paraplegia: a pilot clinical study

paraplegia: a pilot clinical study

Cytotherapy (2009) Vol. 11, No. 7, 897–911 Ex vivo-expanded autologous bone marrow-derived mesenchymal stromal cells in human spinal cord injury/para...

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Cytotherapy (2009) Vol. 11, No. 7, 897–911

Ex vivo-expanded autologous bone marrow-derived mesenchymal stromal cells in human spinal cord injury/paraplegia: a pilot clinical study Rakhi Pal1*, Neelam K. Venkataramana2*, Abhilash Bansal3, Sudheer Balaraju1, Majahar Jan1, Ravi Chandra3, Ashish Dixit3, Amit Rauthan3, Uday Murgod3 and Satish Totey1 Cytotherapy Downloaded from informahealthcare.com by University of Virginia on 11/03/13 For personal use only.

1Stempeutics

Research Private Ltd, Bangalore, India, 2Advanced Neuroscience Institute,

BGS Global Hospitals, Bangalore, India, and 3Manipal Hospital, Bangalore, India Background aims

Results

Spinal cord injury (SCI) is a medically untreatable condition for

At the time of writing, three patients had completed 3 years of

which stem cells have created hope in the last few years. Earlier pre-

follow-up post-BM MSC administration, 10 patients 2 years follow-up

clinical reports have shown that transplantation of bone marrow

and 10 patients 1 year follow-up. Five patients have been lost to

(BM) mesenchymal stromal cells (MSC) in SCI-simulated models

follow-up. None of the patients have reported any adverse events

can produce encouraging results. In a clinical pilot study, we investi-

associated with BM MSC transplantation.

gated the growth kinetics of BM MSC from SCI patients, their safety and functional improvement post-transplantation.

Conclusions The results indicate that our protocol is safe with no serious adverse

Methods

events following transplantation in SCI patients. The number of

Thirty patients with clinically complete SCI at cervical or thoracic

patients recruited and the uncontrolled nature of the trial do not

levels were recruited and divided into two groups based on the dura-

permit demonstration of the effectiveness of the treatment involved.

tion of injury. Patients with  6 months of post-SCI were recruited

However, the results encourage further trials with higher doses

into group 1 and patients with  6 months of post-SCI were included

and different routes of administration in order to demonstrate the

into group 2. Autologous BM was harvested from the iliac crest of

recovery/efficacy if any, in SCI patients.

SCI patients under local anesthesia and BM MSC were isolated and expanded ex vivo. BM MSC were tested for quality control, charac-

Keywords

terized for cell surface markers and transplanted back to the patient

autologous, bone marrow, mesenchymal stromal cells, spinal cord

via lumbar puncture at a dose of 1  106 cells/kg body weight.

injury.

Introduction

seems to be the only hope for the afflicted. However, this is insufficient to help patients resume normal activities of daily living. Thus it is imperative to devise a novel therapy that will help patients regain an independent lifestyle. Stem cell therapy is promising for tissue regeneration. Bone marrow (BM) mesenchymal stromal cells (MSC) have the potential to differentiate into different lineages without being teratogenic [2,3]. Furthermore, BM MSC are easily

The incidence of traumatic spinal cord injury (SCI) in India is high, primarily affecting the younger population of the country. The most common cause of injury is falling from a height, 44.5%, followed by motor vehicle accidents, 34.78% [1]. To date the available standards of care include stabilization of the spine and administration of methyl prednisolone within 24 h of the injury. Post-stabilization, currently neurorehabilitation *Rakhi Pal and Nilam K. Venkataramana contributed equally.

Correspondence to: Dr N. K. Venkataramana, Advanced Neuroscience Institute, Vice-Chairman, BGS Global Hospital, BGS Health and Education City, #67 Uttarahalli Road, Kengeri, Bangalore-560 060, India. E-mail: [email protected] Dr Satish Totey, Stempeutics Research Private Ltd, 9th Floor, Manipal Hospital, Old Airport Road, Bangalore-560 017, India. E-mail: [email protected] © 2009 ISCT

DOI: 10.3109/14653240903253857

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obtained from adult BM and can be expanded ex vivo [4]. Extensive studies have been performed on differentiation of BM MSC into neurons with functional synapses [5–7]. Besides their differentiation, BM MSC can also secrete bioactive molecules, growth factors and cytokines, provide structural support, suppress inflammation and reduce apoptosis [8]. Autologous BM cells have been reported to be effective in the treatment of SCI [9–11], and umbilical cord blood cells have been found to be successful in SCI patients [12]. These results suggest that BM MSC have an ability to heal the injured or damaged spinal cord. In this clinical pilot study we have included data from 30 SCI patients. The study was intended to ascertain the safety and feasibility of BM MSC as a possible therapeutic strategy for SCI. We also studied the effect of SCI on BM MSC culture condition, morphology, population doubling (PD), cell-surface marker profiling and potential to differentiate into various lineages. Three years of follow-up after BM MSC transplantation are described.

