BOLAND CELL MYOBLASTS
The biotechnological aspects of human myoblasts and application thereof are described in this section. The outlook for human suffering from end-stage cardiac disease (heart failure) and declined for heart transplantation, is unfavourable. In patients with established heart failure and ejection fraction below 30%, the prognosis is also unfavourable. Mainstay is beta- blockers and ACE-inhibitors.
Clinical trials in the USA and Europe have reported on the application of biological therapy for heart failure in selected patients' meeting certain inclusion criteria. Although investigational, many trials have shown a positive outcome after autologous skeletal myoblast transplantation provided care is taken to avoid cardiac arrhythmias. Briefly, the process is as follows. The patient is carefully selected. A muscle biopsy is obtained from a thigh muscle. The skeletal myoblasts are cultivated in a specialized laboratory over a 4-6 week period; the cells are refined and injected directly into the heart muscle either at an open operation or via a catheter. Therapy may be stand- alone, or be part of a conventional CABAG procedure.
For the moment myoblasts seem to have superior cardiac enhancement compared to the use of autologous bone marrow. About 25% of myoblast recipients show increased EF after 6 months. With quality assurance, this figure may increase to beyond 45%. Myoblasts can be transplanted or engrafted directly by syringe infusion during CABAG and as a stand alone therapy ( by mini-thoracotomy). The future will lie with percutaneous catheter administration. Menasché has published long-term follow-up with interesting and encouraging results. Arrhythmias are now manageable with fitted defibrillators. See resources Endovasc Ther 2004, 11: 695-704, Cell Transplantation 2005, 14: 11 -19; JAMA 2003, 41: 879-88.
BOLAND CELL : SCIENTIFIC AND ACADEMIC DISCUSION ( MYOBLASTS)
The Paris group under the directorship of Dr Menasche has published extensively in this field. Although the treatment may be controversial, it has been highly successful in some patient's, without question. An earlier side-effect was the appearance of unpredictable cardiac arrhythmias, nowadays mandating an implantable ICD device before skeletal myoblast cell therapy. Myoblasts unfortunately do not form electrical communication with cardiomyocytes of the heart or gap junctions.
MYOBLAST UPDATE 2007:
NEW MYOBLAST INSIGHTS, TECHNOLOGY, APPLICATION AND BEYOND.
BOLANDCELLS has specialized expertise in myoblast and fibroblast technologies ( both theoretical and practical). Cell proprietary, manufacture, quality assurance. The application and use of autologous skeletal muscle myoblasts has now become an ever growing front, challenging all divisions of the medical profession, regarding regeneration of ageing tissue. And this you need to know when working in this field of applied cell biology and cell therapy. Myoblasts have been chiefly used in regenerative cardiology and in patients with advanced ischaemic heart disease and cardiac failure. The trials have been small and results are mixed although positive. But an explosion in knowledge has occurred. In most of the heart trials (obviously uncontrolled, but none the less important), patient selection has been persons with global ischemia following myocardial infarction. But new experimental work from Osaka University ( 2006) show that it is possible to ameliorate cardiac failure in a canine dilated cardiomyopathy model ( see: Jnl of Thoracic and Cardiovascular Surgery. October 2006). This model may open a treatment strategy for the management of patients with end-stage dilated cardiomyopathy. This will place more demand on the profession to be able to deliver and use cultured myoblasts. And it is a massive commercial market. Aspects that have bedevilled the application and transfer of autologous cultured myoblasts include:
- Cell loss or retention ( because we are unfamiliar with the histology of the underlying myocardium and the presence of small venous sinusoids).
- Variation in cell manufacturing standards
- Variation in the biological activity of the transplanted cells
- Variation in the cell carriers and vehicles used to support the cells
- Variation in the fibroblast to myoblast ratio
- Failure to appreciate the importance of the ECM and proteomics
- Failure to appreciate that old muscle can render old cultures of satellite cells.
- Failure to appreciate the literature on the effects of ageing on the muscle.
- Failure to understand culture technology
- Failure to understand the embryology of myoblast replication and development and disregard of the basic sciences relevant to human skeletal muscle morphology and physiology.
- Difficulty in delivering cells into a very thin and dilated left ventricle wall
- Difficulty in ensuring global and even distribution of cells during injection.
