these cellular interactions; however, current protocols are restricted to two, often physically separate, cell populations. Here, we demonstrate a novel, inexpensive in vitro triple co-culture method that facilitates the co-culture of at least three cell populations with some degree of cell-cell contact. Using this method, we determined the effect of macrophages and fibroblasts on myoblast proliferation and migration. A significant increase in myoblast proliferation and migration was observed following co-culture with either macrophages or fibroblasts. However, triple co-culture of macrophages, fibroblasts, and myoblasts revealed that the presence of macrophages prevented fibroblasts from maintaining this positive effect on myoblast migration. Macrophages, on the other hand, continued to promote myoblast proliferation whether in the presence of fibroblasts or not. Our triple co-culture system highlights the significance of multicellular communication in regulating myoblast proliferation and migration and emphasizes the importance of more complex co-culture systems when investigating myogenesis in vitro.

Analysis and quantification of in vitro myoblast fusion using the LADD Multiple Stain.

Myoblast fusion, which is essential for muscle development, regeneration, and repair, can be assessed in vitro via the calculation of a fusion index. Traditionally, this requires use of either immunocytochemistry or fluorescently-labeled cytoskeletal staining, followed by microscopy and laborious analysis. The expense and time-consuming nature of the optimization and application of antibody-based techniques such as immunocytochemistry, as well as the need for specialized analytical equipment such as fluorescence microscopes, presents a barrier to the routine analysis of this crucial step during terminal differentiation. Here, we describe (i) a novel use of the commonly available LADD Multiple Stain for visualization of myoblast fusion in vitro; (ii) the optimization of a simple image analysis method to generate quick, quantifiable data representative of a fusion index; and (iii) the use of a protocol combining these two procedures to investigate in vitro myoblast fusion in a simple and efficient manner as proof-of-concept.

Dose-dependent modulation of myogenesis by HGF: implications for c-Met expression and downstream signalling pathways.

Hepatocyte growth factor (HGF) regulates satellite cell activation, proliferation, and differentiation. We analyzed the dose-dependent effects of HGF on myogenesis. Murine C2C12 and human donor-derived skeletal muscle myoblasts were treated with 0, 2, or 10 ng/ml HGF followed by assessment of proliferation and differentiation. HGF (2 ng/ml) significantly promoted cell division, but reduced myogenic commitment and fusion. Conversely, 10 ng/ml HGF reduced proliferative capability, but increased differentiation. c-Met expression analysis revealed significantly decreased expression in differentiating cells cultured with 2 ng/ml HGF, but increased expression in proliferating cells with 10 ng/ml HGF. Mitogen-activated protein kinase (MAPKs: ERK, JNK, or p38K) and phosphatidylinositol-3-kinase (PI3K) inhibition abrogated the HGF-stimulated increase in cell number. Interestingly, PI3K and p38 kinase facilitated the negative effect of HGF on proliferation, while ERK inhibition abrogated the HGF-mediated decrease in differentiation. Dose-dependent effects of HGF are mediated by changes in c-Met expression and downstream MAPK and PI3K signalling.

Simultaneous isolation of enriched myoblasts and fibroblasts for migration analysis within a novel co-culture assay.

Skeletal muscle injury elicits the activation of satellite cells and their migration to the wound area for subsequent terminal differentiation and tissue integration. However, interstitial fibroblasts recruited to the site of injury promote deposition of fibrotic tissue, which hampers myoblast-mediated muscle regeneration. Currently, analysis of myoblast migration in vitro can be accomplished using chemotactic, cell-exclusion, or wound healing assays. Yet, to investigate cell motility following skeletal muscle damage more accurately, migration assays need to better simulate the repair process. Here we present a protocol for the simultaneous isolation of myoblasts and fibroblasts from the same muscle tissue, ensuring the consistent generation of enriched, purified, and matched cell populations at a low passage number. We then describe a wound assay that uses a novel approach to the co-culture of myoblasts and fibroblasts to mimic the injured environment more closely than other established methods. Using this assay, we demonstrate that fibroblasts are able to increase myoblast migration significantly, validating our new in vitro method. As the observed effect on migration is most likely mediated by secreted factors, our assay could easily be extended to include antibody-based protein analysis of secreted factors in animal or human systems.

