Name of journal: World Journal of Transplantation esps manuscript no: 18452 Manuscript Type: Original Article Retrospective Study



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DISCUSSION

The therapy of SCI with autologous HSCs and HPs demonstrated high efficiency (to 95.1%) of stem (CD34+) peripheral cells mobilization in SCI patients. It is well known that under the conditions of undamaged hematopoiesis, the hematopoietic stem cells circulate in peripheral blood of a human. But their concentration is extremely low (less than 0.01%) that makes their detailed study and transplantation almost impossible. High concentration of hematopoietic stem cells results from the damage of hematopoiesis (usually as a result of chemotherapy), or administration of colony-stimulating factors (CSF). In the clinical practice, the granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) are the most widely used. These factors increase the concentration of hematopoietic stem cells 100-1000 times, thus allowing the harvest of the cells and their use for transplantation. It should be noted that mobilization of the stem cells and precursor cells into peripheral blood vessels in the patients with traumatic disease of spinal cord was efficient in all cases – both absolute number and the percentage of CD34+ in leukoconcentrate received after 1 session of leukapheresis meet the transplantation standard of the number of mononuclears (> 2 × 106/kg).

As seen from the description, we applied the suspension of autologous HSCs and HPs, and not a standard suspension of autologous hematopoietic stem cells (CD34+). We consider this cell suspension to reflect systemic specific response of bone marrow of each patient to the injury of the central nervous system, and the cell composition received in specific stimulation conditions and cryopreservation is unique and obligate. In case of stimulation of an SCI patient with G-CSF in the dose of 10 or 20 mg/kg for 5-6 d as it is recommended in the manuals of hematooncology, we harvest mature differentiated hematopoietic cells, able to restore hematopoiesis, and not injured nervous system. We applied the standard sparing scheme of simulation, which is ubiquitously used in pediatric oncology. This empirically selected mode of stimulation allowed us for new property of cell suspension that conditions its clinical effectiveness.

As seen from Table 3, we refused from cryopreservation with 10% DMSO with polyglycine, although the combination is considered ideal to protect hematopoietic stem cells. We applied lower concentration for cryopreservation, and, namely 5% DMSO and polyglycine that demonstrated its high efficiency and safety for intrathecal transplantation.

The stem cells and committed precursor cells form a so-called pool of hematopoietic stem cells. The expression of CD34 molecule on the surface of a membrane is common to all cells of the pool, and this property enabled use of flow cytometry methods to detect the precursor cells and to provide their quick count in any hematopoietic material. Last decades the peripheral blood was the main source of stem and precursor cells. Thus, for example, transplantation of separated fraction of mononuclear cells of peripheral blood with hematopoiesis stimulation permits considerable reduction of critical cytopenia in patients after high-dose chemotherapy. The phenomenon is conditioned by stem and precursor cells entering peripheral blood under colony stimulating factors influence. Special attention should be given to the composition of subpopulation of CD34+ cells, that is, the number of the cells of different compartments of HSCs and HPs pool.

Subpopulation composition of CD34+ cells was assessed by flow cytometry with triple-labeling method. Our analysis of efficiency of the suspension in SCI patients demonstrated that best motor restoration in SCI cases was observed only when the membrane of an autologous stem cell expressed gp130 protein. Gp130 is a transducing molecule of IL-6 cytokines and a receptor of cell functional condition. Basic pleiotropic action of these receptors is to contribute to cell differentiation, gene expression, stimulation or inhibition of cell growth and control of cell apoptosis. At day 4.5 and 5 of stimulation with G-CSF the abrupt decrease of gp130 expression was observed, which reduced activity of the cell preparation and therapy effect. The HSCs and HPs harvested according to standard protocol at day 6 of stimulation did not lead to any clinical effect. We received the pool of formed mononuclears with highly differentiated and well-diagnosed genuine hematopoietic and mesenchymal stem cells, therapeutic effect of which is disputable in our case. The cell suspension we use for therapy does not contain conventional hematopoietic stem cells, although they are assessed in CD34+ gate, when evaluated in flow cytometer, the suspension contains heterogeneous mixture of mobilized low-differentiated precursors, promoting regeneration of nervous system. In this technique the dose has no relevance and can considerably vary. The standard cell composition is of the key importance, reflecting the level and concentration of the output of non-differentiated PC at the proposed sparing G-CSF stimulation modes in the patients with post-traumatic neurologic deficit. The proposed individual preparation contains the mixture of highly efficient mobilized stem precursor cells of bone marrow, including hematopoietic-like cells. To date, we are unable to accurately identify what exactly type of cells of this pool make the treatment effective, but this seems unimportant for the patients and the clinical practice. We know that using the proposed method of harvest, we receive a standard cell preparation that gives a steady, reproducible and progressing clinical effect.

