08-26-2001, 07:32 AM
The title: " Fetal cell transplantation for spinal cord injury "
The short title: " Cell transplantation for spinal injury"
Samuil S. Rabinovich1, Victor I. Seledtsov2, Olga V. Poveschenko2, Vladimir V. Senuykov2, Vadim Ya. Taraban2, Vladimir I. Yarochno, Nicolay G. Kolosov1, Vladimir Kozlov2, Sergey A. Savchenko1.
1 Novosibirsk State Medical Academy, 2 Institute of Clinical Immunology, 3 34 Municipal Clinical Hospital, Novosibirsk, Russia.
The corresponding author:
Victor Seledtsov M.D., D.Sc.
Institute of Clinical Immunology
Russian Academy of Medical Sciences
14 Yadrintsevskaya str.
630099, Novosibirsk, Russia
Tel/Fax: +7 (3832) 28-26-73
Fax: +7 (3832) 22-70-28
The cells from fetal nervous and hemopoietic tissues (gestational age 16-22 weeks) were subarachnoidally implanted into 15 patients of 18-to-52 year old with severe consequences of the traumatic spinal cord injure (SCI) at cervical or thoracic spine level. The times after SCI were from 1 month to 6 years. The number of fetal cell transplantations (FCT) into a patient varied from 1 to 4. The time intervals between FCT were 14-to-30 days. In 11 of 15 cases FCT was performed immediately after resection of the connective tissue cyst(s) that has been formed within the site of SCI. Before FCT the neurological state of each of the patients was characterized by complete motor and sensory function disorder and was consistent with a grade A of SCI according to Frankel classification. With FCT therapy, the neurological state of 6 patients became clinically consistent with C grade of SCI. These patients received an ability to walk with the aid of crutches or a special hinged apparatus. The less, although noticeable, clinical improvements were noted in other 5 FCT-treated patients. Their state became consistent with grade B of SCI and was characterized by appearance of contracting activity in some muscles and incomplete restoration of sensitivity. Lastly, 4 patients subjected to FCT therapy did not demonstrate any clinical improvements. No serious complications of FCT were noted. The results presented herein point out a clinical relevance of the FCT-based approach to treating severe consequences of SCI.
Key words: spinal cord injure, fetal cell transplantation.
The human adult nervous tissue is well known to have a very limited reparative potential. Therefore, the treatment of the patients with severe injures of the central nervous system (CNS) frequently do not give desired results. Over last decades a considerable progress was reached in the understanding of the mechanisms regulating cell reparative activity in CNS. The accumulating evidence clearly outlines two feasible approaches to improve reconstruction of injured CNS. The first is based on creation in CNS of conditions which are favorable to nerve fiber growth, whereas the second, on replacement of destroyed neurons by new functionally active neural cells. The transplantation of fetal-derived immature cells appears to unite both these approaches. In fact, it has been established that when grafted into adult CNS the fetal-derived cells are able both to elaborate the factors favoring axonal growth from recipient neurons and to provide the generation in CNS of new functionally active donor neurons [reviewed in 1-3]. In this paper we present the results of applying fetal (neural plus hemopoietic) cell transplantation (FCT) therapy in 15 spinal cord injury (SCI) patients.
MATERIALS AND METHODS
The study was performed in the exact accordance with the protocol that has been approved by the Scientific Council and Ethics Committee at the Institute of Clinical Immunology. Informed consent was obtained from each subject who have been enrolled in the study.
The fetal brain neural and hemopoietic liver tissues were isolated from human fetus (gestational age 16-22 weeks) after spontaneous or prostaglandin-induced abortion, and then prepared in the form of cell suspension, as described early . The cells were further cryopreserved in the standard way in RPMI 1640 medium containing 50 % fetal bovine serum and 10 % dimethyl sulfoxide , and stored in liquid nitrogen until use. On the day of transplantation, the cell suspensions were thawed at 370 C, washed extensively, and assayed for cell viability by a trypan blue exclusion method in the routine way. The cell suspension designed for transplantation was composed of equal numbers of cells obtained from three distinct donors. The overall number of viable cells in such suspension was 25 x 107, whereas a ratio of cells from neural tissue to those from liver one was 10.