Methods Patient selection A pilot clinical study was designed to determine the safety and feasibility of BM MSC. As per national guidelines, approval from an institutional ethics committee (IEC) was taken. Informed consent was obtained from every patient who participated in the study. Each patient was screened for HIV: Human Immunodeficiency Virus; HBV: Hepatitis B Virus; HCV: Hepatitis C Virus; CMV: Cytomegalovirus; and VDRL: Venereal Disease Research Laboratory, by a nationally certified testing laboratory before being included in the trial. Any deviations, drop-outs and adverse events were documented and the IEC informed. In total 30 patients were included in the study. The patients were divided into two groups: group 1 comprised 20 patients who had been injured for 1–6 months; group 2 comprised 10 patients who had been injured for more than 6 months. Criteria for patient inclusion in the study were that the patients could be of either sex but must be aged between 18 and 55 years, the level of spinal injury was between C4 and T10 level (neurologic) and the SCI was clinically complete and categorized as per the American Spinal Injury Association (ASIA) impairment scale. Before inclusion of a patient in the study, informed consent was mandatory and obtained. Patients were not included in the clinical pilot study when the size and location of SCI was difficult to determine, the

patient was having gunshot or other penetrating trauma or trans-section, the longitudinal dimension of injury was 3 cm (using magnetic resonance imaging; MRI), the patient was on mechanical ventilation because of neurologic impairment or on ventilator assistance within 24 h of surgery, there were serious pre-existing medical conditions, disease or impairment that precluded adequate neurologic examination, or previous or concomitant treatment with immune modulators or experimental drugs 60 days prior to study enrollment.

Isolation of MSC BM-derived MSC were isolated and expanded using a modification of methods reported previously [13]. Briefly, 60 mL BM were aspirated aseptically from the iliac crest of each patient under deep sedation. Henceforth all processing of samples was done inside a class 100 biosafety hood in a class B cGMP: current Good Manfacturing Practices, facility. The BM was diluted (1:1) with knockout Dulbecco’s modified Eagle’s medium (KO-DMEM; Invitrogen,Carlsbad,CA, USA; www.invitrogen.com) and centrifuged at 1800 r.p.m. for 10 min to remove anticoagulants. The supernatant was discarded and the BM washed once with culture medium. Mononuclear cells (MNC) were isolated by layering onto a lymphoprep density gradient (1:2; Axis-Shield PoC AS). The MNC present in the buffy coat were washed again with culture medium. The mononuclear fractions that also contained MSC were plated onto T-75cm2 flasks (BD Biosciences, San Jose, CA, USA; www.bdbiosciences. com) and cultured in KO-DMEM. The media were supplemented with 10% fetal bovine serum (FBS Fetal Bovine serum: certified Australian origin; Lot no: GQM0049; South Logan, Utah, USA, Hyclone), 200 mm Glutamax (Invitrogen) and Pen-Strep (Invitrogen). The non-adherent cells were removed after 48 h of culture and replenished with fresh medium. Subsequently, the medium was replenished every 48 h.

Subculturing and expansion of MSC Once the cells attained confluency, they were dissociated with 0.25% trypsin/0.53 mm ethylene diamine tetra acetic acid (EDTA; Invitrogen) and reseeded at a density of 5000 cells/cm2 in one-cell stacks (Corning Plastic ware supplied by Sigma Aldrich Chemical Private Limited, Bangalore, Karnataka, India) After 15 days in culture, the cells reached 80% confluency and were subcultured for subsequent propagation or transplantation. The cells were further up-scaled and expanded in order to provide the

MSC in human spinal cord injury

required number of cells for the patient. The PD time was also calculated between the passages.

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Quality control testing In-process test Before releasing the cells for transplantation, in-process quality testing of the cells was carried out for cell-surface marker analysis and endotoxins using the LAL test: Limulus Amebocyte Lysate test and mycoplasma using the reverse transcription–polymerase chain reaction (RT-PCR). At any step if any sample was detected as positive, it was discarded immediately and appropriately. End-product test The final cell suspension that was provided to the clinician for transplantation was tested again for karyotyping, endotoxin, mycoplasma and cell-surface markers in order to confirm the homogeneous population of BM MSC and its clinical eligibility. In this study, patients received either two or three injections 1 week apart. Therefore, cells were at passage 1 (P1; injection 1), passage 2 (P2; injection 2) or passage 3 (P3; injection 3). Before releasing the MSC for transplantation at each passage, they were subject to endproduct quality testing for immunophenotyping, mycoplasma, endotoxin and karyotyping. A certificate of analysis (COA) was prepared for each batch released. This followed the Slaper–Cortenbach criteria and fulfilled the European directive on quality and safety of tissues and cells. Quality system The Organization refers to Stempeutics Research Private Limited has a certified quality system that implements the appropriate consent-taking from patients and relatives, working instructions, maintaining batch-processing records, guidelines, training and reference manuals, reporting formats and donor records. All samples processed were identified through a laboratory identification system and accordingly cryopreserved and archived for future requirements, thereby setting national standards.