This preparation has no prototype, as well as the presence or absence of hematopoietic stem cells (CD34+) is not pivotal. Novelty of this preparation is determined by the presence of the mixture of non-differentiated cell precursors, restoring neurogenesis and regeneration in the damaged brain/spinal cord. The researches in cell medicine mention the facts of using hematopoietic stem cells to treat multiple sclerosis and amyotrophic lateral sclerosis. None of the authors used cryopreservation, they applied a single bolus injection of stem cell preparation. Our experience clearly demonstrated that only multiple and long-term (for 5-8 years) administration of the preparation will provide the maximal benefit of the existing regenerative potential of the cells and the opportunity to restore the damaged brain/spinal cord functions. It is the sparing 4 d long mode of G-CSF administration in the patients with neurologic deficit that provides for the harvest of all necessary nuclear cell precursors.

According to our evaluation of long-term outcomes of SCI cell therapy, the transplantation of autologous HSCs and HPs is an efficient method to repair lost functions in SCI patients, and it is not directly dependant on the dose and number of autologous HSCs and HPs transplantation. The patients with the lesion exceeding 50% of spinal cord cross-wise and 1 segment long-wise, and possibly those with moderate CSF circulation disorders should be excluded from therapy. Presumably, such patients require reconstructive surgical intervention with meningoradiculomyelolysis, spine stabilization and, possibly, tissue engineering of spinal cord. The HSCs and HPs transplantation only will hardly result in the restoration of spinal functions in these cases.

The rehabilitation is a requisite component in the therapy of chronic SCI patients.

Research of the SCI therapy demonstrated that to restore the functions, the conductance along various nervous pathways (pyramidal, extrapyramidal, spinothalamic, etc.) must be restored, and new synaptic links between injured segments of spinal cord must be established. Under these circumstances, the grey matter of spinal cord need not be replaced due to availability of cross innervation of dermatomes and myotomes in humans. Mere surgery and/or rehabilitation do not lead to the expected outcomes, as they do not eliminate the main cause of the disorder and do not restore injured neural structures of spinal cord. Application of the systems of adult stem and progenitor cells confirmed the opportunity to restore spinal cord. The regulatory action of the mobilized progenitors, and not their regenerative potential, seem to be the main mechanism of functional restoration in SCI, activating synaptogenesis in adult brain, increasing plasticity of injured neural tissue of SC and developing functional neurophysiologic bypass. The intensity of HSCs and HPs regulatory potential depends on the size of SCI and directly proportional to intact neural structures of spinal cord.

The analysis of treatment efficiency depending on the level of injury deserves special attention. The reason for better clinical restoration at thoracic level seems to lie in morphological feature of the spinal cord structure and cervical and lumbar intumescence, where great number of neurons is located (second motor neurons, interneurons, etc.). The spinal cord injury at the level of intumescences leads to larger damage of spinal cell components and more intense pathologic processes; hence, the restoration in such cases of SCI is more difficult. The axons of motor neurons are located mostly at the thoracic level, the bodies of them are found in motor cortex of brain, and hence, less number of bodies of neural cells is involved into the injury. Restoration of the motor functions is associated with the increase of regeneration potential of the spinal cord, mainly at the level of cortical influence of HSCs and HPs on the intact bodies of motor neurons. The mechanism of HSCs and HPs effect does not seem to be associated with their differentiation into neurons and glial cells of SC. Most likely, the regulatory influence of HSCs and HPs at the site of SC injury leads to gene expression and secretion of neurotrophic factors, entailing growth and regeneration of axons in the site of injury and restoration of nerve impulse conductance along the intact but functionally inactive axons. As a result, the available ensembles of neurons are differentiated due to the development of new synaptic contacts below and above the injury site. The phenomenon is only observed when the stem cells are transplanted into the injured spinal cord, and it fully agrees with the data offered by Snyder[13]. The development of new synaptic links below and above injury level can serve as an explanation of the clinical results of motor restoration that we have observed.

The analysis of the obtained data indirectly confirms the hypothesis of HSCs and HPs influence on axonal growth in the site of injury or development of conductance along functionally inactive, but anatomically intact fibers, as it is the patients with incomplete injury, who demonstrate maximal restoration of motor functions. Consequently, incomplete SCI is prognostically more favorable for restoration of motor functions of spinal cord. However, to obtain representative results the clinical data have to be compared depending on the level of SCI. In the cases of complete SCI we observed intensively restoring functions, too.

The issue of termination of HSCs and HPs therapy of SCI remains important for us. Many patients, who have completed 3 year and 6 year courses, insist on continuation of the therapy. Their arguments are quite simple: “My own cells cannot hurt me and I see steadily increasing positive effect from them, so it is harmless to continue the therapy”. To date, 15 patients received HSCs and HPs transplantation for 8 years on a regular basis and no negative effects have been observed either at the level of clinical picture, or at the level of thorough paraclinical examination.

