WO2008031448A1

WO2008031448A1 - Preparation of autologous non-hematopoietic progenitor stem cells, method of preparation and use thereof

Info

Publication number: WO2008031448A1
Application number: PCT/EP2006/009008
Authority: WO
Inventors: Andrei Stepanovich Bryukhovetskiy; Olga Palumbo; Georgy Ruslanovich Pugachev
Original assignee: Meditech Industries LLC
Current assignee: Meditech Industries LLC
Priority date: 2006-09-15
Filing date: 2006-09-15
Publication date: 2008-03-20
Anticipated expiration: 2009-03-15

Abstract

The invention belongs to the field of cell transplantology and neurology and can be used to treat the central nervous system injuries. The main idea of the invention is a preparation of non-hematopoietic stem cells (non-HSC) , which is autologous nonHSC obtained from patient's peripheral blood enriched with cells containing CD34 antigen, in the final concentration of (40-100) XlO6 cells/ml. Blood enrichment is performed through intermittent administration of the preparation of the granulocyte colony- stimulating factor (G-CSF) - for example, Neupogen in the glucose solution, according to a certain scheme. Also, the method of production, cryopreservations of the preparation and the method of treating traumatic brain and spinal diseases by the new preparation is proposed here. The developed system allows neurological patients to be helped efficiently with minimal harm.

Description

DESCRIPTION

Preparation of autologous non-hematopoietic progenitor stem cells, method of preparation and use thereof

FIELD OF THE INVENTION [001] This invention belongs to the field of cell transplantation and neurology and namely it covers the preparation of autologous non-hematopoietic stem cells, the method of harvest and cryopreservation of said preparation and the use of the said preparation in the therapy of traumatic diseases of the brain and spinal cord.

BACKGROUND OF THE INVENTION

[002] Despite considerable development of therapeutical methods for various neurological diseases, in many the results are still poor. This applies to neurodegenerative diseases, post-traumatic brain and spinal cord injuries and the consequences of blood supply diseases. The current methods of therapy are frequently unable to restore the lost function, the rates of disability remain high and the prognosis for an active life is unfavorable. Thus it is crucial to develop new methods promoting reparative processes in the neural tissue. [003] Recently cell culture transplants from embryonic neural tissue have been widely practiced to treat neural diseases (cf., Patent of the Russian Federation 2160112, 2000) . However the therapy with embryonic tissue preparations presents serious histocompatibility problems, risk of graft rejection, late immune conflicts and various moral, ethical, legislative and religious challenges.

[004] Presumably the use of autologous neural stem cells for transplantation have great potential, (cf. the Patent of Russian Federation 2191388, 2002) the same holds true for the hematopoietic stem cells, which were used in the transplantation experiment to the myocardium

(the Patent of the Russian Federation 2237440, 2004) .

[005] Similar to our invention is the technology described in the Patent of the Russian Federation 2216336 (2003) , where the preparation of autologous haematopoietic stem cells (AHSC) , expressing SV40 antigen, is used for the therapy of nervous diseases. However this technology uses the stem cells extracted from the bone marrow through puncture, which is traumatic for the patient and rather demanding for the staff.

[006] Therefore the use of hematopoietic stem cells (HSC) obtained from the peripheral blood of the patient for the therapy of the nervous system injuries is potentially valuable approach both theoretically and practically. The cells having great functional plasticity permit to avoid considerable adverse effects when introduced into the spinal fluid or ventricles of the brain, to increase the efficiency of the therapy and to decrease possible complications. Local differentiation of SC in in situ brain tissue has been generally controlled by the "signals" of the microenvironment . Limited clinical observations demonstrated the absence of SC differentiation abnormalities in the graft. However, the use of hematopoietic stem cells has not yet been demonstrated to be really effective in humans affected by brain or spinal trauma. SUMMARY OF THE INVENTION [007] The technical task of the invention is to set up a preparation of autologous stem cells which is able to overcome the above disadvantages and, above all, can effectively be used to prepare a medicament useful to treat patients who have lost motion due to the above trauma. Said task is reached by a preparation of autologous non-hematopoietic stem cells from peripheral blood (Anon-HSCPB) , the method of harvest and cryo- preservation of the preparation and the method of the therapy of the central nervous system injury. BRIEF DESCRIPTION OF THE DRAWINGS [008] The invention will be described in detail hereinbeiow with reference to the following figures:

- Figure 1 shows a graphic comparing the peculiarities of conventional AHSC stimulation for 6-7 days and the claimed stimulation for 4 days;

- Figure 2 shows a histogram representing the efficiency of various therapies in patients with spinal cord and brain trauma.