Immunophenotyping Immunophenotyping of the cultured BM MSC was performed using flow cytometry to identify the presence of specific cell-surface antigens. Briefly, BM MSC were dissociated with 0.25% trypsin–EDTA and resuspended in wash buffer at a concentration of 1  106 cells/mL. Wash buffer consisted of phosphate-buffered saline (PBS) supplemented

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with 1% (v/v) FBS and 1% (w/v) sodium azide. Cell viability was measured by flow cytometry using 7-amino actinomycin D (7-AAD; which can penetrate through the cell membrane of dead cells); 200 μL cell suspension were incubated in the dark for 30 min at 4°C with saturating concentrations of fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibodies (Ab). Appropriate isotype-matched controls were used to set the instrument parameters. After incubation, cells were washed three times with wash buffer and resuspended in 0.5 mL wash buffer for analysis. Flow cytometry was performed on a LSR-II (BD Biosciences). Cells were identified by light scatter for 10 000 gated events and analyzed using FACS DIVA software (BD Biosciences). The following markers were analyzed: CD45–FITC, CD73–PE and CD90–PE (BD Pharmingen, San Diego, CA, USA; www.bdbiosciences. com/pharmingen).

Differentiation The differentiation potential of human BM MSC into osteoblasts and adipocytes was investigated to confirm mesenchymal properties. Differentiation into the astroglial lineage was also carried out to understand the transdifferentiation potential. Osteoblast differentiation was induced by culturing human BM MSC in KO-DMEM supplemented with 10% FBS (Hyclone), 200 mm glutamax (Invitrogen), 10–8 m dexamethasone (Sigma-Aldrich), 30 μgm/mL ascorbic acid (Sigma-Aldrich) and 10 mm E -glycerophosphate (SigmaAldrich Chemical Private Limited, Bangalore, Karnataka, India) for 3 weeks. Fresh medium was replenished every 3 days. Calcium accumulation was assessed by von Kossa staining. The differentiated cells were washed with PBS and fixed with 10% formalin for 30 min. The fixed cells were incubated with 5% AgNO3 for 60 min under ultraviolet (UV) light and then treated with 2.5% sodium thiosulphate for 5 min. Images were captured using an Nikon Eclipse 90i microscope (Nikon Corporation, Towa Optics, New Delhi, India; www.nikon.com) and Image-Pro Express software (Media Cybernetics Inc., Silver Spring, MD, USA; www. mediacy.com). To induce adipogenic differentiation, human BM MSC were cultured for 21 days in KO-DMEM supplemented with 10% FBS, 200 mm glutamax, 1 μm dexamethasone, 0.5 mm isobutylmethylxanthine, 1 μg/mL insulin and 100 μm indomethacin (all Sigma-Aldrich). Inducing factors were added to the replenished medium every 3 days. Cells were

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R. Pal et al.

fixed in 10% formalin for 20 min and 200 μL Oil Red O staining solution added and incubated for 10 min at room temperature. The cells were rinsed five times with distilled water. The images were captured using an Nikon Eclipse 90i microscope (Nikon) and Image-Pro Express software (Media Cybernetics). To induce astroglial differentiation, human MSC were cultured for 2 weeks in DMEM-F12 supplemented with 30 ng/μl thyroxin (Sigma-Aldrich Chemical Private Limited, Bangalore, Karnataka, India) and N2 supplement. Inducing factors were replenished every third day (unpublished data). Immunofluorescence was done using anti-glial fibrillary acidic protein (GFAP) and anti-oligodendrocyte marker 4 (O4) Ab (R and D systems, B Biotech India Pvt Limited). Differentiated cells were fixed in 4% PFA: Para Form Aldehyde for 1 h at 4°C followed by three washes with 0.05% PBS-T: Phosphate Buffered Saline-Tween. To reduce non-specific binding, blocking was done for 30 min at room temperature with 3% bovine serum albumin (BSA). The cells were incubated in the primary Ab (GFAP/O4) overnight at 4°C followed by a 1-h incubation with secondary Ab. This was followed by DAPI staining in the dark for 30 min. Finally the slides were mounted with Vectashield solution and visualized under a microscope. Images were captured using an Nikon Eclipse 90i microscope (Nikon) and Image-Pro Express software (Media Cybernetics).

apart at a dose of 1 million cells/kg body weight at P1 (injection 1), P2 (injection 2) and P3 (injection 3).

Karyotyping

Follow-up schedule

BM MSC cultured in vitro were karyotyped before transplantation. Chromosomes were visualized using a standard G-banding procedure and more than 200 cells were analyzed per cell line and reported according to the International System for Human Cytogenetic Nomenclature.