Summing up, we can conclude that the method is safe, effective and considerably improves the life quality of SCI patients. Administration of the autologous cell systems of hematopoiesis precursors led to real restoration of various movements and improved life quality in major part of our patients. About 15 patients are able to walk independently or with supporting devices, over half of them restored sensation of different types and the function of the bowel and bladder. The therapy was approved for clinical use as the treatment of choice. In terms of the long-term clinical outcomes, we can discuss complexity of the processes, observed in the central nervous system after SCI and under HSCs and HPs therapy, which are often hard to explain from clinical point of view. Being limited by the size of journal article, we are unable to demonstrate the whole range of long-term neurophysiologic and urodynamic paraclinical results, and their correlations with the mentioned clinical data, but we would be happy to offer them in our other works.


COMMENTS

Background

Contemporary healthcare have greatly improved the survival rates in spinal cord injuries (SCI) cases as well as their life expectancy, leading to the overall growth of the national economic burden. However, current healthcare advances have not led to any breakthrough in restoration of the functions of spinal cord after the injury, and ever since the Edwin Smith Papyrus the SCI has been classified as the ailment not to be treated. To date SCI is a verdict that entails impossibility to return to previous way of life, to restore previous working capacity and reproductive functions, resulting in tremendous social and economic losses. Inefficiency of the available SCI therapies used to be explained by the absence of the regeneration potential in adults, and the restoration of the damaged neural cells has been demonstrated only recently.


Research frontiers

By now, the first steps to develop new restorative therapy of SCI have been made, and the cell transplantation is the most obvious choice, although no universally acknowledged methods to restore spinal cord after the injury are observed. The methods of transplantation and the types of cells significantly vary; the evidence gathered is mostly limited by a one or two years follow up. Being involved into stem cell transplantation for SCI for about 25 years in research and in clinical practice we have accumulated substantial experience of achievements and failures in stem cell therapy. In the current work we describe the cell therapy that proved the safest and the most effective both in the short-term period and in long-term follow-up.


Innovations and breakthroughs

The method implies multiple long-term transplantations of the preparation of hematopoietic stem cells and hematopoietic precursors that was harvested from peripheral blood after sparing mode of administration of granulocyte colony-stimulating factor. The composition of the applied preparation is characterized. The cells are administered intrathecally in a subarachnoid space every three months and the transplantation is followed by vigorous specialized rehabilitation. The effects are evaluated by conventional indexes and tests, including somatosensory evoked potentials tests and urodynamic tests, as well as by specifically developed scales. The effects of 20 consecutive transplantations for each case are measured.


Applications

The method is safe, effective and is applicable to chronic SCI cases when no further restoration of the functions is observed. It considerably improves the life quality of the SCI patients. The method received official approval in the Russian Federation in 2005 and in 2006 and is recommended as the therapy of choice.


Terminology

Intrathecal transplantation means the infusion of the cells in the subarachnoid space in the course of lumbar puncture.


Peer-review

This is an important manuscript describing the clinical outcome of cellular therapy for spinal cord injury.


REFERENCES

1 Lee BB, Cripps RA, Fitzharris M, Wing PC. The global map for traumatic spinal cord injury epidemiology: update 2011, global incidence rate. Spinal Cord 2014; 52: 110-116 [PMID: 23439068 DOI: 10.1038/sc.2012.158]

2 National Spinal Cord Injury Statistical Center. Spinal cord injury facts and figures at a glance. J Spinal Cord Med 2014; 37: 243-244 [PMID: 24559421 DOI: 10.1179/1079026814Z.000000000260]

3 Noonan VK, Fingas M, Farry A, Baxter D, Singh A, Fehlings MG, Dvorak MF. Incidence and prevalence of spinal cord injury in Canada: a national perspective. Neuroepidemiology 2012; 38: 219-226 [PMID: 22555590 DOI: 10.1159/000336014]

4 DeVivo MJ. Causes and costs of spinal cord injury in the United States. Spinal Cord 1997; 35: 809-813 [PMID: 9429259]

5 Raisman G. Sniffing out new approaches to spinal cord repair. Nat Med 2000; 6: 382-383 [PMID: 10742141 DOI: 10.1038/74638]

6 Huang H, Chen L, Sanberg P. Cell Therapy From Bench to Bedside Translation in CNS Neurorestoratology Era. Cell Med 2010; 1: 15-46 [PMID: 21359168]