DETAILED DESCRIPTION OF THE INVENTION [009] The technical result is achieved by a new preparation of autologous mobilized non-hematopoietic stem cells used for transplantation for the central nervous system diseases. The preparation consists of autologous mobilized non-hematopoietic stem cells (AMnon-HSC) obtained from the patient's peripheral blood enriched by AMnon-HSC in the final concentration of (40- 100) xlO⁶ cells/ml and characterized by the following markers configuration: CD34+, CD45-, HLA DR±, CD38±, Gpl30+. Said composition has also been characterized by the presence of differentiated lympoid T and B cells .

[010] It has been observed that the use of the preparation comprising autologous mobilized non- hematopoietic stem cells from peripheral blood containing as main antigen CD34 antigen in a concentration of (40-100) xlO⁶ cells/ml for the production of a medicament to be intrathecally or intraventricuiariy injected resulted in surprising and remarkable effective treatment outcomes in the cases of the central nervous system injuries. [Oil] The preparation is obtained by preliminary intermittent administration of granulocyte colony- stimulating factor (G-CSP) - for example, Neupogen^® Filgrastim (Amgem Inc.) or Neilogen^® (La Roche). G-CSF is administered for 4 days according to the scheme: first three days - every 10-12 hours, from 0.5 to 2.5 micrograms/kg; the fourth day - a double dose, following the same time schedule.

[012] Another aspect of the present invention is the method of obtaining the preparation of autologous mobilized non-hematopoietic stem cells to transplant for the injuries of the central nervous system. [013] The improved technical result is achieved through preliminary intermittent administration of a G-SCF preparation, harvest of AMnon-HSC containing CD34 antigen, purification of the cell suspension from erythrocytes and washing in the physiological saline. The received preparation is either reinfused or cryopreserved. [014] According to a particularly preferred embodiment of the invention, the method of obtaining the preparation from the peripheral blood of patients with neurological diseases is performed using the following scheme. [015] To obtain the particularly selected target cells, i.e. autologous mobilized non-heamtopoietic stem cells, and their selected quantity in the peripheral blood, the patient gets 8 injections of granulocyte colony- stimulating factor (G-CSF) - for example, Neupogen in glucose solution - subcutaneously at an interval of 10- 12 hours during 4 days. G-SCF is a medical preparation obtained for instance by genetic engineering, and is absolutely identical to human G-CSF.

[016] In the first three days, the preparation dose is preferably 2.5 micrograms/kg, and the last day the dose is doubled. [017] It has been found that the above described protocol of treatment allows to obtain a very particular selection of autologous stem cells which can be identified, as said above, as autologous mobilized non- hematopoietic stem cells showing the above peculiar markers. It is to be noticed that just the unique identification of said marker, in particular the absence of CD45 marker, differentiates the obtained selection from any other selection that can be obtained by means of any known method and which is usually identified as hematopoietic stem cells. [018] Moreover, said cells results in a composition comprising differentiated progenitor cells as well as unindifferentiated progenitor cells, the differentiated progenitor cells being cells having a differentiation stage between early progenitor cells and hematopoietic cells.

[019] Thus it is important to follow the treatment scheme in order achieve the desired target cells and at the same at a suitable concentration to avoid prolonged treatment which can seriously damage a patient.

[02O]In particular, it is to be noticed that, contrary to what has been usually practiced, the stimulation according to the above treatment scheme is stopped at the fourth day. The fifth day from the peripheral blood the autologous mobilized non-hematopoietic stem cells are collected in a quantity which corresponds to the quantity usually collected the seventh day from stimulation according to the conventional method. Furthermore, the pool of cells obtained are completely different since in the present case they are identifiable as autologous mobilized non-hematopoietic stem cells and following the conventional methods they are identifiable as autologous hematopoietic set cells. [02I]It has also been found that by performing the protocol according to the present invention and by recovering the target cells from the peripheral blood at an earlier stage with respect to the conventional methods, it is possible to block cell differentiation to differentiated progenitor cells and undifferentiated progenitor cell, as explained above. On the contrary, the cells recovered following the known methods present a higher degree of differentiation that can bring to tumor proliferation or at least ineffectiveness of the treatment . [022] As can be seen in Figure 1, the black line represents the peculiarities of conventional AHSC stimulation for 6-7 days, while the grey one shows that the claimed stimulation in specific regimens permits to obtain absolutely different cells-precursors after 5 days. In fact, the aim is to obtain a quantity of cells

(around 5χlO⁶/kg) which is effective for the treatment.