At every follow-up, a patient was assessed clinically using the ASIA scale rating system and with the Barthel’s index (BI) for degree of independence and patient rating. Somatosensory evoked potentials (SSEP), motor-evoked potentials (MEP) and nerve conduction velocity (NCV) were recorded at the end of 1 year post-transplantation and compared with baseline readings. MRI was performed to observe structural changes, if any.

Preparing cells for transplantation Eighty per cent confluent single-cell stacks were selected for transplantation. Each single-cell stack was washed with DPBS: Dulbecco’s Phosphate Buffered Saline 0.25% trypsin– EDTA was added to harvest the cells. Culture medium was added to neutralize the action of trypsin. The cell suspension was centrifuged and the cell pellet washed four to five times with DPBS and once with saline. The entire cell pellet was resuspended in 1 mL saline, the cell number determined (1106 cells/kg of body weight) and suspended in 1 mL saline before being loaded into a syringe for transplantation. Patients received two injections or three injections 1 week

Pre-surgical procedure Each patient was prepared aseptically for intrathecal placement of stem cells. The patient was positioned in the lateral decubitus position, lying on the edge of the table in the operating theater in a knee–chest position with the neck flexed carefully. Local anesthesia was given at the site of injection.

Transplantation of MSC A 26-gauge spinal needle was inserted into the lumbar space below the L3 level. The puncture was confirmed by collecting a few drops of clear cerebrospinal fluid (CSF). The syringe was then loaded on to the needle and the cells injected very slowly at a single site. A dose of 1  106 BM MSC/kg body weight was transplanted into each patient at each time irrespective of the number of injections. The needle was held in position for 2 min and then flushed with 500 μL saline to prevent efflux and ensure that no cells were left behind in the needle or plunger.

Post-operative care The lumbar puncture site was sealed and covered with Tegaderm for 24 h. A patient was usually discharged after 24 h and follow-up visits were scheduled.

Statistical analysis All experimental data are represented as meanSD. Student’s t-test was employed to compare the PD time between group 1 and group 2 and between cervical and thoracic injuries.

Results In this study 30 patients were recruited into two groups. In group 1, 20 patients were included that had had SCI for between 1 and 6 months (six were lost to follow-up); in

MSC in human spinal cord injury

group 2, 10 patients were included that had had SCI for more than 6 months. The details of the patients recruited for this study are given in Table I.

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Growth kinetics of BM MSC from SCI patients Sixty milliliters of BM were aspirated from each patient and BM MSC were isolated and cultured. The average PD time for BM MSC from SCI patients with less than 6 months of cervical injury at P1–P2 was 24.4  0.5 h, and in the case of thoracic injury 20.79  9.9 h. There was no significant difference in PD between these groups. The PD time compared between cervical and thoracic injuries was significantly higher (P  0.05) for BM MSC derived from patients with more than 6 months of injury. The average PD time at P1–P2 for patients with more than 6 months of injury was 35.6  15.29 h for cervical injury and 57.29  28.97 h for thoracic injury (Table II). A significant difference (P  0.01) was also noted between the PD of patients with less than and more than 6 months of thoracic cord injury.

Immunophenotyping The surface-specific expression of CD markers for BM MSC (CD90 and CD73) was comparable. Both these markers were more than 80% positive (bright ve) and negative for CD45 (Figure 1 and Table III). Hence these cells were CD45− CD73  CD90 .

Differentiation BM MSC obtained from all SCI patients were subjected to in vitro differentiation into astroglial cells, adipocytes and osteoblasts. The differentiation potential of the cells derived from BM of SCI patients irrespective of group was comparable (Figure 2).

Karyotyping All the samples were processed for karyotyping before transplantation by a trained cytogeneticist. The samples showed normal karyotypes post ex vivo propagation. A representative ideogram is illustrated in Figure 3.

Clinical assessment Clinical assessment was performed on all patients using the ASIA scale. The ASIA rating scale remained unchanged pre- and post-stem cell therapy. SSEP, MEP and NCV were tested on all patients 1 day prior to stem cell transplantation

