The present invention relates to a method and system for re-imiervation of muscle.

Muscle denervation can result from a variety of conditions and injuries such as spinal disc compression or nerve compression by a tumour. After childbirth, the bladder can lose innervation by the pudendal nerve leading to incontinence. In quadriplegia, the key gripping muscle in the hand can become denervated. This is distinct from paralysis in which the nerve is present but is effectively not connected to the CNS. Denervation reduces the excitability of skeletal muscle and typically results in irreversible denervation atrophy over time. This eventually leads to muscle degeneration and permanent loss of function. In other words, the muscle loses its ability to contract.

Electrical stimulation has been used for many years in an attempt to restore electrical contractility to denervated muscles. This technique is based on conditioning with intense electrical pulses usually applied through surface electrodes. However, deeper muscles are often inaccessible to surface electrodes and it is unclear if such stimuli can be delivered effectively using implanted neural prostheses which are currently available. Typically, electrical stimulation is ineffective in inducing muscle contractions or in preventing deterioration. Furthermore, neural prostheses such as BIONS (Bionic Neurons) require nerves to be present to work. BIONS are wireless electrical devices that can be implanted in muscles that require stimulation and at peripheral nerves. They are powered and controlled via radio waves from a small external controller that can be worn by the patient. The device is compact (2 mm wide by 15 mm long) and includes an integrated circuit chip sandwiched inside an antenna coil. The BION can be iήαplanted non-surgically with the use of a 12-gauge Intracath hypodermic needle.

It is known that muscles can be reinnervated naturally, even after more than a year following dennervation and that reinnervation can reverse atrophic changes and may restore function to some degree. However, this reinnervation does not occur in muscles where all the motoneurones have died, such as in diseases such as amyotrophic lateral sclerosis (ALS), the spinal muscle atrophies or in cases of spinae bifida or injuries to the lumbar/sacral spinal cord. In these cases, the affected muscles have markedly reduced excitability with profound atrophy and loss of function. Function can only be restored if the affected muscle fibres are reinnervated by an alternative source of neurones. In certain circumstances this can be achieved to some extent by grafting a healthy nerve into the dennervated muscle with the consequent loss of its original function. However, such opportunities are limited. In a previous approach by the inventor, small grafts of immature spinal neurons were transplanted into surgically denervated muscles of a rat. The nerves supplying the lateral gastrocnemius-soleus in one hind limb in adult male Wister rats (240-26Og) were cut. Small 1 mm3 neural grafts were removed from the lumbar region of embryonic (E21) rats and placed a few millimetres deep into the mass of the denervated lateral gastrocnemius. The fate of these neurons was examined at intervals up to 3 months post-operatively. Only a limited degree of innervation was observed. This is likely to be due to axotomy since the preparation of the graft involves cutting a piece of the spinal cord causing the axons of many of the neurons to be cut. The majority of axotomised neurons die which results in a large loss of cells in the graft. Thus only a limited degree of excitability to electrical stimulation is restored. There are also ethical issues associated with the use of embryonic tissue.

Recent advances in stem cell technology have provided alternative sources of motor neurons and their support cells. Motor neurons have been derived from embryonic precursor or stem cells. Embryonic stem cells have been used in an attempt to reiήnervate skeletal muscle. Embryonic stem cells are amplified in vitro and then injected into the body. They can be injected intramuscularly or intravenously, and once in the blood stream they pick up molecular signals and travel to the target, differentiating into the target tissue on route. However, this requires the stem cells to be exposed to the correct environment in the body. For example, the cells might be directed into cell types other than neurons. One study involved the injection of embryonic stem cells directly into the nerve stump (Erb DE, Mora RJ, Bunge RP (1993) Reinnervation of adult rat gastrocnemius muscle by embryonic motoneurons transplanted into axotomized tibial nerve, Exp. Neurology, vol 124:372-376, 1993 ) in an attempt to overcome this problem. However, it is technically difficult to find and inject the right nerve and for deep nerves this would involve invasive surgery. The present invention seeks to overcome one or more of the above disadvantages.