Simple silicone chamber system for in vitro three-dimensional skeletal muscle tissue formation.

Bioengineering skeletal muscle often requires customized equipment and intricate casting techniques. One of the major hurdles when initially trying to establish in vitro tissue engineered muscle constructs is the lack of consistency across published methodology. Although this diversity allows for specialization according to specific research goals, lack of standardization hampers comparative efforts. Differences in cell type, number and density, variability in matrix and scaffold usage as well as inconsistency in the distance between and type of adhesion posts complicates initial establishment of the technique with confidence. We describe an inexpensive, but readily adaptable silicone chamber system for the generation of skeletal muscle constructs that can readily be standardized and used to elucidate myoblast behavior in a three-dimensional space. Muscle generation, regeneration and adaptation can also be investigated in this model, which is more advanced than differentiated myotubes.

In vitro myoblast motility models: investigating migration dynamics for the study of skeletal muscle repair.

Skeletal muscle repair requires the migration of myoblasts (activated satellite cells) both to the injury site and then within the wound to facilitate cellular alignment in preparation for differentiation, fusion and eventual healing. Along this journey, the cells encounter a range of soluble and extracellular matrix factors which regulate their movement and ultimately determine how successful the repair process will be. Sub-optimal migration can lead to a number of scenarios, including reduced myoblast numbers entering the wound, poor alignment and insufficient differentiation to correctly repair the damage. It is therefore critical that all aspects of myoblast migration are understood, particularly in response to the changing growth and matrix factor profile prevalent following skeletal muscle injury. Since 1962, when Boyden first introduced his chemotactic chamber, numerous in vitro migration assays have been developed to mimic the wound more closely. These have increased in complexity to account for the complex micro-environment found in vivo during muscle repair and include a range of modified cell exclusion, chemotactic and three-dimensional assays. This review describes and discusses these advances and highlights the importance they have in expanding our understanding of myoblast migration dynamics.

Satellite cell pool expansion is affected by skeletal muscle characteristics.

INTRODUCTION: We investigated changes in satellite cell (SC) pool size after an acute bout of strenuous exercise and evaluated the influence of baseline SC count and fiber type. METHODS: Participants completed a downhill running (DHR) intervention (5 × 8 min, 2-min rest; 80% VO2max ; -10% gradient). Muscle biopsies were taken 7 days before VO2max and 7-9 days after the DHR intervention. Delayed-onset muscle soreness (DOMS) and creatine kinase activity (CK) were measured on days 1, 2, 7, and 9 post-DHR. SCs were identified by Pax7 and laminin staining. Relative distribution of MHC isoforms was determined by electrophoresis. RESULTS: DOMS and CK peaked on day 1 post-DHR (P < 0.01). The SC pool increased (26%) after DHR (P = 0.005). SCs/total myonuclei after recovery correlated with baseline SCs (r = 0.979, P = 0.003) and VO2max (r = 0.956, P = 0.011), whereas change in SC pool (Pax7(+) cells/total myonuclei: recovery minus baseline) tended to correlate with percent MHC II (r = 0.848; P = 0.06). CONCLUSION: Interindividual physiological characteristics affect SC pool expansion after a single bout of DHR and are influenced by VO2max .

TGF-ß isoforms inhibit IGF-1-induced migration and regulate terminal differentiation in a cell-specific manner.