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and 1 year after stem cell transplantation had been completed. Two patients showed significant clinical and functional recovery (i.e. were able to walk with support and sit with support); the rest of the patients have shown variable patterns of recovery. Notable signs included improvement in bladder functions (three patients): two patients were completely off the catheter, i.e. able to appreciate the feeling (sensory) and void (motor) independently, while one patient showed sensory recovery. Additionally, three patients reported patchy sensory recovery that did not fit into any dermatomal pattern. However, comparing SSEP, MEP and NCV before and after stem cell therapy did not reveal any increase in conduction velocity, decrease in latency or increase in amplitude. MRI showed no change between the findings at baseline and 1 year post-stem cell transplantation. No adverse effects of transplantation were detected on the MRI of the patients 1 year after stem cell transplantation. No reported changes in cystic regions or syringomyelia, and no further external compression of the cord or formation of tumorlike masses in and around the injection site or along the cord, were visualized (Figure 4). ASIA scoring by the neurologist did not reveal any significant change or further worsening in the neurologic or functional level in any patients. Sensory and motor functioning remained the same post-stem cell transplantation. Of 30 patients treated for stem cell transplantation, only two reported neuropathic pain after the transplant. Overall there was no further deterioration among the patients. BI was performed on the patients before stem cell transplantation and 1 year after transplantation. All patients with more than 6 months of injury (cervical or thoracic) did not show any appreciable change in scores. Similarly, patients with less than 6 months of cervical injury did not demonstrate any significant improvement. However, patients with less than 6 months of thoracic-level injury showed improvement in the scores (Tables IV and V and Figure 5A,B). Thus there was an improvement in the BI score 1 year following stem cell transplantation in thoracic injury patients, while for cervical injury patients there was no appreciable change in the BI scores. As an example, patient 1 presented with thoracic-level traumatic SCI and underwent stem cell transplantation within 6 months of injury. As is evident from his BI score sheet, his activities of daily living had improved substantially post-stem cell transplantation (pre-score 5 to post-score 55).

31

28

54

56

42

30

25

38

32

24

43

28

55

43

40

21

17

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Age

1

Group 1

Case no.

M

F

M

M

M

M

M

M

M

M

M

M

M

M

M

M

M

Sex

Thoracic

Cervical

Thoracic

Cervical

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Cervical

Thoracic

Cervical

Level of injury

2005 Dec. 2005 Dec. 2005 Dec. 2006 July 2006 June 2006 Dec. 2007 Apr. 2007 Sept. 2007 Sept. 2007 Aug. 2007 Aug. 2007 Sept. 2007 May 2007 May 2007 May 2007 May 2007 May

 6 months  6 months  6 months  6 months  6 months  6 months  6 months  6 months  6 months  6 months  6 months  6 months  6 months  6 months  6 months  6 months

Date of SCT

6 months

Duration of injury

Table I. Details of patients included in the study.

1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight

Dose/no. of cells injected

3

2

2

3

3

2

3

3

3

3

2

2

2

2

2

2

2

No. of injections given

Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture

Route of administration

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Lost to follow-up Lost to follow-up













MRI

C

A

A

A

C

C

A

A

A

A

A

A

A

A

A

C

C

C

A

A

A

C

C

A

A

A

A

A

A

A

A

C

ASIA scale

902 R. Pal et al.

25

27

19

20

29

30

23

31

23

28

51

28

46

2

3

4

5

6

7

8

9

10

F

F

M

M

M

M

M

M

M

M

M

M

M

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Thoracic

Cervical

Cervical

Cervical

2006 Feb. 2006 March 2006 April 2006 April 2006 May 2007 Feb. 2007 Oct. 2006 July 2006 May

6 months 6 months 6 months 6 months 6 months 6 months 6 months 6 months 6 months

2007 May

 6 months

2005 Dec.

2007 May

 6 months

 6 months

2007 May

 6 months

1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight

1  106 cells/kg body weight 1  106 cells/kg body weight 1  106 cells/kg body weight

3

3

3

2

2

2

2

2

2

2

3

3

3

Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture Lumbar puncture

Lumbar puncture Lumbar puncture Lumbar puncture





 









 





 





 



Lost to follow-up Lost to follow-up Lost to follow-up









M, male; F, female;  indicates data present; A and C, A and C two scores as part of  the ASIA scoring system for Spinal cord injury (which ranges from A to  D).

26

1

Group 2

22

18

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C

A

A

A

A

A

A

A

A

A

A

A

A

C

A

A

A

A

A

A

A

A

A MSC in human spinal cord injury 903

R. Pal et al.

Table II. PD time in hours for SCI patients with less than 6 months and more than 6 months of injury. P3–P4 (h)

24.4  0.5 20.79  9.93

49.26  9.98 41.6  16.25

38.46  14.4 38.141  14.3

35.6  15.29 57.29  28.97

52.67  28.15 77.64  41.49

50.86  22.642 78.831  41.072

Pattern of recovery

The PD time is represented as the mean  SD for each group. The PD values between less than 6 months and more than 6 months of thoracic injury were statistically significant ( 1P  0.01) but comparable for cervical injury. Also, for more than 6 months of injury, there was a significant difference in PD time between cervical and thoracic injuries ( 2P  0.05).

There seemed to be an interesting pattern of recovery in these patients. First to recover was bladder sensation, followed by variable degrees of regulation of bladder and bowel function. Subsequent improvements were in the sensory tracts. The last to recover was motor function, with the muscles proximal to the injury first followed by the rest. It appears that the central fiber tract responded first, followed by sensory tracts, and minimal recovery was seen in the anterior horn cell function. However, these findings did not correlate with electrophysiologic testing.