7 Lima C, Escada P, Pratas-Vital J, Branco C, Arcangeli CA, Lazzeri G, Maia CA, Capucho C, Hasse-Ferreira A, Peduzzi JD. Olfactory mucosal autografts and rehabilitation for chronic traumatic spinal cord injury. Neurorehabil Neural Repair 2010; 24: 10-22 [PMID: 19794133 DOI: 10.1177/1545968309347685]

8 Zorin VL, Cherkasov VR, Zorina AI, Deev RV. The Characteristics of World Market Cell Technologies. Kletochnaya Transplantologiya i tkanevaya engineeriya 2010; 3: 96-115 (in Russian)

9 Bryukhovetskiy AS. Transplantatsiya nervnikh kletok i tkanevaya engineeriya mozga pri nervnikh bolezniakh [Transplantation of nerve cells and tissue engineering of brain in nerve diseases]. Moscow, ZAO Neurovita, 2003: 1-400 (in Russian)

10 Bryukhovetskiy AS. Travma spinnogo mozga: kletochniye tekhnologii v lechenii i reabilitatsii [Spinal cord injury: Cellular technologies in the treatment and rehabilitation]. Moscow, Prakticheskaya meditsina, 2010: 1-341 (in Russian)

11 Belova AN. Neiroreabilitatsiya: Rukovodstvo dlya vrachei [Neurorehabilitation: A Manual for Physicians]. Moscow: Antiodor, 2000: 1-736 (in Russian)

12 Frolov AA, Bryukhovetskiy AS. Effects of hematopoietic autologous stem cell transplantation to the chronically injured human spinal cord evaluated by motor and somatosensory evoked potentials methods. Cell Transplant 2012; 21 Suppl 1: S49-S55 [PMID: 22507680 DOI: 10.3727/096368912X633761]

13 Snyder E. Neural stem cells: Developmental insights may suggest therapeutic options. Proceedings of the 7th International Congress of the Cell Transplant Society; 2004, November 17-20; Boston, MA, USA, 2004: 53


P-Reviewer: Liu L, Tanabe S S-Editor: Ji FF L-Editor: E-Editor:
Table 1 Sex, age, level of injury distribution of patients with traumatic disease of spinal cord (main group)

No. of patients

202 (1008 case histories)

Age

From 19 to 51 yr

Gender

Males - 156, females - 46

Years post injury

Less than 1 yr - 11

From 1 to 5 yr - 144

Over 5 yr - 47


Level of spinal cord injury

Cervical level - 93

Thoracic level - 98

Lumbar level - 11


Type of injury

Complete - 43

Incomplete - 159



No. of transplantations

No less than 20 HSCs and HPs transplantations

Average number of transplanted cells

5.8 ´ 106

HSCs: Hematopoietic stem cells; HPs: Hematopoietic precursors.



Table 2 Sex, age, level of injury distribution of patients with traumatic disease of spinal cord (control group)


No. of patients

20 (62 case histories)

Age

From 18 to 44 yr

Gender

Males - 13, females - 7

Years post injury

< 1 yr - 6

From 1 year to 5 - 10

Over 5 yr - 4


Level of spinal cord injury

Cervical level - 14

Thoracic level - 4

Lumbar level - 2


Type

Complete - 12

Incomplete - 8





Table 3 The characteristics and basic differences of the hematopoietic stem cells and hematopoietic precursors preparation from the preparation of hematopoietic stem cells used for bone marrow transplantation


Technique

G-CSF dose

Period of administration (d)

Stimulation regimen

Cell markers

Cryopreservant

Administration of HSCs in blood

10-20 µg/kg

6-7

1 in 24 h

CD34+, CD45+

HLA DR+, CD38+

Gp130±


10%-20% DMSO

Administration of HSC and HPs in CSF

5 µg/kg; double dose at day 4

5

2 in 24 h

CD34+, CD45-

HLA DR±, CD38±

Gp130+


5%-10% DMSO +

polyglucin


HSCs: Hematopoietic stem cells; HPs: Hematopoietic precursors; G-CSF: Granulocyte-colony stimulating factor.



Table 4 Clinical symptoms of the complications and side effects in spinal cord injury patients

Symptoms

Stages of research

Control group patients




1 stage

2 stage

3 stage




Increased spasticity

46%

49.9%

54.5%

0

Fever

15%

19%

18.8%

0

Post-puncture headache

11%

14.9%

12.2%

14.9%

High blood pressure

10%

8.1%

14.5%

0

Coordination disturbance

2.3%

1.3%

0

0

Dizziness

2%

3.4%

0

4.2%

Sleepiness

2.1%

1.7%

0

0

Emotional lability

1.6%

1.7%

0

0

Disordered consciousness

1.2%

0.8%

0.8%

0

Meningism

3.7%

2.95%

0.8%

0

Low blood pressure

1.68%

2.95%

5.9%

0

General % of the patients with complications

63.5%

72.9%

75%

19.1%



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