When it was reasonable, the FCT was performed immediately after operative resection of connective tissue cyst that has been formed within the site of SCI. In the several cases, along with the above described cell suspension, a fetal spinal cord fragment together with olfactory ensheathing cells (2 x 105) was implanted into spinal cord lesion. The olfactory ensheathing cells have been previously reported [reviewed in 1] to be effective stimulators of nerve fiber growth and myelination.
Before FCT therapy, all 15 patients with SCI enrolled in the study had complete motor and sensory function disorder. The patient characteristics ( age, time after SCI, lesion level, number of performed FCT) are shown in Table 1. The neurological status of the patients was examined in terms of modified Frankel definitions. For the Frankel score, a five scale subdivision was used: A = complete motor and sensory function disorder; B = motor complete, sensory incomplete function disorder; C = motor and sensory incomplete function disorder; D = useful motor function with or without auxiliary means; E = no motor or sensory function disorder . The dynamic examination of motor functions in the patients was conducted with usage of ASIA (American Spinal Injury Association) motor scale .
As shown in Table 1, clinical improvements was noted in 11 of FCT-treated 15 patients. Six patients changed their neurological state from A to C grade of SCI. These patients received an ability to walk with the aid of crutches or orthoses. The less, although noticeable, clinical improvements were noted else in FCT-treated 5 patients. Their state became consistent to grade B of SCI and was characterized by appearance of contracting activity in some muscles and incomplete restoration of sensitivity. Lastly, 4 FCT-treated patients did not demonstrate any clinical improvements. Three cases with most improvements are described in detail below.
Case 1. 52-year-old male patient was admitted to the Emergency City Hospital 6 hours after a vehicular accident. On admission the patient was in grave condition: he was stuporous, although followed instructions and correctly answered simple questions; his puls rate was 110 b min -1, arterial blood pressure 90/60; his respiration was self-dependent with the of 22 min -1 ; the functions of his granial nerves were not compromised; there was teraplegia and apparent muscle hypotonia; his tendon and periosteal bone reflexes were flaccid in both upper and lower extremities (D=S), his abdominal reflexes were absent; there was profound disorder of all kinds of sensitivity on both sides of his body lower Th1 level, but also vesical disfunction; the palpation of _3-to-C6 vertebras was painful. X-ray examination failed to reveal any traumatic bone changes in cervical spine. Magnetic resonance imaging (MRI) showed the rounded area (0.7 x 0.5 cm) of hypointensity with clear-cut contours at C3-C4 junction (see Figure 1, A) and the centro-dextral spindle-shaped area of 5.8 x 0.3 cm, giving signal of increased intensity at T2 regime along the whole length from C2 to C5 level. A diagnosis was made out: a cord contusion at the C3-C5 level and a syndrome of complete SCI.
The standard therapy aimed for brain dehydration was started immediately after the cervical spine fixation. The cell suspension (10 ml) composed of cells obtained from fetal neural and hemopoietic tissues was grafted subarachnoidally (via lumbar puncture) into patients 4 days after trauma (day 0). On 28 days after FCT the first signs of spinal function restoration were noted: the level of sensory disturbance lowered down to Th4 and some motor activity of the left foot appeared. A clinic examination of the patient performed on 40 day after FCT revealed the following: a complete restoration of pain and touch sensitivity; kinesthesia restoration (S>D); the appearance of some sighs for vesical reflex; active leg motion with the strength of 3 and 2 points (according to the ASIA scale) at the left and the right side, respectively. The muscle strength in arms was 2 points at the left side and absent at the right one. Tendom and periosteal reflexes were active. MRI scan showed the intramedullar heterogeneous formation with indistinct contours (the cyst might presumably be embedded in neural cell graft) at C7, as well as the normal thickness and structure of the spinal cord bellow the level of SCI (Th1-Th3) (see Figure 1, B). On 96 day after FCT, the complete restoration of all kinds sensitivity and functions of pelvis organs was noted. The strength in arms and legs was found to reach for 4 and 5 points, respectively. The patient began walking with crutches.