Prior to stem cell transplantation, he required partial assistance for feeding himself, complete assistance for bathing, grooming and dressing, and had no control over bladder and bowel. He was even unable to sit up independently or balance himself, transfer himself from bed to wheelchair or vice versa or move with/without assistance on a flat surface or stairways. At the 1-year follow-up post-stem cell transplantation, his bladder and bowel management had improved. He could feed, bathe, groom and dress himself independently, required minimal assistance for personal hygiene and

Discussion SCI affects many people, resulting in local and distant damage to the cord followed by a range of cellular disturbances,

C

103

104

105

FITC-A

Isotype control FITC

D

CD 45-FITC 3.5%

105

E

250

104

P3

200

200

SRPL/001-CD 90

250

300

105

SRPL/001-CD 73

P3

50 103

104

105

PE-A

Isotype control PE

F

0

0 102

P3

50

100

150

Count

200

250 104

FITC-A

103

7AAD 98.3%

100 103

200

(x 1,000)

102

Control

0 102

150

SSC-A

50 105

7AAD-A

50

50 0 102

104

7AAD-A

150

Count

200 150

Count

P4

100

100 50

Count

P4

103

SRPL/001-180

300

SRPL/001-CD 45 250

SRPL/001-180

102

150

B

150

200

A

P5

100

250 200 150

P5

50

SSC-A

SRPL/001-7AAD

100

(x 1,000)

SRPL/001-AUTO

Count

6 months Cervical Thoracic

P2–P3 (h)

0

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P1–P2 (h)  6 months Cervical Thoracic

bed to chair transfers, could sit up alone and maintain sitting balance without assistance and could walk on a flat surface with an aid for more than 50 yards. This indicated that he may be able to lead an independent lifestyle, which is of paramount importance to SCI patients, both psychologically and socially. Similarly, patients 3–7 showed noticeable improvements on their BI scores, indicating that they too were able to lead an almost independent lifestyle.

100

904

102

103

104

PE-A

CD 73 PE 98.3%

105

G

102

103

104

105

PE-A

CD 90 PE 98.9%

Figure 1. A representative photomicrograph of in vitro-cultured BM MSC from an SCI patient. Morphologically there was no difference in cultured BM MSC from the SCI patient and a healthy donor (A). BM MSC harvested for transplantation showed more than 90% viability using 7-AAD (B). A representative histogram of BM MSC confirming the immunophenotypic expression of cell-surface markers showing isotype control FITC (C), CD45 () (D), isotype control PE (E), CD73–PE () (F) and CD90–PE () (G).

MSC in human spinal cord injury

Table III. Immunophenotyping of MSC isolated from SCI patients and propagated in vitro. CD45 (%)

CD73 (%)

CD90 (%)

 6 months Cervical Thoracic  6 months Cervical Thoracic

0.75  0.21 0.55  0.21

93.8  4.1 96.2  3.82

97.3  3.54 93.90  1.98

0.25  0.31 0.57  0.003

83.95  4.74 89.53  0.002

95.35  1.06 84.50  0.001

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Each CD marker is expressed as its mean values  SD for each group.

hemostatic imbalance and ionic and neurotransmitter derangement [14]. SCI patients show abnormal secretion and accumulation of neurotransmitters, resulting in excitotoxicity that leads to severe cellular damage and cell death [15]. These cellular damages and cell death lead further to secondary injury and ultimately there is formation of glial scar and cavitations that leads to regenerative failure [16]. Therefore, stem cell therapy has emerged as an intriguing and attractive possibility in the field of transplantation medicine, especially for SCI. The objective of our clinical pilot study was to evaluate the safety and efficacy of BM MSC in the treatment of SCI.

A

C

905

Our strategy was to inject BM MSC through an intrathecal route into the lumbar space for two reasons. Firstly, it is an easy, routinely practiced and non-invasive procedure. Secondly, our preliminary pre-clinical data confirmed that BM MSC injected through the intrathecal route in an SCIsimulated rat model did migrate to the site of injury and showed significant improvement in behavioral testing (data not shown). In the present study we have found that autologous BM MSC transplanted through an intrathecal route into the lumbar space is safe, feasible and beneficial. BM MSC transplantation in patients with less than 6 months of thoracic-level injury showed noticeable improvement in the patients’ daily activities and quality of life (BI), including restoration of bladder and bowel sensation. The patients were able to lead an almost independent lifestyle post-BM MSC transplantation. Neurologic assessment revealed moderate recovery in the sensory and motor levels but not sufficient to elicit a positive electrophysiologic response. These sensory responses may be because of the early recovery of central fibers that may have retained some anatomical integrity. The long tract recovery could be because of remyelination. Despite these improvements in thoracic injury patients, there was no change in MRI and electrophysiologic parameters such as SSEP, MEP and

B

D

E

Figure 2. Differentiation of human BM-derived MSC from SCI patients into osteogenic (A) and adipogenic lineages (B) using specified inducing agents (10 magnification). Transdifferentiation of human BM-derived MSC from SCI patients into astroglial lineage (C) (10  phase contrast) using specified inducing agents as evidenced by expression of O4 (D)and GFAP (E) (20  magnification).

