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Intravenous Application of Allogenic Peripheral Blood-Derived Mesenchymal Stem Cells: A Safety Assessment in 291 Equine Recipients Sarah Broeckx1,#, Bizunesh M Borena2,3,#, Marieke Zimmerman4, Tom Mariën4, Bert Seys5, Marc Suls5, Luc Duchateau2 and Jan H. Spaas1,* 1
Global Stem cell Technology (GST), Geeneindestraat 1, B-3560 Meldert-Lummen, Belgium; 2Department of Comparative Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium; 3Department of Veterinary Laboratory, College of Agriculture and Veterinary Science, Ambo University, Ethiopia; 4Equitom Equine Hospital, Venusbergstraat 1, B-3560 Meldert-Lummen, Belgium; 5Equine Veterinary Practice Dr Suls, Kazernelaan 144, NL-6006 SP Weert, The Netherlands Abstract: It has been reported that mesenchymal stem cells (MSCs) have homing capacities and immunomodulating effects after an intravenous injection. However, transplanting MSCs in murine tail veins can result in pulmonary reactions and even death of the animals. Unfortunately, only a few intravenous MSC transplantations have been reported in large animal species and these were performed in a limited number of individuals. To assess the safety of MSC transplantations, a large study on 291 recipient horses is reported here. MSCs were isolated from the peripheral blood (PB) of a 4-year-old and 6-year-old donor horse after having tested their PB for a wide range of transmittable diseases. The MSC samples from both donor horses were characterized and resuspended in 1ml of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Dimethyl Sulfoxide (DMSO). After hand-thawing in the field, 291 horses with ages ranging from 3-months to 33-years were directly injected into their jugular vein. 281 horses (97%) received a single injection of a physiological dose of 0.2 x106 MSCs, 5 horses (1.7%) were re-injected after approximately 6 weeks (using the same dose and donor cells) and a single superphysiological dose of 106 MSCs was administered to 5 horses as well. In total, 176 recipients were injected with MSCs from the 4-year-old donor and 115 recipients received MSCs from the 6-year-old donor. From all the injected horses (n=291) no acute clinical adverse effects were noticed. Apart from one horse that died of colic 7 months after the treatment, no deaths were registered and all the horses were monitored for 1 year after the injection. In conclusion, no adverse effects were noticed in 291 recipients after an intravenous injection of allogenic PBderived MSCs. Nevertheless, further research is warranted in order to verify the immunogenic properties of these cells after allogenic transplantation into various (patho)physiological sites.
Keywords: MSCs, peripheral blood, horse. INTRODUCTION The allogenic intravenous application of human mesenchymal stem cells (MSCs) has been previously reported to restore the hearing in guinea pigs [1] and to prevent corneal graft transplant rejections in mice [2]. Intravenous infusion of allogenic MSCs also clinically improved chronic spinal cord injury in rats [3] and enhanced the outcome in experimental ischemic stroke in rats [4]. Moreover, autologous MSCs have the capacity to home throughout the vasculatory system towards an induced fracture site and enhance the healing process [5]. An intravenous injection of a mixture of autologous MSCs and hematopoietic stem cells has been reported to improve multiple sclerosis (MS) symptoms in humans [6]. In addition, therapy-resistant graft-versushost diseases have been successfully treated in human patients using allogenic MSCs [7-9]. In horses, an intravenous *Address correspondence to this author at the Global Stem cell Technology (GST), Geeneindestraat 1, B-3560 Meldert-Lummen, Belgium; Tel: +32 13 55 61 06; Fax: +32 13 55 61 07; Email:
[email protected] #Contributed equally first author 1574-888X/14 $58.00+.00
injection with autologous stem cells improved ophthalmological diseases [10] and could be applied safely in combination with a local stem cell treatment for dermatological [11] and tendon pathologies [12]. To date, most intravenous applications report the use of autologous MSCs. However, harvesting MSCs of the patient itself is not always feasible under field practice. The subsequent cell isolation may then take so long that it compromises the treatment. Also the quality of the MSCs should be taken into account, because most of the individuals that require such autologous treatment are in fact elderly patients and it has been reported that they yield less potent stem cells [13]. Therefore, each MSC isolate should be characterized before clinical application, which would be costly, timeconsuming and not always feasible. In view of the difficulties with autologous transplantation, research into allogenic therapies is being performed. In this regard, a double blinded placebo-controlled study recently demonstrated significant changes in several hematological parameters after an intravenous injection of allogenic equine peripheral blood (PB)-derived MSCs. Indeed, the safe © 2014 Bentham Science Publishers