This can be done following the conventional AHSC stimulation. However, if the stimulation is carried out following the above scheme the desired quantity can be recovered at an earlier time. In addition, as clearly shown in figure 1, the recovered cells differ from the cells recovered according to the conventional stimulation since they are precursor autologous stem cells and not hematopoietic autologous stem cells. [023] In other words, the present invention is based on a particular stimulation protocol and early recovering so that the recovered cells consist of autologous mobilized non-hematopoietic stem cells undifferentiated identified by the above specific markers in a quantity effective to be used for preparing a composition for treating patient suffering from brain or spinal trauma.

[024] The cell harvest is performed on the 5^th day after the beginning of the G-CSF stimulation using the blood separator Cobe Spectra and the disposable system for WBC (white blood cell, catalogue number 777-006-000) separation thereof and standard solutions. The concentration of CD34+ cells in the cell suspension obtained during cytapheresis, is determined by flow cytofluorometry on the FACScan (Becton Dickinson, USA) . [025] In the peripheral blood the stem cells and differentiated progenitor cells form a so-called stem cell pool (SCP) . Expression of CD34+ antigen molecules on the cell membrane is common to all the cells of this pool. The subpopulation structure of CD34+ cells was determined by flow cytofluorometry by means of monoclonal antibodies. Further markers, as stated above, are determined by means of conventional detecting methods using known monoclonal antibodies. [026] It is important to note that all the technologies applied to determine and quantify the target ceils of the invention are conventional technologies. LOGISTICAL SUPPORT - Flow laser cytofluorometer;

- Software (Cell Quest Lysix ProCount for WinMDI) ;

- Monoclonal antibodies (MCA) : to antigen CD34 : HPCA-2a (8G12) , isotype IgGl, marked with phycoerythrin (PE) or pyridinchlorophill (PerCP) produced by Becton Dickinson (USA) ; for CD45 antigen, isotype IgGl, fluorescein marker (FITC) ;

Isotypic controls: murine immunoglobuline of Gl isotype with the appropriate mark (PE, FITC, PerCP) ;

- Sodium azide (NaN₃): 2.5% of the mother solution of sodium azide is prepared with the physiological solution. Sodium azide is added to the nutrient solution and phosphate-buffered saline (PBS) until the final concentration of 0.25% is achieved;

Lysing reagent to clean up SCP suspension from erythrocytes. Formula: NH₄Cl - 8.26g, potassium bicarbonate - Ig, Na₄-ethylene diamine tetraacetic acid

- 0.37g, should be dissolved in 10Og of purified water;

- Phosphate-buffered saline (PBS) ;

- Bovine serum albumine (BSA) ; - PBS with 0.1% of BSA (PBS-BSA) or medium 199. The number of progenitor cells was determined by a direct immunofluorescence test (IFT) .

[027] The double-tracer method should be considered the most appropriate, as it simultaneously stains the cell substrate with monoclonal antibodies for the CD34 antigen - the main marker of cells in SCP - and for the CD45 molecule, a common leucocyte antigen which detects all hematopoietic cells. This technique allows to immediately determine the quantity of CD34+ cells for all hematopoietic CD45+ cells in the material. The absence of CD45 marker (CD45-) just identifies the stimulated cells as non-hematopoietic cells. To evaluate the levels of nonspecific binding, some cells are stained by isotypic control. Murine immunoglobuline of IgGl isotype (IgGl) , marked with the same stains used for monoclonal antibodies (PE, FITC, PerCP) , are normally used as isotypic control. Before setting up IFT, the examined cells should be cleaned from erythrocytes by standard lyses procedure and consequent washing in the PBS-BSA with centrifugation at lOOOg for 5-7 minutes.