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1

2

6

7

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19

3

4

8

14

20

9

15

10

16

21

5

22

11

17

12

18

X

Figure 3. A representative karyotype of MSC from an SCI patient following ex vivo expansion. The ideogram shows the normal karyotype of an adult female SCI patient after in vitro culturing.

NCV. In contrast, BM MSC transplantation in patients with more than 6 months of thoracic- or cervical-level injury failed to show any improvement. Failure of engraftment of

BM MSC in older patients may be because of the lack of plasticity in the cord as the injury becomes older, along with formation of thick glial scars making any significant axonal sprouting on the injury side almost impossible. We have seen that diseased conditions significantly affect the growth of MSC and proliferation rate, but it is unlikely that this difference really affects engraftment at the site of injury [17]. BM MSC injected into the subarachnoid space of the lumbar spine have been seen to migrate toward injured thoracic spinal cord tissue rather than the intact spinal cord and differentiate into glial cells [9,18,19]. These results strengthen our findings that SCI patients with less than 6 months of thoracic-level injury show remarkable improvement. This also confirms that the timing of stem cell transplantation in SCI patients is one of the most critical factors. Some animal studies have reported that early transplantation immediately after injury does not promote recovery as effectively as 7 days after injury [5]. Perhaps in chronic cases this route of transplantation may not be as effective as early injury and needs to be tackled differently. Scars that form after SCI are generally believed to be inhibitory and

Figure 4. MRI scan of an SCI patient at the time of injury (A), before stem cell transplantation as a baseline (B) and 1 year after stem cell transplantation (C). Radiologic findings suggested that, in comparison with baseline MRI, there was no significant change in imaging features. The myelomalacia and cystic change in the cord at C5 and C6 levels were unchanged. No spinal canal stenosis was seen. The rest of the spine and cord were unremarkable. The white arrow marks the site of injury.

Bathing 0  dependent 5  independent (or in shower) Grooming 0  needs help with personal care 5  independent face/hair/teeth/shaving (implements provided) Dressing 0  dependent 5  needs help but can do about half unaided 10  independent (including buttons, zips, laces, etc.) Bowels 0  incontinent (or needs to be given enemas) 5  occasional accident 10  continent Bladder 0  incontinent, or catheterized and unable to manage alone 5  occasional accident 10  continent Toilet use 0  dependent 5  needs some help, but can do something alone 10  independent (on and off, dressing, wiping) Transfers (bed to chair and back) 0  unable, no sitting balance

2

8

7

6

5

4

3

Feeding 0  unable 5  needs help cutting, spreading butter, etc., or requires modified diet 10  independent

1

Activity score

BI

0

0

0

0

0

0

0

5

5

0

0

5

5

5

10

Patient 1

0

0

0

0

0

0

5

5

0

0

0

0

0

10

Patient 4

0

0

0

0

0

0

5

5

5

0

0

5

5

10

10

Patient 5

Table IV. BI scores of patientsa with less than 6 months of injury at the time of transplantation.

0

0

0

0

5

0

0

5

5

0

0

10

5

5

10

Patient 6

0

5

0

0

0

0

0

10

5

5

0

0

5

5

10

Patient 7

Cytotherapy Downloaded from informahealthcare.com by University of Virginia on 11/03/13 For personal use only.

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Patient 8

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Patient 9

0

0

0

0

0

0

0

(Continued)

0

0

0

0

0

0

0

Patient 10

MSC in human spinal cord injury 907

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0 0 0 0 5 0

0 0 0 0 0 0

0 0 0 0 0 0

50 15

Thoracic Cervical

Post Stem cell Transplantation

Barthel's Index Score-Thoracic level < 6 months

100 90 80 70 60 50 40 30 20 10 0

Thoracic

Pre Stem cell Transplantation

Post Stem cell Transplantation

Figure 5. BI scores of thoracic and cervical SCI patients with less

40

0

0

10 0 0

0

5 10 5

than 6 months of injury (A) and more than 6 months of thoraciclevel injury (B) before and after stem cell transplantation. (Patients

10

0

0

Patient 6

Barthel's Index Score < 6 months of Injury

Pre Stem cell Transplantation

B

Patient 7

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Patient 8

Patient 9

Patient 10

A 100 90 80 70 60 50 40 30 20 10 0

45 10 1–7 were thoracic injury patients and patients 8–10 were cervical injury patients.

55 5

10 aPatients

10

9

0 on the BI scale and hence are not shown on the graph).

