Muscle-derived stem cells for tissue engineering and regenerative therapy

Skeletal muscle has been recognized as an essential source of progenitor or satellite cells, which are primarily responsible for muscle regeneration. Recently, muscle has also been identified as a valuable source of postnatal stem cells that appear to be distinct from satellite cells and possess the ability to differentiate into other cell lineages. These cells, named muscle-derived stem cells, possess a high myogenic capacity and effectively regenerate both skeletal and cardiac muscle. Remarkably, when genetically modified ex vivo to express growth factors, these cells can differentiate into osteogenic and chondrogenic lineages and have been shown to promote the repair of bone and cartilage. Muscle stem cell-based regenerative therapy and tissue engineering using ex vivo gene therapy, are promising approaches for the treatment of various musculoskeletal, cardiovascular, and urological disorders.     

Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells

The anterior cruciate ligament (ACL) usually fails to heal after rupture mainly due to the inability of the cells within the ACL tissue to establish an adequate healing process, making graft reconstruction surgery a necessity. However, some reports have shown that there is a healing potential of ACL with primary suture repair. Although some reports showed the existence of mesenchymal stem cell-like cells in human ACL tissues, their origin still remains unclear. Recently, blood vessels have been reported to represent a rich supply of stem/progenitor cells with a characteristic expression of CD34 and CD146. In this study, we attempted to validate the hypothesis that CD34- and CD146-expressing vascular cells exist in hACL tissues, have a potential for multi-lineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured ACL. Immunohistochemistry and flow cytometry analysis of hACL tissues demonstrated that it contains significantly more CD34 and CD146-positive cells in the ACL ruptured site compared with the noninjured midsubstance. CD34+CD45- cells isolated from ACL ruptured site showed higher expansionary potentials than CD146+CD45- and CD34-CD146-CD45- cells, and displayed higher differentiation potentials into osteogenic, adipogenic, and angiogenic lineages than the other cell populations. Immunohistochemistry of fetal and adult hACL tissues demonstrated a higher number of CD34 and CD146-positive cells in the ACL septum region compared with the midsubstance. In conclusion, our findings suggest that the ACL septum region contains a population of vascular-derived stem cells that may contribute to ligament regeneration and repair at the site of rupture.     

Gamma interferon as an antifibrosis agent in skeletal muscle.

Muscle injuries are a common problem in sports medicine. Skeletal muscle can regenerate itself, but the process is both slow and incomplete. Previously we and others have used growth factors to improve the regeneration of muscle, but the muscle healing was impeded by scar tissue formation. However, when we blocked the fibrosis process with decorin, an antifibrosis agent, we improved the muscle healing. Here we show that gammainterferon (gammaINF)--a cytokine that inhibits the signaling of transforming growth factor beta1 (TGFbeta1), a fibrotic stimulator--reduces fibrosis formation and improves the healing of lacerated skeletal muscle. With gammaINF treatment, the growth rate of muscle-derived fibroblasts was reduced and the level of fibrotic protein expression induced by TGFbeta1 (including TGFbeta1, vimentin, and alpha-smooth muscle actin) was down-regulated in vitro. In a mouse laceration model, the area of fibrosis decreased when gammaINF was injected at either 1 or 2 weeks after injury. More importantly, the injection of gammaINF at either 1 or 2 weeks post-injury was found to improve muscle function in terms of both fast-twitch and tetanic strength. This study demonstrates that gammaINF is a potent antifibrosis agent that can improve muscle healing after laceration injury.     

Pharmacological characterization of the receptors involved in the β-adrenoceptor-mediated stimulation of the L-type Ca2+ current in frog ventricular myocytes