ERYTHROCITE LYSES TECHNIQUE

- Add 2 ml of lysing reagent to 0.2 - 0.5 ml of cellular deposit, stir and incubate until the solution is clear; - Wash the cells twice in 199 medium (Sigma-Aldrich) or in PBS-BSA with centrifugation at lOOOg for 5-7 minutes. [028] It is to be noticed that this technique is very- important as it permits to avoid the dangerous presence of erythrocites in the preparation. In fact, the presence of erythrocites, and thus iron, can cause meningitis. However the above mentioned technique can be replaced by other suitable techniques that permit to remove the erythrocytes or at least iron from the preparation.

[029] IFT is performed on the obtained cells in a 96- wells tray. To determine the quantity of SCP in any material, a 3-wells panel is used:

- unstained cells; - cells stained with isotypic controls with the mark correspondent to the mark of used MCA; cells stained with both MCA for CD34 and CD45 antigens .

[030] It is important that MCA for CD34 is used preferably with the phycoerythrin (PE) or pyridinchlorophill (PerCP) mark. These fluorochromes have a higher level of a specific signal compared to the

FITC. [03I]It is optimal to use antibodies for antigen CD34 of clone HPCA-2 (8G12) , Isotype IgCi, to determine the number of CD34+ cells.

SETTING UP IFT [032] Cells are introduced in the wells at not less than

500,000 per basin.

[033] The mix of the antibodies named above is introduced into each well and resuspended carefully with the dropper. Each MCA should be taken in the volume of 10 microliters per well; the total volume of MCA in the well should be 20 microliters.

[034] Cells are incubated with the antibodies for 30 minutes at +4°C.

[035] After the incubation is completed, the cells should be washed twice from unbounded antibodies by centrifugation for 5-7 minutes at 100Og.

[036] The cells are moved to plastic tubes in order to determine the number with the flow cytometer.

[037] The volume of cell-rich fluid in each tube should be brought up to 200-500 microliters by adding the PBS-

BSA.

[038] The cell population of CD34+ cells in the peripheral blood is low. Under the condition of preliminary stimulation of non-hematopoietic SC migration to the peripheral blood, the percentage of CD34 expressing cells is about 1-3%. The final concentration of CD34+ cells in the preparation is (40- 100) xlO⁶ cells per ml.

[039] It is not always that the autologous non-HSCPB preparation rich in CD34 antigen cells which was obtained from a neurological patient can be used immediately after the harvest. The pattern of use of the preparation depends on the patient's state and other objective and subjective conditions. For example, it may be necessary to treat the patient with the preparation several times.

[040] Consequently the issue of preserving the preparation in order to hold its properties until the moment of usage has come up. Another aspect of this invention is therefore the technique of cryopreservation of the preparation, viewed as the best way to solve this problem.

[04I]A traditional technique for cryopreservation is to add dimethyl sulfoxide to the cell-rich fluid in a final concentration of 10%, to freeze it at a rate of 1°C per minute down to -80⁰C or -120⁰C, using an electronic freezer, and to store it in liquid nitrogen or liquid nitrogen vapors [Adrian P. Gee "Bone marrow processing and purging: a practical guide", 1991, 332-337]. However it has been observed that the technique is not appropriate to preserve the preparation of the present invention. The cells rupture if frozen under this technique .

[042] Accordingly, a technical task of the present invention is to prolong the storage life of the new preparation. The technical result is achieved by means of a technique of cryopreservation of the autologous non-HSCPB preparation, wherein the preparation is mixed with dimethyl sulfoxide (DMSO) and consequently frozen down with liquid nitrogen in the temperature gradient, the technique being characterized by the fact that the preparation is frozen in the mixture of DMSO at the initial concentration of 10-12% with polyglucine, the temperature gradient is 1. I⁰C per minute when freezing down to -4O⁰C and the final temperature is -165°-170° C. This technique permits to store the preparation of the invention for an unlimited time.

[043] The following original technique of cryopreservation in the vapors of liquid nitrogen, using low concentrations of DMSO mixed with polyglucin, permits to store the HSCPB preparation almost without losses at all stages of cryopreservation. [044] The cryopreservation procedure includes: - Adding cryophilactic to the cell-rich fluid; - Freezing down the cells. [045] Highly purified DMSO is used as a cryophilactic for hematopoietic ceils. An equal volume of a DMSO mixture with polyglucin is added to the cell-rich fluid being constantly stirred. Mixing DMSO and polyglucin is an exothermic reaction, releasing moderate amount of heat. The initial concentration of DMSO in the polyglucin is 10-12%.