Case 2. 24-year-old male patient with SCI at _7 was admitted to the neurosurgical department 6 days after an incidence. In the view of bursting fracture of C7 vertebra and spinal cord compression the operation was made: a partial resection of C7 vertebral body and ventral spondylodesis at _6-Th1 with a titan-nickel (Ti-Ni) implant. Seven weeks later patient was again admitted to the neurosurgery department for further his management. The overall health status of the patient on admission was of average severity. His consciousness was clear, cardiovascular and respiratory systems were without any pathological changes. Neurological parameters according to the ASIA scale was described as follows: inferior flaccid paraplegia, superior paraparesis (strength of arms at distal and proximal parts was 3 and 5 points, respectively), conductive type of disorders of all kinds of sensitivity lower Th1 level, tendon and periosteal reflexes active on upper extremities and flaccid on lower ones (D=S). Urination was via cystostoma. The patient condition was complicated by 26 _ 18 _ 5 cm necrotic pressure ulcer over the sacrum. MRI examination revealed deformation and size diminution of C6-_7 vertebral bodies. At C6-_7 level Ti-Ni implant was visualised. The spinal cord was thin at the injure level, but its continuity was not broken. The intramedullary cyst of 1.01 _ 0.2 cm was clearly visualized at _7 segment (Figure 2, A). At Th1-3 level the spinal cord heterogeneity with the hyperintensive areas of up to 0.12 cm were detected in T2 regime.
The patient was subjected to three FCT with 14 day intervals. At the first and third FCT, the suspension of viable cells in a volume of 10 ml was injected subarachnoidally via a lumbar puncture. The intermediate second FCT was performed immidiately after surgery: laminectomy at C7, extraction of intramedullary cyst and implantation into the made cavity of a fetal spinal cord fragment enriched with olfactory ensheathing cells.
One and a half month after the last FCT, the apparent clinical improvements in the patient were noted: complete restoration of sensitivity and vesical reflex , the presence of strength of 5 points in arms from both sides and some motions in foots and in knee joints, and complete healing of pressure ulcer. On MRI scan in the field of a former cyst the areas of various densities with indistinct contours were seen (see Figure 2, B). Those areas might be visible manifestation of reparative activity of the grafted cells. Ten month after the FCT therapy the patient entirely controlled the functions of pelvis organs. He was able to stand, leaning on crutches, and to walk, using a special hinged apparatus.
Case 3. 25 year-old male patient had received bursting fracture of C7 vertebra with spinal cord compression, as a consequence of a road traffic accidence. Some days later he underwent liminectomy Th6-Th7 and 4 months later he was again admitted into the neurosurgical department for further his management. On admission the patients was in condition of middle severity. His consciousness was clear, cardiovascular and respiratory systems were without any pathological changes. Neurological status had the following characteristics: inferior flaccid paraplegia, conductive disorders of all kinds of sensitivity lower Th5 level. Urination was via cystostoma. MRI scan showed deformation and size diminution of Th6 vertebral body. The spinal cord was thin at the injure level, but its continuity was not broken. At Th6 level, a 3.0 _ 0.5 cm intramedullary cyst was clearly seen. The spinal cord heterogeneity with the hyperintensive areas of up to 0.7 cm at were also visualized in T2 regime.
The patient was subjected to two FCT with 30 days interval. The first FCT was preceded by the surgery: repeated laminectomy at Th5-Th7 level, extraction of intramedullary cyst and implantation into the formed cavity of a fetal spinal cord fragment enriched with olfactory ensheathing cells.
Four months later the last FCT the patient demonstrated complete restoration of all kind of sensitivity, overall control of the functions of pelvis organs and evident improvements in motor sphere: he was able to stand, leaning on crutches.
By present there is already a valid experimental basis for applying FCT-based therapy in treating patients with CNS injures [reviewed in 1-3]. It seems quite reasonable that realization of the reparative potential of fetal-derived immature cells in such patients may greatly improve outcome of their disease. Importantly, novel techniques of propagation of immature multi- and unipotent cells in vitro, which are being now actively developed, allow to solve not only technical, but also ethical problems confronting progress in transplantology [reviewed in 7,8] and, thereby, may promote widespread adoption of FCT-based advances in clinical practice. An effective CNS repair appears to require the presence in injured sites of not only neural cells potentially able to provide axonal growth, but also the other cells capable of creating the microenvironment favorable to both growth and myelination of nerve fibers. In our own investigation we transplanted into the SCI patients not only the cells from fetal nervous tissues, but also the fetal liver cells belonging mainly to erythroid and myeloid differentiation lineages. In fact, evidence is accumulating that 1) fetal hematopoietic tissues contain the stem cells capable of differentiating into cells forming nervous tissue [7, 9]; 2) hemopoietic cells can elaborate the mediators able to support the growth and viability of distinct cells including the cells constituting nervous tissue [reviewed in 7]; 3) these cells possess a potent natural suppressor activity  that may be directed against developing cell transplantat-induced immune processess; and 4) they may contribute to neovascularization of ischemized tissues [11, 12]. Lastly, there are publications indicating a capacity of immature hemopoietic cells not only to inhibit but even to reverse the development of scar connective tissue [13,14] that is known to represent a insuperable obstacle to axonal growth. Thus, it is reasonable to believe that the fetal hemopoietic tissue-derived cells may be capable of markedly contributing the repair of adult CNS.