1. The whole-cell patch-clamp was used for studying the effects of various β₁- and β₂-adrenoceptor agonists and antagonists on the L-type Ca current (I_Ca) in frog ventricular myocytes.
2. Dose-response curves for the effects of isoprenaline (non selective β-agonist), salbutamol (β₂-agonist), dobutamine (β₁-agonist) on I_Ca were obtained in the absence and presence of various concentrations of ICI 118551 (β₂-antagonist), metoprolol (β₁-antagonist) and xamoterol (partial β₁-agonist) to derive EC₅₀ (i.e. the concentration of β-agonist at which the response was 50% of the maximum) and E_max (the maximal response) values by use of a Michaelis equation. Schild regression analysis was performed to examine whether the antagonists were competitive and to determine the equilibrium dissociation constant (K_B) for the antagonist-receptor complex.
3. Isoprenaline increased I_Ca with an EC₅₀ of 20.0 nM and an E_max of 597%. ICI 118551 and metoprolol competitively antagonized the effect of isoprenaline with a K_B of 3.80 nM and 207 nM, respectively.
4. Salbutamol increased I_Ca with an EC₅₀ of 290 nM and an E_max of 512%. ICI 118551 and metoprolol competitively antagonized the effect of salbutamol with a K_B of 1.77 nM and 456 nM, respectively.
5. Dobutamine increased I_Ca with an EC₅₀ of 2.40 μM and an E_max of 265%. ICI 118551 and metoprolol competitively antagonized the effect of dobutamine with a K_B of 2.84 nM and 609 nM, respectively.
6. Xamoterol had no stimulating effect on I_Ca. However, xamoterol competitively antagonized the stimulating effects of isoprenaline, salbutamol and dobutamine on I_Ca with a K_B of 58–64 nM.
7. We conclude that a single population of receptors is involved in the β-adrenoceptor-mediated regulation of I_Ca in frog ventricular myocytes. The pharmacological pattern of the response of I_Ca to the different β-adrenoceptor agonists and antagonists tested suggests that these receptors are of the β₂-subtype.

     

Role of cyclic nucleotide phosphodiesterase isoforms in cAMP compartmentation following β2-adrenergic stimulation of ICa,L in frog ventricular myocytes

The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the β₂-adrenergic stimulation of the L-type Ca²⁺ current ( I _Ca,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 μM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of I _Ca,L were used to determine the gradient of local vs. distant cAMP concentration (α). IBMX (100 μM), a non-selective PDE inhibitor, reduced α from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 μM) or PDE4 inhibition by Ro 20-1724 (3 μM) reduced α by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 μM), a non-selective phosphatase inhibitor, or H89 (1 μM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of I _Ca,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon β₂-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of β₂-adrenergic receptors with nearby Ca²⁺ channels via local elevations of cAMP.     

Intradermal influenza vaccination of healthy adults using a new microinjection system: a 3-year randomised controlled safety and immunogenicity trial

Background

Intradermal vaccination provides direct and potentially more efficient access to the immune system via specialised dendritic cells and draining lymphatic vessels. We investigated the immunogenicity and safety during 3 successive years of different dosages of a trivalent, inactivated, split-virion vaccine against seasonal influenza given intradermally using a microinjection system compared with an intramuscular control vaccine.

Methods

In a randomised, partially blinded, controlled study, healthy volunteers (1150 aged 18 to 57 years at enrolment) received three annual vaccinations of intradermal or intramuscular vaccine. In Year 1, subjects were randomised to one of three groups: 3 μg or 6 μg haemagglutinin/strain/dose of inactivated influenza vaccine intradermally, or a licensed inactivated influenza vaccine intramuscularly containing 15 μg/strain/dose. In Year 2 subjects were randomised again to one of two groups: 9 μg/strain/dose intradermally or 15 μg intramuscularly. In Year 3 subjects were randomised a third time to one of two groups: 9 μg intradermally or 15 μg intramuscularly. Randomisation lists in Year 1 were stratified for site. Randomisation lists in Years 2 and 3 were stratified for site and by vaccine received in previous years to ensure the inclusion of a comparable number of subjects in a vaccine group at each centre each year. Immunogenicity was assessed 21 days after each vaccination. Safety was assessed throughout the study.

Results

In Years 2 and 3, 9 μg intradermal was comparably immunogenic to 15 μg intramuscular for all strains, and both vaccines met European requirements for annual licensing of influenza vaccines. The 3 μg and 6 μg intradermal formulations were less immunogenic than intramuscular 15 μg. Safety of the intradermal and intramuscular vaccinations was comparable in each year of the study. Injection site erythema and swelling was more common with the intradermal route.

Conclusion

An influenza vaccine with 9 μg of haemagglutinin/strain given using an intradermal microinjection system showed comparable immunogenic and safety profiles to a licensed intramuscular vaccine, and presents a promising alternative to intramuscular vaccination for influenza for adults younger than 60 years.

Trial registration

Clinicaltrials.gov NCT00703651.