[046] To freeze tubes with cell-rich fluid, they should be put in an appropriate container for example made of multiple layers of plywood 10mm wide. The lid should be tight, and the container should match the tubes' size. Then the container is placed into the vapors of liquid nitrogen until the temperature of -165°-170° C is reached. The speed of freezing down is 1.1⁰C per minute when freezing down to -40⁰C. The final temperature is - 165°-170° C and the preparation is stored at that temperature. After 1.5 - 2 hours of freezing, the container can be moved into the storage where it is stored till transplantation. This method of cryopreservation permits to retain the properties of the preparation for an unlimited time. Advantages of freezing in the vapors of liquid nitrogen:

- Safe storage of the containers;

- Reduced consumption of liquid nitrogen; - More convenient handling of the containers. [047] The obtained new non-HPBSC preparation has been used for the voluntary treatment of the patients .

Another aspect of this invention is the technique of treating patients with the brain and spinal cord injuries.

[048] Earlier, the pharmacotherapy was applied to treat such patients, and recently the cell therapy with embryonic stem cells was used (cf. the Russian Federation Patents # 2152038 dated 20.06.2000 and #2152039 dated 27.06.2000). However as it was mentioned above, the therapy with embryonic tissue preparations holds serious histocompatibility problems, the risk of graft rejection, suspended immune conflicts and also moral, ethical, juridical and religious restrictions. [049] The technical task of the given invention is to eliminate these disadvantages and to increase the efficiency and decrease the treatment time, increase the speed of treatment sessions and decrease any injury done to the patient. [050] The technical result is achieved by the therapy of the central nervous system diseases consisting in the injection of a new autologous cell preparation obtained from the patient's own peripheral blood rich in stem cells with the CD34 antigen. The dose of the injected cell at one time is (0.005-16.8) xlO⁶ CD34-containing cells per 1 kg of body weight, preferably 2.7 xlO⁶. The preparation should be preliminarily mixed with the carrier, which is acceptable for intrathecal or intraventricular injection, in the proportion of 1 ml of cell preparation per 1.0-2.0 ml of the carrier. The cryopreserved and then thawed preparation is also used in the therapy. The preparation is thawed immediately before infusion in a water-bath at the temperature of 37-40⁰C. The preparation should be washed twice in the physiological solution, and the preparation can be used only during the first 6 hours after thawing, since the thawed preparation destroys rapidly and may cause dangerous toxic effect .

[051] Therefore, according to a further object of the present invention, a pharmaceutical preparation comprising (0.005-16.8) xlO⁶ CD34-antigen autologous non- hematopoietic stem cells, preferably 2.7 xlO⁶, mixed with a pharmaceutically acceptable carrier suitable for intrathecal or intraventricular infusion is obtained. [052] It is to be noticed that the concentration of the autologous non-hematopoietic stems cells of the preparation has been carefully selected to avoid any possible adverse effects. In fact, the number of stem cells is much higher (100-folds) in the conventional cancer therapies, than the number of cells selected for the present case. Higher concentration of cells used for brain or spinal injuries resulted in grave adverse effects. Surprisingly the therapy is efficient at lower concentrations and no adverse effects were aroused. [053] As stated, it is just the scheme of the stimulation and the earlier collecting period that allow to obtain a different pool of stem cells characterized by being undifferentiated. This peculiar aspect of the invention has been demonstrated to be fundamental to recover specific target cells and in the suitable quantity to avoid the risk of causing tumor proliferation. [054] As stated above, the goal of the present invention is to provide effective therapy for the central nervous system injuries that would lack all drawbacks of the prior ones and would be simple to perform with the minimum discomfort for the patients. Therefore the present invention provides also for the use of the above preparation to manufacture a formulation for the treatment of central nervous system diseases. [055] The efficiency of the present therapy was tested on patients with chronic brain and spinal cord injuries and compared to transplantations and transfusions of embryonic stem cells (ESC) and the conventional chemotherapy. In particular, the present invention can be used in the treatment of injury or damage caused for instance by physical trauma, hypoxia, chemical agents, drug abuse, stroke, perinatal ischaemia, cerebral palsy, Alzheimer's and Parkinson's diseases, Huntington's disease, Korsakoffs disease and Creuzfeld-Jacob disease. [056] Special scales have been used to verify the results: ASIA (Scale of the American Spinal Injury Association) and FIM (Scale of the Functional Independence) (C.Grander, 1979, L. Cook, 1994) , the data from MR-imaging, electroneuromyography, cerebral mapping, encephalographic examination, complex urodynamic examination, immunochemical examination of blood and SCF. 312 patients with brain and spinal cord injuries were included into the study. They were divided into three groups: 1^st group (main group, 80 patients) included the patients who received the transfusion of the autologous HSCPB preparation; 2^nd group (112 patients) consisted of the patients who received the transfusion of embryonic stem cells (ESC) ; 3^rd group (control group, 120 patients) was the patients who received the conventional chemotherapy and the patients who received the diagnostic rachicentesis with the injections of 1 ml of 0.9% physiological solution NaCl. [057] The distribution of the patients according to their sex and age is shown in Table 1. The diagram of the treatment efficiency of the central nervous system injuries (the brain and spinal cord) according to different methods is shown on Fig. 2 where the comparison between the efficiency of a new autologous non-HSCPB preparation transfusion to a patient with the brain or spinal injury, with similar transplantations and transfusions of ESC is demonstrated. The additional table 1, displays the number of the patients with different efficiency of treatment. The transplantation and transfusion of a new autologous HSCPB preparation proved to be the most efficient as compared to the group which received ESC and to the control group. Table 1

