Intramuscular Injection of Bone Marrow Stem Cells in Amyotrophic Lateral Sclerosis Patients: A Randomized Clinical Trial

Abstract

Background: Preclinical studies suggest that stem cells may be a valuable therapeutic tool in amyotrophic lateral sclerosis (ALS). As it has been demonstrated that there are molecular changes at the end-plate during the early stages of motorneuron degeneration in animal models, we hypothesize that the local effect of this stem cell delivery method could slow the progressive loss of motor units (MUs) in ALS patients.

Methods: We designed a Phase I/II clinical trial to study the safety of intramuscularly implanting autologous bone marrow mononuclear cells (BMMCs), including stem cells, in ALS patients and their possible effects on the MU of the tibialis anterior (TA) muscle. Twenty-two patients participated in a randomized, double-blind, placebo-controlled trial that consisted of a baseline visit followed by one intramuscular injection of BMNCs, follow-up visits at 30, 90, 180, and 360 days, and an additional year of clinical follow-up. In each patient, one TA muscle was injected with a single dose of BMMCs while the contralateral muscle was given a placebo; the sides were selected randomly. All visits included a complete EMG study of both TA muscles.

Results: Our results show that (1) the intramuscular injection of BMMCs is a safe procedure; (2) ALS patients show heterogeneities in the degree of TA injury; (3) a comparison of placebo-injected muscles with BMMC-injected muscles showed significant differences in only one parameter, the D50 index used to quantify the Compound Muscle Action Potential (CMAP) scan curve. This parameter was higher in the BMMC-injected TA muscle at both 90 days (placebo side: 29.55 ± 2.89, n = 20; experimental side: 39.25 ± 3.21, n = 20; p < 0.01) and 180 days (placebo side: 29.35 ± 3.29, n = 17; experimental side: 41.24 ± 3.34, n = 17; p < 0.01).

Conclusion: This procedure had no effect on the TA muscle MU properties, with the exception of the D50 index. Finding differences in just this index supports the fact that it may be much more sensitive than other electrophysiological parameters when studying treatment effects. Given the low number of patients and their heterogeneity, these results justify exploring the efficacy of this procedure in further patients and other muscles, through Phase II trials.

Clinical trial registration: www.clinicaltrials.gov (identifier NCT02286011); EudraCT number 2011-004801-25.

Keywords: ALS; CMAP scan; D50; MUNE; MUNIX; fiber density; motor units.

Copyright © 2020 Geijo-Barrientos, Pastore-Olmedo, De Mingo, Blanquer, Espuch, Iniesta, Iniesta, García-Hernández, Martín-Estefanía, Barrios, Moraleda and Martínez.

Figures

FIGURE 1

Determining the recording and injection point. CMAP recordings. To determine the recording and injection point for each TA muscle, we mapped the CMAP with five surface recording electrodes placed on the surface of the muscle. We always used the same type of surface electrodes (Ambu Neuroline 700 Single Patient Surface Adhesive Electrodes; Ref. 70010-k/C/12). The left panel illustrates the position of the five active electrodes, as well as the reference electrode, on the right leg of one patient. The five recording electrodes were placed using the same bone references in all patients: 1 cm lateral to the tibial crest on the line linking the tibial tuberosity and the midpoint of the bi-malleolar line (blue lines in the left panel). The distance between the tibial crest and the most proximal electrode, as well as the distance between the successive four electrodes was 10% of the distance between the tibial tuberosity and the bi-malleolar line. A bipolar stimulation skin electrode (Medelec; Ref. FT 296.180 TP, not shown in the picture) was placed on the fibular neck. The ground electrode (Ambu Neuroline Ground Neurology Surface, not shown in the picture) was placed between the stimulus electrode and the uppermost electrode. The right panel shows the CMAP recordings elicited by supramaximal stimulation and recorded simultaneously by the five recording electrodes (3–3000 Hz bandpass filter, without a 50-Hz notch filter). Note the progressive increment of the distal latency of CMAP and different amplitudes in each channel. The recording point was determined as the position of the electrode where the maximum amplitude of CMAP was recorded with minimal contamination from other muscles (electrode no. 2 in this case). This position was marked with indelible ink so it could be used as a reference for the intramuscular injections and recordings in successive visits. Calibration: 50 ms/5 mV dot intervals. The CMAP was recorded using standard motor nerve conduction techniques at the basal and follow-up visits. After a supramaximal superficial stimulation of the peroneal nerve in the fibula neck, three or four artifact-free recordings were superimposed to obtain the best response.