According to a first aspect of the present invention, there is provided a method of preparing terminally differentiated electrically excitable cells from stem cells in vitro, the excitable cells being capable of developing functional connections with muscle fibres, including the steps of adding media conditioned with nerve tissue to the stem cells, the conditioned media having been previously incubated with nerve tissue or extracts thereof.

Terminal differentiation is defined as the state in which a cell has acquired specialised properties and has ceased proliferating and dividing permanently. This is in contrast to non-dividing cells which are resting and can be stimulated to divide.

In a preferred embodiment, the stem cells are bone marrow derived mesenchymal stem cells. The cells can be prepared from adult bone marrow tissue which may be donor tissue obtained from a tissue bank.

Preferably the method further comprises the step of preparing the conditioned media by incubating the nerve tissue in tissue culture media and subsequently isolating the media.

The media may be conditioned with extracts of nerve tissue. Advantageously the nerve tissue comprises motor neurons. The nerve tissue may be spinal or peripheral nerve tissue. The tissue can be taken from the patient below the site of a lesion. Preferably, the nerve tissue is human. This avoids cross species infection. The tissue may be from a donor bank.

The nerve tissue may be incubated for at least 12 hours at about 37° C. Advantageously, the nerve tissue is incubated for at least 24 hours. Preferably, the method includes the step of centrifuging the collected media at about 2,000 to 4,000 rpm. This removes large particles such as dead cells. The centrifugation step may last for 5 to 30 minutes.

The method preferably comprises the step of collecting and storing the supernatant. The supernatant may be stored in a variety of ways such as by cold storage (at about 4° C) or by freezing. Alternatively, the conditioned media may be stored prior to centrifugation. Of course, the conditioned media may instead be used straight away.

Preferably the nerve tissue and the stem cells are derived from the same individual.

The conditioned media added to the stem cells may be diluted by 30 to 70%, and preferably by about 50% with non conditioned culture media.

Advantageously the method includes culturing the stem cells in the conditioned media for at least 24 hours and preferably for at least 48 hours.

The method may include the step of removing the conditioned media and replacing it with non-conditioned media.

Preferably the method further comprises the step of storing the cultured cells.

According to a second aspect of the present invention there is provided electrically excitable cells capable of developing functional connections with muscle fibres obtainable by a method according to any of claims 1 to 19. According to a third aspect of the present invention there is provided a pharmaceutical composition for the reinnervation of muscle comprising electrically excitable cells according to claim 20 and a pharmaceutically acceptable carrier.

Preferably the composition comprises more than about 250,000 excitable cells and more preferably more than 500,000 excitable cells.

The cells may be suspended in a medium supplemented with serum, glucose and nerve growth factor.

According to a fourth aspect of the present invention there is provided method of reinnervating muscle including the step of administering to a patient a pharmaceutical composition as claimed in any of claims 21 to 24.

Preferably the pharmaceutical composition is injected into a denervated muscle. The muscle may be skeletal or smooth muscle. The patient may receive more than one injection of cells which may be at different sites in the muscle. The additional* injections do not have to be carried out at the same time.

The method may further comprise the step of implanting a device for electrically stimulating the administered neurons. The device may be a neural prosthesis such as a BION, or another stimulator such as part of a FES (functional electrical stimulation) system. More than one device may be implanted. This maximises the electrical contractibility of the muscle.

Preferably the device is implanted simultaneously with the excitable cells. This is advantageous as the patient is only injected once. In addition, it ensures the stimulator is in the same location as the excitable cells that need to be stimulated. Preferably the excitable cells are prepared from stem cells harvested from the patient to which the excitable cells are administered. This autologous transplantation reduces the likelihood of an adverse immune reaction.

According to a fourth aspect of the present invention there is provided a method of making conditioned media for converting adult stem cells to excitable cells comprising the steps of incubating motor nerve tissue in tissue culture media, and isolating the media following incubation so that once isolated it can be used in a method of preparing excitable cells.