Table 2

[066] Table 2 shows the results of the limited clinical use of transplants and transfusions of a new autologous HSCPB preparation used intrathecally in the patients with the brain and spinal cord injuries. High efficiency of new preparation is demonstrated.

Advantages of obtaining and use of the preparation of autologous blood stem cells: i) The opportunity to obtain enough cells from peripheral blood without anesthesia or bone marrow puncture and with minimum damage done to the patient; ii) The opportunity to obtain the preparation multiply; iii) Relative quickness of obtaining; iv) The opportunity to store the preparation for an unlimited time, at certain conditions, without changing the properties of the preparation; which allows multiple injections; v) Unlimited opportunities for genetic engineering.

[067] To obtain autologous HSCPB preparation 80 sessions have been carried out according to the new technology. No complications or intolerance that might lead to the failure or termination of the session was registered. The duration of the session has not exceeded 3 hours, at the average speed of taiking blood of 60 ml/min, depending on the weight of a patient - which means time reduction of the treatment procedure. Example 1 [068]Patient S., 40 years old. Diagnosis: chronic vertebrospinal injury. Consequences of a compressive fracture C6-C7 with the spinal cord damage expressed by intramedullary cyst on C5-C6 level, with lower spastic paraplegia and paraparesis of upper limbs and dysfunction of the pelvic organs. The spinal injury on the cervical level was received in a road traffic accident in 1994. The patient has been long treated in specialized clinics and rehabilitation centers for the spinal patients. The symptoms of lower spastic paraplegia and deep paraparesis of the upper limbs are noted, pelvic organs dysfunction expressed in inability to control urination and defecation. For 10 years he has been adapting to his state and could be independent at home. After full evaluation according to the protocol of a branch program "New Cell Technologies for Medicine" , the course of new autologous HSCPB preparation therapy was administered to him. After the stem cells stimulation with G-CSF for 4 days, the HSC harvest session was performed through the peripheral veins on the COBE Spectra separator. Duration of the session was 60 minutes, with blood flow 60 ml/min. The preparation was obtained in the dose of 2.7 (0.005)xl0⁶ CD34 antigen-containing cells per 1 kg of the patient's weight . The preparation was processed with the DMSO with polyglucin and cryopreserved. In 3 days, the preparation was thawed, and 1 ml of the preparation was mixed with 2 ml of a pharmaceutically acceptable carrier and injected intrathecally to the patient. No adverse effects were observed. In 7 days after transfusion, positive changes were observed: movements in the fingers of the left and right hands appeared; small movements were recovered

(can use fingers and hold small objects) ; the range of movement in the forearms increased, more in the left; several areas of normal sensitivity appeared in the legs; small movements in the right hallux and control of the abdominal muscles appeared. After 1.5 months, abducent and adducent movements in both legs appeared, and the patient could move the foot up and down. The control electroneuromyography showed increased electroconductivity in the arms and legs, more in the left. Functions of the pelvic organs were restored after 3 months: the patient could control defecation and urination, which was proved by complex urodynamic examination. Example 2

[069] Patient G., 22 years old. Clinical diagnosis: consequences of permanent spinal cord injury and fracture of Thχ₂ spondyl . Dysfunction of pelvic organs . [070] Secondary diagnosis: nidal gastratrophia. Chronic cystitis. Chronic pyelonephritis. Urolithiasis. Condition after epicystostomy (2003) . Condition after operation - transurethral endovesical mechanical cystolithotrypsy, cystlithotapsy (2004) . [07I]He had complaints about the absence of movement and sensibility in the lower limbs, dysfunction of defecation and urination.