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use of allogenic multipotent MSCs has been described for the treatment of different pathologies in multiple independent human and equine case reports [7, 14-18]. On the other hand, it should be mentioned that severe pulmonary reactions and even death have been previously reported in mice after intravenous human [19] and murine [20] MSC transplantations. Also vomiting and respiratory distress have been described after allogenic intravenous MSC infusion in cats [21]. A clinical evaluation in a large cohort of horses would therefore be advisable in order to assess the safety of a new possible human treatment in a large animal model. Of course, the equine animal model also directly benefits veterinary patients. MATERIALS AND METHODS Isolation of Mesenchymal Stem Cells (MSCs) As approved by the Ethical Committee of Global Stem cell Technology (EC_2012_001), 50 ml of blood was collected in sterile EDTA tubes from the vena jugularis of a 4year-old and 6-year-old donor gelding for mesenchymal stem cell (MSC) isolation. At the same time serum was collected and sent to Böse laboratory (Harsum, Germany) for testing on different transmittable diseases as previously reported [15]. Then, the blood was centrifuged at 1000g for 20 minutes and the buffy coat was collected and diluted 1:2 in phosphate buffered saline (PBS) 1x. Afterwards, this suspension was gently layered on an equal amount of Percoll® density gradient (GE Healthcare). The subsequent centrifugation steps were performed as previously described [15]. After that, 20x106 peripheral blood mononuclear cells (PBMCs) were seeded per T75 flask in 3 flasks and cultured in culture medium, as previously described [22]. The medium was refreshed twice a week and the cells were maintained at 37°C and 5% CO2 . At 60% confluency the cells were trypsinized
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with 0.25% trypsin-EDTA and subcultured until passage 5 in expansion medium, consisting of low glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco), 20% fetal bovine serum (Gibco) and 1% antibiotics – antimycotics (Sigma). The isolated cells were characterized as previously described [22, 23]. At passages 3-5, MSCs were trypsinized and resuspended in DMEM low glucose with 10% of Dimethyl Sulfoxide (DMSO, Sigma). The MSCs were prepared and frozen for intravenous application in 1ml per 1.4ml cryotube containing 0.2 x106 MSCs as a physiological dose (Veno-Cell®) or 106 MSCs as superphysiological dose. The 1.4ml innertubes were placed in 3.5ml outertubes and stored in a temperature-controlled ultralow temperature freezer (80°C). Samples were shipped on dry-ice before clinical application. Thawing and Injecting Mesenchymal Stem Cells (MSCs) A total of 291 acceptor horses with ages ranging from 3months to 33-years old received a single intravenous injection with 1ml of allogenic PB-derived MSCs from a 4-yearold (n=176) or 6-year-old (n=115) donor horse (Fig. 1). Five horses received a repeated injection of 0.2 x106 MSCs 6 weeks later and 5 other horses received a superphysiological dose of 106 MSCs (Fig. 1). The Ethical Committee of Global Stem cell Technology approved this experiment (EC_2012_001; EC_2013_002). Under field circumstances the thawing process is not always easy. Since it has been previously reported [24, 25] that hand thawing had similar viability percentages than temperature-controlled thawing, the veterinarians were asked to thaw the MSCs in the palm of their hand. After thawing, the entire 1ml sample was aspirated in a 5ml syringe using a 21 Gauge (G) needle measuring 40mm in length without performing any cell washing steps. Subsequently the vena jugularis was
Fig. (1). Mesenchymal stem cells (MSCs) were cultured from passage (P)1 to P5 for clinical application and characterized at P2. Donor 1 (4years-old) MSCs were intravenously injected into 176 recipients using 0.2 x106 cells (). Donor 2 (6-years-old) MSCs were transplanted in 115 recipients, using a single injection of 0.2 x106 cells (1x) in 105 horses, 2 injections of 0.2 x106 cells (2x) in 5 horses or a single injection of 106 cells (1x) in 5 horses.