Following muscle injury, the damaged tissue and influx of inflammatory cells stimulate the secretion of growth factors and cytokines to initiate repair processes. This release of chemotactic signaling factors activates resident precursor cells and stimulates their mobilization and migration to the site of injury where terminal differentiation can occur. The three transforming growth factor-ß (TGF-ß) isoforms, and insulin-like growth factor-1 (IGF-1) are among the known regulatory factors released following muscle damage. We investigated the effect of recombinant active TGF-ß1, -ß2, -ß3 and IGF-1 on C2C12 skeletal muscle satellite cell and P19 embryonal carcinoma cell terminal differentiation and migration. C2C12 myoblast fusion as well as P19 embryoid body formation and myogenic differentiation was assessed following 72 h TGF-ß treatment (5 ng/ml), whereas the effect of the TGF-ß isoforms on migration was determined following 7 h incubation. Our results showed that TGF-ß decreases C2C12 myoblast fusion in an isoform-independent manner, whereas in the P19 cell lineage, results demonstrate that TGF-ß1 specifically and significantly increased P19 embryoid body formation, but not expression of Connexin-43 or Myosin Heavy Chain. IGF-1 significantly increased migration compared to TGF-ß isoforms, which, on their own, had no significant effect on the mobilization of either C2C12 or P19 cells. TGF-ß isoforms decreased IGF-1-induced migration of both cell lineages. By distinguishing the factors involved in, and the molecular signals required for, myoblast recruitment during repair processes, strategies can be developed towards improved cell-mediated therapies for muscle injury.

Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues.

The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.

Potential myogenic stem cell populations: sources, plasticity, and application for cardiac repair.

The ability of unspecialized stem cells to differentiate into mature, specialized cell types has made them attractive as potential agents for enhanced tissue repair and regenerative medicine. This is especially true of diseases and disorders for which no or only partially effective treatments are currently available. Recently, increased focus has been placed on the regenerative potential of satellite cells (myogenic precursor cells found in the adult skeletal muscle) in various muscular disorders, such as dystrophy and myocardial injury following ischemia. Animal studies and clinical trials are in progress using satellite cells as cellular candidates; however, this early rollout in the clinical setting has deflected attention from the potential of other less specialized, but potentially more maliable, stem cell sources. Published data is still lacking on the best methods for identification, isolation, and further expansion or nuclear manipulation of these cells in vitro. Also, although differentiation capacity has been proven in terms of protein expression patterns characteristic of myogenesis, proof of contractile and energetic compatibility between graft and host is more difficult to establish. In this regard, although future animal model studies will be invaluable, they must be designed with short- and long-term functional outcomes in mind. This review moves beyond initial excitement regarding the acknowledged potential of cell therapy and provides a realistic exposition of the themes and specific issues that should be considered in current experimental research study designs.

TGF-beta's delay skeletal muscle progenitor cell differentiation in an isoform-independent manner.

Satellite cells are a quiescent heterogeneous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-beta (TGF-beta) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-beta on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-beta1, -beta2 and -beta3 on wound repair in other tissues. In the current study we compared the effect of TGF-beta1, -beta2 and -beta3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-beta increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-beta1, -beta2 and -beta3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-beta promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner.

The changing AMPK expression profile in differentiating mouse skeletal muscle myoblast cells helps confer increasing resistance to apoptosis.

AMP-activated protein kinase (AMPK) functions as a alpha/beta/gamma heterotrimer to preserve ATP levels and so cell viability during stressful conditions. However, its role in aiding survival of adult skeletal muscle precursor cells is unclear. Using the differentiating mouse C2C12 postnatal skeletal muscle myoblast cell line, we have determined that proteins for the AMPK subunit isoforms alpha2 and gamma2 are constitutively expressed, while those for alpha1, beta1 and beta2 are undetectable in undifferentiated myoblasts but increasingly expressed with differentiation to myotubes. Although the gamma3 subunit is expressed at a low level in myoblasts, it too is expressed increasingly with differentiation to myotubes. The p50 but not the p72 isoform of the embryonic alpha subunit homologue MELK is expressed only in proliferating myoblasts, while the ARK5 alpha subunit homologue is increasingly expressed with differentiation. Myotubes displayed higher basal and stimulated alpha1/alpha2 AMPK activation than myoblasts. Furthermore, serum starvation resulted in less apoptosis of differentiated myotubes than of undifferentiated myoblasts. This reflects, in part, the increased expression of functional AMPK in the myotubes, since specific inhibition of AMPK activity with 6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-alpha] pyrimidine (Compound C) exacerbated the apoptosis resulting from serum withdrawal. If these in vitro events can also occur in vivo, they could have implications for pathologies such as muscle wasting, in which undifferentiated satellite stem cells may be easier apoptotic targets than their differentiated counterparts. Furthermore, these results suggest that when interpreting results from in vitro or in vivo experiments on AMPK, the subunit expression profile should be taken into account.