BUT WHAT POSITIVE FINDINGS HAVE BEEN RECORDED IN PATIENTS AND LABORATORY MODELS RECEIVING AUTOLOGOUS MYOBLAST CELL TRANSFER FOR ISCHAEMIC HEART DISEASE?
BOLANDCELL , with laboratory and clinical experience regarding myoblast technology provides details from uncontrolled trials for the interested reader.
- Improvement of one class of NYHA (symptomatic improvement and possibly reduced number of hospitalizations after engraftment.).
- Improvement of ventricular ejection fraction in the range of 9-10% in about 30% of recipients ( see work of Siminiak)
- Regional wall contractility improvement ( see studies of Herrero's)
- Improvement of akinetic and non-viable segments (see studies of Menaschè): JACC 41:1078-83,2003.
- Left ventricular ejection fraction increase in dogs ( see JTCS October 2006.): experimental.
- Improved left ventricle fractional shortening
- Increase in viability and perfusion 12 months after transplantation in humans as assessed by 18 fluorodeoxyglucose and nitrogen 13-ammonia positron emission tomography ( see JTCS 131:799-804, 2006). The importance of PET scans must be stressed.
- Safety and feasibility: including addressing the incidence of post-transplantation arrhythmias.
BASIC HISTOLOGY REGARDING SATELLITE CELLS : Bolandcell emphasizes the importance of histology before this technology is attempted.
Skeletal muscle satellite cells in immature growing muscle are responsible for providing additional myonuclei to enlarging myofibrils ( Mauro , 1961). Muscle satellite cells have long been considered a distinct myogenic lineage responsible for postnatal growth, repair, and maintenance of skeletal muscle ( Seale & Rudnicki. Dev Biol, 218,115-124,2000). Of great importance is that the overall population of satellite cells decreases with increasing age in rodents and man ( Gibson & Schultz, 1983, Grounds, 1998). Kieszenbaum (2002) has emphasized that muscle development involves the chain like alignment and fusion of committed muscle cell precursors, the myoblasts, to form multinucleated myotubes. During myogenesis Myf5 and MyoD, including Pax7 ( a transcription factor) play important roles. Satellite cells, are a distinct population of cells compared to myoblasts ( Kieszenbaum). These cells are situated beneath the basal lamina of the myotube. The process of muscle regeneration is enumerated below:
- Stress risers can stimulate mitotically quiescent satellite cells.
- Proliferation of satellite cells is by expression of the transcription factor, MyoD .
- Hepatic growth factor ( HGF) is associated with the satellite cells by the c-Met receptor.
- Daughter cells of activated satellite cells are referred to as myogenic precursor cells. These undergo cell division. Kieszenbaum has described how the HGF-cMet binding induces the proliferation of satellite cells.
- The same author has demonstrated the expression of myogenic regulatory factors Myf15 and MyoD . The myogenic precursor cells fuse with existing or new myotubes. Pax7 , also a transcription factor is down regulated during this process. Other regulatory factors such as myogen and MRF4 are also important. From an embryological point of view, the muscle-specific gene, Myo-D is important for the development of limb and body wall musculature. ( Sadler, 2004). This is important for the development of myotomes. The muscle-specific gene , Myf5, plays a critical role in the development of epimeric musculature. As previously mentioned, the precursor cells ( myoblasts), fuse and form long, multinucleated skeletal muscle fibers ( Sadler, 2004). Molecular regulation of skeletal muscle development is by BMP4 and FGFs . WNT proteins of ectodermal origin are also important in this activation process. (Sadler, 2004).
RELEVANT SATELLITE CELL INFORMATION THAT IMPACTS ON MYOBLASTS CULTURING:
- Lorenzon et al 2004, has indicated that the ageing process causes a reduction in the regenerative potential of skeletal muscles eventually leading to diminished muscle strength. So a very old skeletal muscle donor, say 75+ may not be able to render the same numbers of myoblasts in culture as a younger person. This has to be taken into account when selecting patients for cardiac myoblast transplantation. Diabetes mellitus also has an effect on myoblast proliferation ex-vivo.
- The work of Renault et al, 2000 also needs to be taken into account: " Satellite cells have a limited capacity to divide entering a state of irreversible growth arrest after a finite number of cell divisions". This contributes to the condition of sarcopenia age-related muscle weakness). Some workers have shown a dysfunctional excitation-contraction mechanism in aged myotubes developed from human satellite cells. So, if myoblast technology is being considered, it is important to understand the probability of exhausted proliferative capacity of aged satellite cells( see studies of Lorenzon).