FIGURE 2

CMAP scan stimulus–amplitude recordings and D50 index. (A) The CMAP scan stimulus–amplitude curve shows the relationship between CMAP size and stimulus intensity, plotted across the range of stimulus intensities from the threshold (S0) to the supramaximal intensity (S100). Each plot shows 500 consecutive values of the CMAP area (mVms) plotted against their stimulus intensities (mA) (0.1 ms duration; delivered at 2 Hz frequency), equally distributed between S0 and S100 in a downward direction. Recordings were made on the control (black symbols) and experimental (red symbols) TA muscles of patient #17 at the + 30-day visit. The comparison of the two curves shows that there are many more CMAP scan discontinuities (marked by horizontal arrows) in the control TA muscle, i.e., more numerous, larger steps during the CMAP scan of the sequential recruiting of the whole muscle fiber population. These discontinuities are caused by a decrease in MU number and an increase in MU size, and may be quantified by the D50 index. The inset shows the shape of the maximum CMAP recorded on both sides. (B) Calculation of the D50 parameter. Each line (control side black, experimental side red) shows the cumulative sum of the increments in the CMAP area between successive responses after ranking these increments from largest to smallest; the D50 parameter is the number of increments necessary to reach 50% of the total sum. For the experimental side (left leg), D50 = 50; for the control side (right leg), D50 = 25. This increase in D50 index on the experimental side with respect to the control side was associated with an increase in axon excitability (measured as the maximum slope of the CMAP scan curve: 0.24 mV/mA on the experimental side and 0.07 mV/mA on the control side).

FIGURE 3

Flowchart of the study. It was impossible to complete the EMG study in some patients, or some data could not be recovered: for this reason, the number of cases shown in the figures may not be the same as the number of patients examined at each visit.

FIGURE 4

Muscle strength and electrophysiological parameters measured in control and experimental TA muscles. Values of MS (A), CMAP peak amplitude (B), D50 (C), FD (D), MUNIX (E), MUSIX (F), statistical MUNE (G), and SMUP (H) measured in the TA muscles of all patients studied. Asterisks in panels (A) and (D) show the presence of statistically significant differences between the basal visit and the 180- or 360-day visit on the control (black asterisks) or on the experimental (red asterisks) side. Comparisons with the Wilcoxon signed rank test for paired samples. Downward arrows (↓) in panel (C) show differences between the control and experimental sides at the 90- and 180-day follow-up visits (Wilcoxon signed rank test for paired samples). In panels (A), (C), and (D): single symbol: p < 0.05, two symbols: p < 0.01, three symbols: p < 0.001. Numbers of measurements for each parameter in the successive visits are as follows: MS (control and experimental sides): 20, 18, 19, 17, 12; CMAP control side: 19, 20, 20, 17, 12 and experimental side: 20, 20, 20, 17, 12; D50 (control and experimental sides): 22, 21, 20, 17, 12; FD (control and experimental sides): 22, 21, 20, 15, 12; MUNIX (control and experimental sides): 19, 19, 19, 16, 11; MUSIX (control and experimental sides): 20, 20, 20, 17, 12; MUNE control side: 19, 19, 17, 12, 8, and experimental side: 15, 17, 17, 12, 9; SMUP control side: 19, 17, 17, 9, 8, and experimental side: 17, 16, 16, 12, 9.

FIGURE 5

Heterogeneity of TA muscles in ALS patients. Values of CMAP peak amplitude (A) and MUNIX (B) measured on the control side for all patients studied. Blue lines represent patients who completed the study from the basal to the 360-day visit; either the remaining patients did not finish the study (due to death or abandonment) or the data from a visit could not be recovered. The red arrowheads point to the time of the five visits (basal, 30, 90, 180, and 360 days). (C) Correlation between the slope of the CMAP peak amplitude measured on the control side in the nine patients that completed the study and the CMAP peak amplitude (left) or MUNIX (right) measured on the control side at the basal visit. The slope of the peak CMAP amplitude was taken from the linear regression of the CMAP values measured on the control side at all five visits. (D) Dendrogram produced by hierarchical cluster analysis using CMAP, MUNIX, D50, and FD at the basal visit and the slope of the CMAP measured on the control side. (E) Mean ± SEM of the CMAP peak amplitude and MUNIX values of patients #10, #12, #16, and #22 (filled symbols) and patients #4, #13, #14, #18, and #20 (open symbols).