In our view, FCT into a SCI patient is most optimal before the formation in the injured site of the fibrous connective cyst that may be a major obstacle for restoration of spinal nerve communications. It is reasonable that in the cases with the formed cysts, FCT should be performed immediately after resection of the cyst and restoration, as far as possible, of canals for nerve fiber growth.
The clinical success of FCT is believed to be significantly defined by survival of the cells implanted in the body. In fact, the fetal neural cells have been previously demonstrated to be able to survive and function in major histocompatibility complex (MHC)- incompatible adult CNS for relatively long period of time [reviewed 1-3]. It should be had in mind, however, that immune privileges of CNS cannot yet guarantee immune unresponsiveness of the host to the allogeneic cells grafted into CNS. Therefore, we transplanted into a recipient only those cells whose alloantigens failed to induce the activation of recipient's T lymphocytes in vitro assays which have been performed before FCT. Moreover, to our opinion, the transplantation of cells from more than one donor may markedly lessen a risk for prompt immune-mediated rejection of all donor cells. Actually, such cell transplantation might lead to the situation when host's immune responses might be directed against only a part of the most antigenically incompatible cells, while another part of the cells might survive and be involved in CNS repair. Consistent with this proposition, we, indeed, found that 4 of 12 patients with severe cranial traumas, who were subjected to more than one FCT in the above-described way, developed both cell and humoral immune responses directed against the part of, but not all, transplanted cells (data not presented).
The cases reported herein indicate that the FCT therapy of SCI patients may result in apparent clinical improvements. Only 4 of 15 FCT-treated patients with time after SCI of 2, 3, 4, and 6 years did not show any improvements in their neurological status. Nevertheless, a time boundary, behind which FCT therapy become inefficient, was not yet determined. In fact, this boundary may be quite blurred and dependent on the individual features of patients. In this connection, it should be paid attention to the appreciable clinical improvements in 4 FCT-treated patients with times after SCI from 2.5 to 3 years.
In all 15 cases described in this paper, FCT therapy led to noticeable improvements in patient's psychological status, which were noted by both the patients, themselves, and their nearest relations.
FCT into CNS appears to be safe and well tolerated. Meningisms, but also raises of body's temperature up to 38.5 C, were noted in the part of patients during 24-to-48 h after FCT. Those occurrences disappeared, by themselves, not requiring any additional medicament interventions. A long-term (3 year ) follow-up of 18 patients (mainly with brain injures) subjected to FCT therapy did not reveal any serious complications which might be related to grafted fetal cells (manuscript in preparation) .
The results presented herein suggest that FCT-based therapy may be successfully applied in treating neurological sequences of SCI. Although much greater clinical experience is needed to determine place and clinical relevance of this therapy in overall complex treatment of the patients with CNS injures.
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Table 1. Characteristics of SCI patients and results of their FCT therapy in the term of Frankel definitions.
No. Patient, age (years) Level of SCI Time after SCI Number of FCT Neurological status in terms of Frankel definitions
before and after FCT therapy
1 K, 52 _6-7 1 month 2 _ _
2 Z, 24* _6 3 months 3 _ _
3 G, 28* Th11 3 months 2 _ _
4 _, 25* Th6 4 months 3 _ _
5. V, 18 Th4 4 months 2 _ _
6. _, 34* Th10 1.5 years 2 _ _
7. _, 34* _6 1.5 years 2 _ _
8. _, 48 Th3 2 years 2 _ _
9. Sh, 28* _6 2.5 years 3 _ _
10. S, 32 * Th9 2.5 years 3 _ _
11. D, 32* _6 3 years 1 _ _
12. S, 18* _7 3 years 2 _ _
13. _, 21 Th4 3 years 2 _ _
14. Kh, 46* _4-6 4 years 4 _ _.
15. _, 38* Th6 6 years 1 _ _
*the case with operative cyst resection