- Workers from Sweden , have also confirmed that skeletal muscle satellite cells represent undifferentiated myogenic precursor cells that can generate new muscle fibers or provide new myonuclei to the parent fiber ( see Kadi et al 2003). But skeletal satellite cells can also generate new satellite cells. Post mitotic myonuclei and cells do not have these attributes.
- Again, it must be emphasized that satellite cells are located between the sarcolemma and the basal lamina of the muscle fiber and remain in a nonproliferative quiescent state. In the clinical setting a stimulant such as Marcaine will be needed to stimulate such cells prior to muscle biopsy. Satellite cells can be studied by light microscopy by the application of the monoclonal antibody against the neural cell adhesion molecule ( NCAM/CD56). For the moment the anti-NCAM antibody is a good marker for the assessment of satellite cells in human skeletal muscles ( Kadi et al 2003). This facility should be in place if a myoblast transplantation programme is to be started. But, it is also important to understand that the satellite population is not static, and can be influenced by exercise in the young and elderly. This concept is important if myoblasts are to be harvested and proliferated from bed-ridden and very immobile cardiac patients. So to ensure success in an autologous myoblast programme if going, knowledge of the effects of ageing on the satellite cell population and stressed by BOLANDCELL.
- The proportion of satellite cells may differ per muscle i.e. masseter, tibialis anterior, vastus lateralis, biceps brachii. So the transplant surgeon must take into account the possibility of a reduced pool of satellite cells and this reduction may be reflected in activation, proliferation or differentiation of satellite cells. This is important when studying proliferative dynamics of satellite cells. Research workers from the University of Washington ( Jnl Histochem & Cytochem, 47: 23-42,1999) have pointed out that addition of HGF to cultured fibers isolated fro old rats can potentially accelerate the progression of satellite cells. Satellite cell proliferation ex-vivo by growth factors is important. Of great importance is that some of the growth factors needed for satellite cells are derived from the myofibers in culture and this is going to impact on how satellite cells are proliferated. So, satellite cell activity contributes to postnatal muscle growth. Furthermore, Maier et al 1999, have indicated that these mononucleated myogenic cells reside in the GO phase of the cell cycle in the basal state. What is the size of the satellite cell pool? Maier et al of Germany have indicated that in the normal adult muscle, satellite cells account for 3% to 10% of all peripheral nuclei. It is a relatively small number of cells and can be quickly deleted by harsh enzymes such as collagenase during the processing and digestion of a muscle biopsy. Cell attrition is the outcome and the correct tissue exposure time to the enzymes is critically important and will eventually affect the yield of myoblasts in culture. The number is reduced in fatigued, atrophied and ischaemic skeletal muscle of patients with cardiac failure, presumably by the effects of TNF.
- After autologous myoblast transplantation coupling of myoblasts and cardiomyocytes does not occur. This is important to understand if a programme of cell transplantation for cardiac repair is considered. More work is needed to study myoblast integration with the host myocardium after cell transfer. The hormone, relaxin, may facilitate intercellular coupling between the donor and host cell. This may facilitate reversal of remodelling after transplantation. Arrhythmias after myoblast transplantation is a potential concern but can be controlled by the insertion of AICDS. The application of autologous cell culture procedures may well reduce the problem of post transplantation ventricular arrhythmias. Recent studies from Japan show great promise for the application of myoblasts for the treatment of non-ischemic dilated cardiomyopathy.