c-Fos immunoreactivity in selected brain regions of rats after heat exposure and pyrogen administration.

We determined c-Fos immunoreactivity (Fos-IR) in selected hypothalamic nuclei, the organum vasculosum of the laminae terminals (OVLT) and somatosensory cortex of rats after hyperthermia induced by exogenous heat exposure, Gram-negative or Gram-positive pyrogen administration. The magnitude of Fos-IR was similar in thermoregulatory hypothalamic nuclei of rats after heat exposure or lipopolysaccharide (LPS) injection, despite the different origins of the hyperthermias. Heat-induced hyperthermia was associated with increased Fos-IR in the somatosensory cortex. LPS, but not heat exposure or injection of killed Staphylococcus aureus cells activated OVLT neurons. The OVLT may thus not be a port of entry for humoral mediators of Gram-positive bacterial fevers.

Efficient transient genetic labeling of human CD34+ progenitor cells for in vivo application.

Genetic labeling of human hematopoietic progenitor cells (HPC) and their consecutive fate-mapping in vivo is an approach to answer intriguing questions in stem cell biology. We recently reported efficient transient genetic labeling of human CD34+ HPC with the truncated low-affinity nerve growth factor receptor (DeltaLNGFR) for in vivo application. Here we investigate whether HPC labeling with DeltaLNGFR affects lineage-specific cell differentiation, whether DeltaLNGFR expression is maintained during lineage-specific cell differentiation and which leukemia cell line might be an appropriate cell culture model for human CD34+ HPC. Human CD34+ peripheral blood stem cells and various leukemia cell lines were characterized by immunophenotyping. Cells were transfected using nucleofection. Hematopoietic differentiation was studied by colony-forming assays. DeltaLNGFR expression was assessed using reverse transcription-PCR, immunofluorescence and flow cytometry. Nucleofection was efficient and did not significantly reduce hematopoietic cell differentiation. Mature myeloid cells (CD66b+) derived from human CD34+ HPC and Mutz2 cells maintained DeltaLNGFR expression at a high percentage (70 +/- 2% and 58 +/- 2%, respectively). Mutz2 cells may serve as an in vitro model for human myeloid HPC. The method described herein has been adopted to Good Manufacturing Practices (GMP) guidelines and is ready for in vivo application.

Old dogmas and new hearts: a role for adult stem cells in cardiac repair?

The vast developmental repertoire of embryonic stem cells is well recognised. These primitive stem cells can differentiate in vivo and in vitro into cells of all three embryonic germ layers (endoderm, mesoderm, ectoderm), making them attractive potential agents to target for enhanced tissue repair and regeneration. Adult stem cells on the other hand are considered more restricted in their lineage differentiation capabilities. Recent research has challenged this dogma with the finding that bone marrow-derived stem cells can differentiate into a wide variety of cell types including muscle (skeletal and cardiac). Furthermore, although the myocardium has for decades been regarded as a post-mitotic organ, a series of studies has indicated that a population of stem cells exists which is capable of at least partial reconstitution of the myocardium following an ischaemic insult. It is therefore now accepted that adult stem cells could be used to enhance myocardial repair. This review discusses the current status of adult stem cell research in the light of its potential for improving myocardial repair.