Source(s) of support: Global Medical Education & Research Centre, Hyderabad.

Introduction

Human mesenchymal stem cells (hMSCs) derived from adult bone marrow represent a potentially useful source of cells for cell repl acement therapy after nervous tissue damage. They are expanded in culture and reintroduced into patients as autografts/allografts. This is a preliminary report on early successful results obtained during treatment of five patients with chronic spinal cor d injury (SCI). In the last decade, adult stem cells have preceded over investigational embryonic stem cell therapy due to their plasticity and possible differentiation into numerous cell types, including neural cells [₁₂₃₄₅]. The two most important co mmon findings are that the mesenchymal stem cells demonstrate significant promise in tissue regeneration and immune modulation. The limited therapeutic option in the management of severe neurological deficits with the resulting permanent invalidity require s new therapeutic approaches. Autologous bone marrow derived stem cells are ideal candidates for treating SCI in emerging clinical studies, because there are no ethical obstacles to their use, and the health risk for patients with SCI is rather small. Nume rous electrophysiological and histological preclinical studies have revealed that the implantation of stem cells from bone marrow or umbilical cord blood in animal models of SCI results in spared white matter and gray matter, neuronal and axonal regeneration, astrocyte proliferation, myelination, neovascularization, and functional improvement [_1678910]. The reports on successful animal model experiments derive the scope of translation to human clinical trials. In this clinical trial, autologous cultured bone marrow derived mesenchymal stem cells were infused at the site of injury; the results of the 12 -month clinical follow -up are described.

Material and Methods

Ethical approval for this study was obtained from the Institutional Review Board

(IRB) and Institutional Ethics Committee (IEC). Patients with traumatic SCI (Total Transectional Injury) with complete motor and sensory disorder were enrolled for the study, and high-risk informed consent was obtained from each patient. The selection and exclusion criteria is summarised in Table.1

Figure 1

Table 1: Inclusion and exclusion criteria for the selection of patients.

Bone marrow aspirates

MSC Cultures

Stem Cell therapy

Results

MSC Cultures

Human bone marrow -derived MSCs were successfully culture -expanded from all donors. As reported by others, the cultures underwent an initial lag phase of about 5 days. A morphologically homogeneous population of fibroblast -like cells with more than 90% confluence was seen at a median of 13 days. After the first passage, the cells grew exponentially, requiring weekly passages (Fig.1). The cultures were contamination free.

Figure 2

Figure 1:MSC culture expansion on the day 2,4,8 and 14.Confluent cultures were obtained between 13 -15 day.

Phenotypic characterization

The cultured mesenchymal cells, iso lated from second passage, were found to comprise a single phenotypic population for surface -expressed antigens by immunohistochemical analysis. MSCs were uniformly positive by IHC for Vimentin (Fig 2) and negative for CD34 (lipopolysaccharide receptor), CD 45 leukocyte common antigen), HLA -DR suggestive of non -contamination with haematopoietic lineage.

Figure 3

Figure 2:The stromal cells stained with H&E, Giemsa and Vimentin.They demonstrated positivity to Vimentin and negative to CD34&CD45;.

The cells were immunophenotyped for the presence of CD106, a marker for mesenchymal stem cells by Flowcytometry. Briefly, the cells were incubated with 20μl of anti CD106 monoclonal antibody (BD Biosciences, USA) for 15 minutes at 4o C. The cells were washed off excess antibody and suspended in 250 l of sheath fluid and the FACS calibur was run to demonstrate the positivity of CD106 marker along with IgG isotype control.

Figure 4

Figure 3:CD106 positivity of the bone marrow stromal cells. Green histogram represents isotype control and purple CD106.

Clinical details of the patients studied are summarised in Table 2.

Figure 5

Table 2

Volume of Bone marrow aspirated, mononuclear count, viability and CD106 positive count with the pre and po st ASIA score are summarised in Table 3.

Figure 6

Table 3

After MSC cell infusion, there were no reports on the occurrence of pain, neuropathic irritation during the procedure and in the immediate post -procedure in any of the patients. Continuous patient monitoring was car ried out during the first 24 hours post - procedure in an intensive care unit, with no alterations of these parameters.