[072] Disease history: in 2001 fell down from a height of about 10 m; after the accident, he was unconscious for a long time, according to the words of relatives; was taken to the hospital immediately. Having recovered consciousness, he felt he could not move and sense his legs. On the third day in the hospital, decompression laminectomy of Th₁₂ was performed, and a titanium stabilizing system was installed. Still he could not move and sense his legs. He was examined and treated in different clinics of Germany and Romania, with no significant effect. He was admitted to the Neurovita clinic to be examined within the frame of a branch program "New Cell Technologies for Medicine" . [073] Neurological status at admission: clear consciousness. Equal pupils, of average size. Live photoreaction, DES. Right nasolabial fold is partly smoothed. Small deviation of the tongue to the right. Strong deviation of the uvula to the right. Throat reflex is present. Muscle tonus in the limbs is reduced. No movement and sensation disorders in the upper limbs. Pathologic Rossolimo's symptom on both sides. No abdominal reflexes. Lower spastic paraplegia. Leg reflexes are vivid, especially on the right side. Clonic movements of lower limbs when exploring passive movements. Block anesthesia of all sensation types with the dermatoma of Th₁₂ on the left side and Ll on the right. Babinski's symptom, Pussep's Gordon's and Sheffer's symptoms are observed on both sides. Dysfunction of pelvic organs, central type. No meningeal signs. Since the last hospitalization, neurological status progressed - the patient can move his hip joint by medial and lateral muscles .

[074] MRI of the thorax-lumbar region of spinal cord and brain; conclusion: condition after rachitomy Thi_2/ installation of a titanium fixer on Th₁₁-Th₁₂ level. V- shaped deformations of the Th₁₁ and Th₁₂ bodies. Uneven lowering of height and change or MR-signal of Th₁₁-Th₁₂ intervertebral are observed with small (~0.2 cm) protrusion to the back. No pathologic changes in lumbar department were found. The brain examination showed no pathologic signal in the brain tissue. There is some moderate asymmetric distention of the front horn of left side ventricle: small distention of convecontal subarachnoid cavities in the frontoparietal region.

[075] Conclusion: condition after laminectomy Th₁₂, titanic fixer Th₁₁-Th₁₂, compression fracture of Th₁₂ body. No signs of the block of SCF system. [076] Reinfusion of the autologous HSCPB preparation into subarachnoid space was performed. Each time the dose was calculated according to the principle 6.3xlO⁶ CD34 antigen-containing cells per 1 kg of patient's weight. [077] Thus the patient with a permanent spinal injury and lower paraplegia was repeatedly admitted to the clinic to continue the treatment under the program "New Cell Technologies for Medicine" and to receive rehabilitation treatment. During the examination, indications for injection of autologous HSC were detected: absence of antibodies to neurospecific proteins in the blood serum and SCF, negative result of tests for neurotropic viruses in blood serum and SCF, clinical tests of SCF and blood are normal. At the same time, the patient performed physical training, exercised in a gym

(including the treadmill) , received massage and physiological procedures. Also the patient received symptomatic conservative therapy.

[078] Catamnesis after one year. The muscle bulk in the legs increased. The shin increased by 3 cm and the hips by up to 2.5 cm from the left side and 5 cm at the right side. Now the patient can walk by himself with the help of a walker, sexual function restored, he can fully control defecation, but urinated only with the help of a catheter. Mosaic sensation appeared in the legs and breech. [079] Thus a new system has been developed which includes the preparation itself, the production, storage and usage of the method of treatment of the central nervous system injuries. This permits to considerably help the patients with spinal and brain injuries in acute states as well as in chronic .

Claims

1. Preparation of autologous mobilized non-hematopoietic stem cells (AMnon-HSC) from a patient's peripheral blood containing CD34 antigen in the final concentration of (40-100) xlO⁶ cells/ml and characterized by the following markers configuration: CD34+, CD45-.