35

0 0

15 0

0

0

0

0

10

0

5 0

5 10 5 10

10  minor help (verbal or physical) 15  independent Mobility (on level surfaces) 0  immobile or  50 yards 5 wheelchair independent, including corners, 50 yards 10  walks with help of one person (verbal or physical)  50 yards 15  independent (but may use any aid, e.g. stick)  50 yards Stairs 0  unable 5  needs help (verbal, physical, carrying aid) 10  independent Total (0–100)

5  major help (one or two people, physical), can sit

BI Activity score

Table IV. (Continued)

Patient 1

Patient 4

Patient 5

with cervical-level injuries for more than 6 months had a score of

prevent the integration of transplanted stem cells. Recently, transplantation of stem cells by laminectomy in combination with intravenous infusion in chronic SCI cases showed remarkable recovery [20]. Because of the small patient population in this study, it is difficult to prove and correlate that BM MSC injected via lumbar puncture undeniably migrate to thoracic spinal cord tissue and hence promote recovery. Treatment of SCI by cell therapy revolves around the replacement of multiple cell types and hence the most important aspect is to choose the appropriate kind of stem cells at the right stage of development. Randomized and non-randomized clinical trials using different kinds of adult stem cells have shown variable success, therapeutic potential and safety in SCI patients [20–26]. Recently, it was demonstrated that co-transplantation of two different populations of stem cells is beneficial for SCI. Perhaps some synergistic cross-talk effect of one cell type to others is possible [27]. In the present study autologous BM MSC were used for the treatment of SCI because we have established that BM MSC have the capability to differentiate into neuronal cells and astrocytes and can migrate to the site of injury. In vitro studies conducted so far have shown that BM MSC possess an intrinsic capacity to differentiate into functional oligodendrocytes and neurons following

8

7

6

5

4

3

2

1

Feeding 0  unable 5  needs help cutting, spreading butter, etc., or requires modified diet 10  independent Bathing 0  dependent 5  independent (or in shower) Grooming 0  needs help with personal care 5  independent face/hair/teeth/shaving (implements provided) Dressing 0  dependent 5  needs help but can do about half unaided 10  independent (including buttons, zips, laces, etc.) Bowels 0  incontinent (or needs to be given enemas) 5  occasional accident 10  continent Bladder 0  incontinent, or catheterized and unable to manage alone 5  occasional accident 10  continent Toilet use 0  dependent 5  needs some help, but can do something alone 10  independent (on and off, dressing, wiping) Transfers (bed to chair and back) 0  unable, no sitting balance 5  major help (one or two people, physical), can sit

Activity score

BI

10

10

10

10

0

5

10

10

10

10

10

0

5

10

Patient 17

10

0

0

5

0

5

10

10

0

0

5

0

5

10

Patient 18

Table V. BI scores of patients1 with more than 6 months of injury at the time of transplantation.

10

0

0

10

0

5

10

10

5

0

10

0

5

10

Patient 19

10

0

0

5

0

5

10

10

0

0

5

0

5

10

Patient 20

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0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Patient 21

5

0

0

0

5

5

0

0

10

0

5

10

(Continued)

10

0

5

10

Patient 22

MSC in human spinal cord injury 909

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40 35 0 30 70 45

0 0 0

60

15

17–22 were thoracic injury patients, except for patient 21 who was a cervical injury patient.

45 80 80

0 0 0

15  independent (but may use any aid, e.g. stick)  50 yards Stairs 0  unable 5  needs help (verbal, physical, carrying aid) 10  independent Total (0–100) 10

Declaration of Interest: The authors report no conflicts of interest and are responsible for the content and writing of the paper.

1Patients

10 10

10 0 0 Mobility (on level surfaces) 0  immobile or  50 yards 5  wheelchair independent, including corners,  50 yards 10  walks with help of one person (verbal or physical)  50 yards 9

0

0 0

0 0 0 15 15 15 15 15 15 10  minor help (verbal or physical) 15  independent

demyelinating lesions, thus demonstrating the ability of adult BM progenitors to generate self-renewing, functional stem cells, including cholinergic phenotypes that are critical for SCI [28–35]. BM MSC also stimulate glial cells to produce neuroregulatory molecules, such as brain-derived neurotrophic factors (BDNF) and nerve growth factor ( -NGF) [36], and promote axonal regeneration by secreting extracellular matrix [37] through release of soluble mediators and cytokines that elicit the observed biologic response [24]. In conclusion, we have shown that BM MSC transplantation is safe; none of the patients showed any adverse reaction and no further deterioration was noted in any of the patients. Despite the safety factor established with this pilot study and the remarkable improvement in BI in a few patients, these results need to be corroborated with a double-blind placebo-controlled study with a larger number of patients.

30

0

5 0

Patient 21 Patient 20 Patient 18 Patient 17 Activity score

BI

Table V. (Continued)

Patient 19

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0

5

Patient 22

910

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