One advantage of the methods and systems of the present invention is that the adult stem cells are directed into a terminal state (whereby they can no longer differentiate into a different cell type) outside the body. In contrast, prior art methods use embryonic cells which are directed naturally inside the body. The embryonic stem cells have a 'homing instinct' and so may travel elsewhere according to the molecular signals they receive. For example, the cells may travel towards the heart and become myocytes. In contrast, transplanted neurons will remain at the site of transplantation. There are also concerns that due to repeated division of the embryonic stem cells within the body, the cells.may become immortal i.e. cancerous. In contrast, adult stem cells lose their ability to divide. Furthermore, use of embryonic stem cells may elicit an immune reaction in the patent as the cells are not autologous. In addition, the use of embryonic stem cells poses ethical and moral concerns and there is a large proportion of the public which are against using such technology.

Embodiments of the present invention are described below, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 shows different view of control plate; and Figure 2 shows the change in normal stem cell morphology to a neuronal morphology.

In summary, the embodiment of the present invention described below involves autologous transplantation of "excitable cells" or motor neurons produced from adult bone marrow derived mesenchymal stem cells taken from a host. The stem cells are amplified in culture and directed into terminally differentiated motor neurons. These cells are then transplanted in suspension into the dennervated muscles (skeletal or smooth muscles) using a hypodermic needle. These cells survive and grow to form functional synapses (or connections) with the muscle fibres and cause these fibres to contract when an electrical pulse is applied. A neural prosthesis is used to stimulate the transplanted cells. These cells are expected to remain at the site of injection which helps to locate the stimulating electrodes of the neural prosthesis. Without such "excitable cells" the muscle would only respond for a limited period of time after dennervation to an electrical pulse of much higher intensity (orders of magnitude more) making neural prostheses impracticable.

Preparation of Stem Cells

Details can be found in "Re-innervating muscle for FES using neurones derived from ad luulltt m meesseenncchhyymmaall s stteemm c ceellllss"",, 99tthh A Annnnuuaall ( Conference of the International FES Society, 345-347, Sept 2004, Bournemouth, UK.

Bone marrow stromal (mesenchymal) stem cells were obtained from adult Sprague Dawley rats. The technique is based upon the protocol of Owen and Friedenstein in "Stromal stem cells: marrow derived osteogenic precursors" CIBA Foundation Symposium 136. 42-60.1988, and represents a typical established adult stem cell source suitable for expansion in vitro. Following schedule one killing (cervical dislocation), tibia and femora were excised within 5 minutes of death. All connective and muscular tissue was removed from the bones and all further procedures were conducted under sterile conditions. Marrow was expelled from the bones by flushing the bones with media (α-MEMS, Gibco Invitrogen Co. UK) containing 10% foetal calf serum, and 1% penicillin/streptomycin. Flushing was achieved by inserting a 25-gauge needle attached to a 5 ml plastic barrel into the neck of the bone (cut at both distal and proximal end) and expelling 2 ml of media through the bone. The media and bone marrow sample were collected in sterile universal containers.

Bone marrow derived stem cells were subsequently dissociated by gentle trituration through a 19-gauge needle approximately 10 times. One ml of aspirate was then placed in six well plates (obtained from SLS Ltd, UK). Two ml of fresh α-MEMS was then added to each well giving a plating density of approximately 12,000-15,000 cells per ml.

The plates were then incubated at 370C, in 5% CO2 in air and left undisturbed for 24 to 48 hours, following the process described by Harrison, MA. & Rae, IF. (1997) General Techniques of Cell Culture. Cambridge Univ. Press. Cambridge.

Following this time period, marrow derived stem cells were isolated from non-plastic adherent cells by aspirating the culture media from the plate. Plastic adherent marrow stromal stem cells remained, and were supported by the addition of 2 ml of fresh α-MEMS (10% foetal calf serum and 1% penicillin/streptomycin). In order to amplify the stem cells, new media was applied every 48 hours until the plate was confluent with colony forming units (CFU' s) confirmed by microscope analysis (as per Owen & Friedenstein, 1988). This required 7 days at 370C. Resultant cells were confirmed as stromal stem cells morphologically and immunohistochemically. Ten 6 well plates were maintained. Five experimental plates received conditioned media, and five control plates receiving untreated media on a routine media change on day 8.