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used to slowly (approximately 30 seconds) administer the MSCs into the circulation after aspirating 2-3ml of blood into the cell containing syringe. Follow Up of the Acceptor Horses The horses were closely monitored for 10 days by clinically examining them on a daily basis (breathing, temperature, pulse, lymph nodes, mucosae). Particular attention was directed to any possible adverse effect or hypersensitivity reaction, which would be noticeable in the form of wheal formation (physical reaction), sweating, increased respiration rate or fever. At 6 months and 1 year after the injection, the treating veterinarians were asked to reassess the horse as mentioned above. RESULTS Isolation of Mesenchymal Stem Cells (MSCs) Approximately 17 days after peripheral blood (PB) sampling, the first spindle shaped cells were noticed in the culture flasks and at 21 days after isolation the cells were trypsinized at approximately 60% confluency. The characterization experiments were performed on the mesenchymal stem cell (MSC) isolates from both horses at initial passages, as previously described [22, 23]. Briefly, the cells were positive for CD29, CD44, CD90, CD105 and Ki67, low in major histocompatibility complex (MHC) type I and negative for MHC II and p63. Moreover, the formation of adipocytes,
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osteoblasts and chondroblasts confirmed their trilineage differentiation potential. In addition, population doubling time (PDT) calculations demonstrated a normal self-renewal capacity during the entire cultivation period. This revealed that the isolated cells fulfilled all the requirements to be typed as MSCs according to the proposed guidelines by Dominici in 2006 [26]. For the present study, MSCs from passage (P) 3 to 5 were used (Fig. 1). Thawing and Injecting Mesenchymal Stem Cells (MSCs) It has been reported that frozen equine PB-derived MSCs possess the same stem cell characteristics as freshly isolated cells [27]. Moreover, fresh equine PB-derived MSCs dramatically decline in cell number after 12 hours of transport and have a higher risk to become senescent after 24 hours of transport [28]. Therefore, the use of frozen samples and transport in dry ice is justified to add to the product shelf life (Fig. 2A). Thawing in the field was performed by keeping the inner tube in the hand palm (Fig. 2B). In order to avoid an excessive decrease in cell viability due to the cytotoxic effect of Dimethyl Sulfoxide (DMSO, 100l/1ml), the MSCs were injected into the vena jugularis within 10 minutes after thawing (Fig. 2C & 2D). One Year Follow-Up of the Acceptor Horses After the allogenic MSC treatment, the horses were closely monitored for 10 days and no acute adverse clinical effects could be noticed by clinical examination either by the
Fig. (2). Samples were transported on dry ice (A) and immediately before hand-thawing the inner tubes were removed from the outer tube (B). A 21 Gauge needle measuring 40mm in length was used to intravenously inject 1ml of MSCs into the vena jugularis (C). At least 2ml of blood from was aspirated into the cell containing syringe (D). Subsequently the MSCs were slowly administered into the circulation.
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attending veterinarian or by the caretakers. Indeed, no wheal formation (physical reaction), sweating, increased respiration or fever was observed. Moreover, no signs of a hypersensitivity reaction could be observed within this time period, which is crucial for ischemic symptoms due to emboli formation. When repeating the allogenic intravenous injection after approximately 6 weeks (using MSCs of the same donor for the second treatment) in 5 recipients, no adverse effects were noticed during the 1 year follow-up period. Also horses injected with the higher dose of 106 MSCs/ml failed to demonstrate any adverse effects during the entire study. In addition, no deaths were registered within 10 days after the injection. Only one horse died at 7 months after the injection due to colic and the rest of 290 horses showed no abnormal features or adverse clinical effects during the 1 year follow-up period. DISCUSSION In the present study, 291 horses were clinically evaluated after an intravenous injection of allogenic peripheral blood (PB)-derived mesenchymal stem cells (MSCs). No acute adverse effects were noticed during the daily follow-up in the first 10 days after injection. In addition, all the horses except 1 could be evaluated during the entire follow-up period of 1 year and failed to show any clinical adverse effect. It has been postulated that approximately 60% of transplanted autologous MSCs reside in the lungs at 1 hour after intravenous administration and that this amount is strongly reduced within 24 hours [29]. The first 24 hours after intravenous injection could be considered as crucial regarding acute respiratory or even lethal effects. Therefore, clinically evaluating the horses during the first 10 days after injection, covered an initial safety assessment caused by possible acute adverse effects. Unfortunately, one recipient died at 7 months after the intravenous injection as a result of a severe colic. Recently, a study of 690 horses (varying from 1- to 20-years-old) demonstrated that colic was the most common health problem in horses, occurring in 33% (226/690) of the presented patients in that particular equine hospital [30]. When looking at the incidence of colic in horses owned by clients of a general equine practice (= field study), a summary estimate of 0.22 cases/horse-year are at risk. In the present study, this would implicate that 64 horses (291 x 0.22) would be at risk of developing colic [31]. When extrapolating the reported overall colic mortality rate of 25% in horses (n=208) that were referred to an equine hospital [32], 16 colic-related deaths out of the 64 possible colic cases could be expected during the present 1 year follow-up study. However, it should be taken into account that the aforementioned study [32] reports the death rate in referral patients, which concerns horses that were in a further stage of illness. Therefore, the theoretically calculated 16 deaths overestimates the death rate of colic horses in field circumstances. Nevertheless, at least 1 death due to colic could be expected when clinically assessing 291 healthy horses during a longer period of time. Recently, it has been reported that an intravenous injection of allogenic MSCs into cats with chronic kidney disease induced vomiting in 2 out of 5 (40%) and respiratory distress in 4 out of 5 (80%) patients [21]. In the latter study, the effects could be noticed within 30 minutes after infusion