2. The preparation according to claim 1, further characterized by the following marker configuration: HLA DR±, CD38±, Gpl30+.

3. The preparation according to claim 1 or 2, comprising said autologous mobilized non-hematopoietic stem cells as undifferentianted progenitor cells.

4. The preparation according to any one of claims 1 to 3 obtainable by the method of production which comprises preliminary, intermittent administration to the patient of granulocyte colony-stimulating factor (G-CSF) for 4 days, according to the following schedule: first three days - every 10-12 hours, in the dose from 0.5 to 2.5 micrograms/kg,- on the 4^th day - a double dose, following the same time schedule.

5. The preparation according to any one of claims 1 to 4 , comprising a carrier suitable for intrathecal or intraventricular infusion.

6. The cryopreserved preparation according to any one of claims 1 to 5, mixed with a blend of DMSO and polyglucine, in the initial concentration of DMSO 10- 12%.

7. The cryopreserved preparation according to claim 6, wherein the cryopreservation means is liquid nitrogen, preferably vapors of liquid nitrogen.

8. A pharmaceutical preparation comprising (0.005- 16.8) XlO⁶ CD34-antigen containing autologous mobilized non-hematopoietic stem cells per 1 kg of body weight of a patient, preferably 2.7 xlO⁶, mixed with a pharmacutically acceptable carrier suitable for intrathecal or intraventricular infusion.

9. Use of a preparation according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of diseases of the central nervous system.

10. The use according to claim 9, wherein the said preparation is obtained through preliminary, intermittent administration of G-CSF Neilogen or Neupogen in a glucose solution for 4 days, according to the following schedule: first three days - every 10-12 hours, in the dose from 0.5 to 2.5 micrograms/kg; on the 4^th day - a double dose, following the same time schedule .

11. A method of production of the preparation according to any one of claims 1 to 8, comprising the step of: purifying autologous non-hematopoietic stem cells derived from patient's peripheral blood from erythrocytes .

12. The method of production of the preparation according to claim 11, comprising in order the steps of:

- preliminary, intermittent treatment of a patient with granulocyte colony-stimulating factor (G-CSF) for 4 days, according to the following schedule: first three days - every 10-12 hours, in the dose from 0.5 to 2.5 micrograms/kg; on the 4^th day - a double dose, following the same time schedule;

- taking peripheral blood from the said patient;

- purifying autologous mobilized non-hematopoietic stem cells derived from patient's peripheral blood from erythrocytes.

13. The method according to claim 11 or 12, further comprising the step of harvesting the autologous mobilized non-hematopoietic stem cells on the fifth day after the beginning of the treatment with the white blood cell (WBC) separation method.

14. The method according to claim 13 , further comprising a concentration step after harvesting of the cells by means of cytapheresis .

15. The method according to any one of claims 12 to 14, wherein the step of purifying autologous mobilized non- hematopoietic stem cells from erythrocytes is carried out by:

- adding a lysing reagent to a cellular deposit of the autologous mobilized non-hematopoietic stem cells; - stirring and incubating until the solution is clear;

- washing the cells twice in a suitable medium by means of centrifugation.

16. The method according to claim 15, wherein the lysing reagent is an aqueous solution of NH₄Cl, potassium bicarbonate and Na₄-ethylene diamine tetraacetic acid.

17. The method according to claim 15 or 16, wherein the washing medium is selected from medium 199 or PBS-BSA.

18. The method according to claim 12, further comprising the step of determining the concentration of the cells by means of flow-through cytofluorimetry.

19. A method of cryopreservation of a preparation of autologous mobilized non-hematopoietic stem cells deriving from periferal blood, comprising the steps of:

- mixing said preparation with dimethyl sulfoxide (DMSO) at an initial concentration of 10-12% and polyglucine;

- freezing down the mixture by means of liquid nitrogen with a temperature gradient of l.l°C per minute till reaching -40⁰C;

- further freezing down the mixture until -165° -17O⁰C is reached.

20. The method according to claim 19, wherein the freezing is carried out with vapors of liquid nitrogen.

21. A method of treating patients suffering form central nervous system injuries with a preparation comprising (0.005-16.8)xlO⁶, preferably 2.7 xlO⁶, CD34-antigen autologous haematopoietic stem cells per l kg of body weight of a patient .