Preparation of Conditioned media

The method follows a conditioned media approach with our own formulation. In this example, 3 ml of media (α-MEMS - containing 10% foetal calf serum, and 1% penicillin/streptomycin) was placed in each well of 6 well plastic tissue culture plates. A 1 mm length of neonate rat spine was then placed in each well. All plates were maintained for 24 hours at 370C, in 5% CO2 in air and left undisturbed.

After 24 hours, media conditioned with neonate spine was aspirated from the wells and placed in plastic centrifuge tubes. Pooled media was then centrifuged at 3,000 rpm for 10 minutes to remove any solid material e.g. dead cells etc. The supernatant was then aspirated and maintained at 370C.

The media does not need to be incubated with the tissue for 24 hours or more, though this is preferred. Positive results are achieved if the incubation lasts for more than 12 hours. Similarly, it will be apparent to the skilled worker that the centrifugation parameters may be varied. The actual duration and speed is not important so long as solid material is removed.

Preparation of terminally differentiated motor neurons

Conditioned media was added to the 5 experimental plates at a dilution of 1 ml of supernatant in 2 ml of culture media. Control plates received 3 ml of culture media. Plates were examined under phase microscopy using an inverted microscope every 24 hours.

All cultures thrived in both groups. At 48 hours there was a clear change of phenotype in all experimental plates whilst control plates (see Figure 1) maintained a stem cell morphology.

In the experimental plates, cells had changed from the normal stem cell morphology to a neuronal morphology (see Figure 2). These neurons appear to be very similar to motor neurons (for example they sprout extensions and are attracted to muscle fibres) and show extensive connectivity. Furthermore, they express key protein markers which are expressed by motor neurons, for example Ch AT-positive and MAP.-2. Other markers for motor neurons include VAChT, LIM homebox transcription factors and REG 2.

The change in morphology in all experimental plates was of further interest as it was stable and persisted through at least 14 further media changes using normal, unconditioned, culture media.

It is not essential that the excitable cells produced are 'real' neurons and display all their characteristics. If fact they are likely to be abnormal neurons and therefore are unlikely to fit the strict anatomical, neurophysio logical and neuropharmalogical definitions of motor neurons. The main requirement is that they are safe and lower the electrical threshold for excitation of the cell and muscle fibre combination.

Indeed, 'normal' naturally occurring motor neurons innervating skeletal muscle have unsheathing schwami cells to allow propagation of action potentials from the spinal cord. Our "excitable cells" probably do not have these cells. However, the "excitable cells" produced by the method of the present application do not need to propagate action potentials from the spinal cord. Instead they relay signals from the neural prosthesis to the muscle fibres with which they have formed a functional connection.

The cell bodies of motor neurons that innervate skeletal muscle normally reside in the protected environment of the spinal cord and may not survive outside this environment. In contrast, the cells produced by our method survive and grow in the abnormal environment of the muscle.

Naturally occurring neurons form special synapses with other neurons and other support cells (motor and non motor neurons) and form intelligent neural circuits. Since the excitable cells we have produced are 'controlled' by the electrical stimulator, they do not require this property. Naturally occurring motor neurons make only one connection (a neuromuscular junction) with one skeletal muscle fibre. It does not matter how many connections our cells make since they will all be in synchronism with the electrical pulses from the neural prosthesis.

Re-innervation of skeletal muscle

The prepared neurons are injected in suspension into the denervated muscle. For skeletal muscle, typically over 250,000 neurons are injected in one or more injections. Preferably, more than 500,000 neurons are injected. In smooth muscle, fewer neurons are needed. Typically more than 100,000 neurons are injected. The neurons can be suspended in a variety of media including Eagle's minimum essential medium supplemented with 10% heat activated rat serum, glucose and nerve growth factor. For human application, we an animal serum or other product free formulation is preferred to avoid transferring any animal virus.