MYOBLAST IMPLANT TECHNIQUE WILL AFFECT OUTCOME. NEED FOR TRAINING
BOLANDCELL emphasizes that myoblasts in the Petri-dish, bioreactor or incubator, whatever, are living cells. Most clinicians have never been in a tissue culture lab. Surgeons and cardiologists do not know how to communicate with scientists and top lab people, and vice-versa. As clinicians we know about blood transfusions, but when it comes to live cell therapy and biotechnology we fall far behind. Cell therapy is very specialized treatment. Far more complex than whole organ heart transplantation and immune suppression. Because an in-depth knowledge of biotechnology is needed. And cardiologists and thoracic surgeons are poorly equipped with the skills and instruments of cell culturing and the knowledge of cell proliferation.. This is an Achilles heel. It is not part of their training and these skills are difficult to acquire. Most cardiologists are not schooled in the use of living cell therapy. It is just not injecting cells into the heart ! Success and failure are determined by how the cells are delivered into the myocardium. Cells are affected by the releasing process, storage after release and the way the cells are delivered. The cells are very fragile and viability is easily adversely affected. For the moment, cell therapy should be administered by a few specialized units that are up to date in the advances of cell therapy. There are not many top dogs in the field and most departmental heads in academic units are hopelessly ill-equipped to handle cell therapy. They need more training, not in the theatre, but in the research lab. Not all chest surgeons and interventionalist appreciate the detailed anatomy of the left ventricle that is needed for successful cell therapy. It is not always the fault of the lab, that the cells fail to improve the EF of the patient. Results are not instant. Cardiologists and thoracic surgeons do not have a foggy idea of what goes on inside cells, proteomics, the importance of biomarkers, not to talk about the importance and functioning of the extracellular matrix ( ECM). Most lack training in advanced histological technology and immunocytochemistry. This opens up their defences and contributes to the failure of cell therapy. Cells need to engraft in the host organ and is a process we do not fully understand. It is not just a paracrine effect. Mechanical and anatomical aspects also play a great role in cell engraftment.
Factors that must be taken into account when applying myoblast cell transfer as treatment for ischemic heart disease and heart failure:
- Do I have enough cell biology training and background? Do I know what cell therapy is? Do I need more training? What outcome do I want?
- Do I know what cultured myoblast cells look like under the microscope? How do the cells actually grow? Singly, monolayers? What is a cell culture cell medium?
- Can the cells be just put into the coronary sinus, or just injected under the epicardium? Catheter or direct injection? Will the cells not get stuck on the sides of a long catheter? Can we prevent this?
- How soon must I give the cells after cell release by the lab?
- Will the cells still be viable tomorrow, if the patient is cancelled to-day?
- What dilution must I use of cells?
- What diluents can be used?
- What do I do if the antero-septal aspect of the left ventricle is very thin? Can I inject the cells safely in these cases.? What about paper thin ventricular walls?
- How can I maximize cell retention in theatre? How can I avoid venous sinusoids that will simply wash away the cells?
- Do I understand the embryology, morphology, phenotype, differentiation and biomarkers of satellite cells, myoblast and fibroblasts? Do I understand growth factors and the effect on angiogenesis? Are these stem cells? How do they differ from progenitor cells? How do precursors differ from progenitor cells? Can I define what a progenitor cell is?
- Must I inject the septum?
- How can I avoid missing an adynamic or akinetic area in theatre or lab?
- How many cc of cells can the left ventricle tolerate?
- What happens if I inject a papillary muscle, the Purkinje fibers or trabeculae carnii? What about the apex of the heart?
- How do I assess outcome. 24-slice CT scan, MRI, ultrasound, PET scan? Do I need a nuclear scan ? Thickness, movement?
- Do I know how to take a muscle biopsy properly. Which muscle? Can I just cut through the fibers? Do I want short or long fibers? How does the biopsy affect muscle proteomics? Is the muscle inter-Z space important? Do I know what proteomics is? Is it relevant to cell therapy?
- Do I understand angiogenesis? How do I apply PET technology to angiogenesis? How long before cell therapy should I have an assist device placed to prevent arrthymia?
- How does myoblast therapy differ from bone marrow cell therapy? How does bone marrow work? Do you get angiogenesis? If I use myoblasts, do I get electromechanical coupling of the donor myoblasts and the host cardiomyocytes?
OTHER IMPORTANT APPLICATION OF AUTOLOGOUS CULTURED SKELETAL MYOBLASTS AND SATELLITE CELLS: BOLANDCELL 2007 UPDATE:
Currently studies are underway to use myoblast in other areas of cell regeneration. This is because the myoblast is easy to proliferate in culture, is resistant to ischemia and contractile. Myoblast cell transplantation is currently being studying in the following fields.
- Treatment of oesophageal reflux disease( GERD)
- Treatment of urinary stress incontinence ( periurethral injections to enhance the bladder neck)
- Treatment of muscle tears and atrophy
- Treatment of pelvic floor prolapse due to levator failure
- Muscle dystrophy ( results disappointing)