The ASIA score and nerve conduction study reports are summarized in fig 4, fig 5.

Figure 7

Figure 4:The two representative cases with improved motor function have had mononuclear therapy elsewhere with a window period of 6 weeks prior to MSC therapy.

Figure 8

Figure 5:The nerve conduction study carried but before and 3 months post stem cell therapy.

Discussion

The treatment approaches in SCI h ave not been successful with single strategy alone.

A number of studies report a poor prognosis; only about 5 -6% of patients with complete SCI (ASIA grade A) improve after 1 year. Spinal cord injury represents a complex event, and therefore effective thera peutic strategies will consist of a series of interventions. First, secondary tissue loss should be prevented through early neuroprotective, anti -inflammatory, or immunomodulatory interventions. Subsequently, strategies to promote the regrowth of axons and the restoration of function will involve multiple approaches: reducing scar formation, overcoming additional inhibitory molecules, stimulating damaged nerve cells to regenerate axons, facilitating axonal growth across the site of injury, and enabling the formation of new connections.

Postnatal bone marrow has traditionally been seen as an organ composed of two main systems rooted in distinct lineages: the haematopoietic tissue proper and the associated supporting stroma – marrow stromal cells. Unlike haem atopoietic stem cells, whose role in the treatment of hematopoietic diseases has been known for a long time. MSCs were originally examined only because of their critical role in the formation of the hematopoietic microenvironment. Besides, the neuronal pro tective role of MSCs, hematopoetic stem cells also fosters neuroprotection. There is little doubt that bone marrow stem cells represent one of the most accessible sources of stem cells for therapeutic use. The ease with which they are harvested and the simplicity of the procedures required for their extensive growth in culture, together with easy expansion in vitro, may make them ideal candidates.

In our present study, the initial results are encouraging in that the two patients have demonstrated functiona l recovery in less than 2 months post transplantation. These two cases prior to admission at our hospital have had bone marrow derived mononuclear cell therapy elsewhere. The lag phase for MSC therapy included an average period of 6 weeks. The dualistic th erapy of BMMNC and BMSC had significant benefit in the functional recovery as assessed by the masked scoring of ASIA and nerve conduction reports. The other group of patients persisted on the same ASIA score. The synergistic effect of mononuclear cells and mesenchymal stem cells provided with BDNF niche may prove to be a new approach to the treatment of SCI. The study presented by Moviglia et al., demonstrates the regeneration phenomenon based on the controlled inflammatory activity at the injured site [₁₁] . Similar reports from Yaniv et al substantiates the contention that the local immune response plays a crucial role in recruitment of adult neural progenitor cells to the lesion site, and suggest that similar immunological manipulations might also serve a s a therapeutic means for controlled migration of stem/progenitor cells to other acutely injured CNS sites[₁₂]. The proposed study owing to no adverse events envisages a safe and effective transplant procedure. The method of administration at the site of l esion by various researchers documented varied success rates. Satake et al transplanted the MSCs into the subarachnoid space of the lumbar spine and reported the migration to the injured thoracic spinal cord tissue [₁₃]. They have also reported the differentiation of MSC’s to Nestin positive, immature neurons or glial cells in the rat model experimentation. The site -specific stem cell transplantation may be more advantageous for rapid myelination of axons with greater viability and number of cells available for the tissue repair and regeneration. The results of the present study concur with other reports for the therapeutic time window of 6 months to 12 months for the SCI therapy. MSC’s transplantation within 12 months period has proved more effective as aga inst greater time window with least possible success rates. Furthermore, there are several issues that need to be addressed, such as implanted cell – host interaction in terms of immune response to transplanted cells, homing mechanism and differentiation t o desired cell phenotype even before stem cell therapy being a routine clinical intervention. The dualistic therapy of olfactory ensheathing Glia (OEG) and MSC’s are reported for accelerated myelination and functional recovery. The clinical study has been initiated at our centre is under evaluation.

Correspondence to

Dr. Lakshmi Kiran Chelluri., PhD Global Hospitals, Hyderabad –500 004 (A.P), India E-mail: apparusu@hotmail.com ; lkiran@globalhospital.net Ph:0091-40-30244501 Mob: 9848112342 Fax: 0091-40-2324 4455