An electrical stimulator (or neural prosthesis) is also implanted in the patient. The stimulating electrodes are directed to the site of reinnervation.

Neural prostheses are devices that restore or supplement the function of the nervous system lost during disease or injury. One form of neural prosthesis has stainless steel wire electrodes coated in Teflon. The free ends of the electrodes are bared and bent back towards the neural prosthesis. The neural prosthesis can be inserted using a hypodermic needle. The neural prosthesis is loaded into the needle with the bared electrode located ends outside the needle. The needle is pushed into a muscle and the bared ends act as a fish hook, attaching to the muscle. As the hypodermic needle is removed, the neural prosthesis is retained in the muscle. A plurality of neural prosthesis can be injected into the muscle. It is possible to inject the neural prosthesis and the neurons at the same time using the same needle. This is advantageous as the electrodes are easily located in situ with the neurons. With certain neural prosthesis the electrode wires are up to a metre in length. The neural prosthesis can be conveniently inserted under the skin and the electrodes tunnelled to the target tissue. This is advantageous as it is a minimally invasive procedure.

Another neural prosthesis known as the BION may be used. This neural prosthesis is approximately 16mm long by 2mm wide. It has a stimulating electrode at either end and an electronics circuit in the middle. The electronics circuit directs stimulus pulses via the stimulating electrodes to the muscle. The electronics contains an RF (radio frequency) circuit which picks up power from an external RF antenna coil. Some neural prosthesis also include batteries which can be recharged via an external RF recharging coil. As a minimally invasive technology, BIONs offer an advantage over functional-electrical- stimulation systems that require surgical implantation of a stimulator or that apply electrical currents at the surface of the skin. BIONs enable the application of currents directly to one or more muscles at widely varying levels of intensity, depending on the clinical need.

When the capacitor electrode of the BION is fully charged, a discharge of that electrode through a controlled current source produces well-controlled stimulation pulses of any intensity which are able to stimulate a peripheral nerve or even a relatively large muscle (via its motor axons).

Once the muscle has been reinnervated with the motor neurons and the neural prosthesis is in place, the neural prosthesis can be stimulated to trigger a signal in the neurons which is conducted to the muscle fibres causing contraction. As the implant controls the nerve signalling, the transplanted neurons do not need to connect with the CNS and can simply reside in the muscle. Typical stimulation parameters are 5-60 Hz and 50 microamperes to 20 milliamperes.

Instead of reinnervating skeletal muscle, this technique could be applied to smooth muscle. Conditioned media for preparing human motor neurons from stem cells can be prepared in a variety of ways. For example spinal tissue harvested from below a lesion can be used to condition media. Alternatively, human motor neurons can be used. A different, less important muscle could be denervated and the motor neurons excised and used. A preferred option is the use of donor human motor neurons from tissue banks. Mammalian neurons can also be used although this is not preferred due to concerns relating to cross species infection. An advantage of using human tissue to condition the media is that the media is free of animal products and viruses. Preferably, the cells are autologous to further reduce the likelihood of an immune reaction.

In use, a service may be provided whereby bone marrow samples are collected from patients in a clinic and sent to a laboratory for direction into neurons. A preparation of neurons in suspension for each individual patient is then sent back to the clinic for injection. The preparation may be supplied in loaded syringes. Alternatively, conditioned media may be supplied. Both the neuron preparation and the conditioned media can be readily stored (including frozen) and transported. Instead of collecting bone marrow samples from patients, donor cells from a tissue bank may be used.

There are many potential applications for prosthetic control of denervated muscle including: restoration of grasp function in brachial plexus injuries, restoration of continence where the sphincters are denervated, breathing when the phrenic nerve is lost, restoration of locomotor functions in spinae bifida, and restoring vocal chord crycoarytenoid. Other biomedical applications include: muscle transfers where it is not possible or inconvenient to preserve the original innervation, and providing a source of internal power using a transferred muscle or a tissue engineered muscle construct. In addition to medical applications, this can be used in veterinary applications.