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with 4 x106 (in 20ml HBSS = 0.2 x106/ml) allogenic cryopreserved adipose-derived MSCs. After reducing the dose to 2 x106 MSCs or using freshly prepared cells, only limited adverse effects (n=6) could be noticed by the authors, indicating at least a dose-related effect. In the present study, the same cell concentration of 0.2 x106/ml (and even 106/ml in 5 recipients) with frozen cells was used without noticing any of the mentioned adverse effects. This is probably due to the fact that a physiological dose was used in the present study. In horses, it has been described that around 0.0001% of the PB mononuclear cells (PBMCs) (8 on 10,000,000) were able to form a colony in adhesion [22] and the reported number of PBMCs varies between 106/ml [22] and 3.41 x106/ml PB [33]. Taken the aforementioned together, equine PB contains between 1 and 3.41 MSCs/ml PB and since the average blood volume of an adult horse varies around 45 liter of blood [34], the physiological amount of MSCs in the PB of the average horse used in the present study varies between 45,000 (1 MSC x 45,000 ml PB) and 153,450 (3.41 MSCs x 45,000 ml PB). Since 0.2 x106 covered the physiological amount of MSCs in the PB of larger horses as well and most of the horses in the present study were mature, we decided to use this dose in 98% of the patients (286/291). In addition, it has been reported that very small suspended human MSCs (±12.6 μm) caused no microinfarcts in rats, whereas the medium (±17.9 μm) and larger (±30.4 μm) sized 2D cultured MSCs caused a microinfarct in 1/5 and 5/5 of the animals, respectively [35]. It should be noted that the reported diameter of suspended murine MSCs varies between 15 to 19 m [36], whereas the 2D cultured MSCs are larger [37], as described for human MSCs. Indeed, a larger animal species might have approximately the same MSC size as smaller animals after in vitro cultivation, yet they possess a larger vasculatory network and a lower metabolism, both facilitating MSC migration throughout the circulation. In this regard, it has been reported that autologous MSC homing towards fracture sites in mice would be dosedependent as well [5]. Interestingly, the latter study described an ED50 of 0.3 x106 MSCs, with a plateau at 0.7 x106 MSCs. This clearly indicates that MSC migration to the injured site is saturated at one point and high doses would not be necessarily correlated with better clinical results. In fact, intravascular infusion of human adipose tissue-derived MSCs caused death in 40% of the mice (n=10) at a dose of 1 x106 MSCs, whereas 0.2 x106 MSCs still caused death in 25% of the animals (n=8) [19]. In the present study, an average dose of 0.2 x106 MSCs was used. Although this seems low for a large animal species, such as horses, a placebocontrolled study recently demonstrated significant effects on several hematological parameters after MSC administering using the same dose [25]. In addition, the aforementioned feline study, also described statistically significant effects using the lowest dose of MSCs, whereas less hematological parameters significantly changed when doubling the dose [21]. In mice, it has been postulated that the suspension medium influences pulmonary toxicity after autologous MSC transplantation [20]. Resuspending MSCs in heparin was highly beneficial, avoiding clinical symptoms in 95% of mice, whereas replacing heparin with PBS/EDTA or control
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buffer caused severe pulmonary reactions and even death. It should be mentioned though, that another study reports the use of heparin in all the samples and noticed side effects in 80% of the animals at a high dose [21]. However, HBSS (PBS analogue) was used for the intravenous infusion. In the present study, no adverse clinical effects were found when the MSCs of two different donor horses were transplanted at the given dose using the carrier fluid consisting of DMEM and 10% DMSO.
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In conclusion, we can state that the intravenous application of allogenic PB-derived MSCs initiated no clinical adverse effects in 291 recipient horses.
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CONFLICT OF INTEREST
[13]
The authors MS, TM and JHS declare competing financial interests as shareholders in Global Stem cell Technology (GST). SB and JHS are both employed by GST and inventors of several pending patents owned by GST (PCT/EP2013/070247 and PCT/EP2013/070257). The content of this manuscript contains a product under development owned by GST.
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ACKNOWLEDGEMENTS The authors would like to thank Nathalie Gijbels for her technical assistance. Moreover, the Federal Public Service of Health should be acknowledged for providing GST with a laboratory recognition number (LA1700607), allowing us to perform this study.
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SOURCES OF FUNDING This study was supported by a grant (number 130543) to SB and JS from the agency for innovation by science and technology Flanders (IWT Vlaanderen). Furthermore, the authors would like to acknowledge Global Stem cell Technology and the sources of private funding that have provided the basis for this study. REFERENCES [1]
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Received: December 26, 2013
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Revised: February 18, 2014
Accepted: February 18, 2014
