Preclinical Safety Evaluation of Intranasally Delivered Human Mesenchymal Stem Cells in Juvenile Mice

Simple Summary The concept of utilizing mesenchymal stem cells for the treatment of central nervous system disorders has progressed from preclinical studies to clinical trials. While promising, the effectiveness of cell therapy is hampered by the route used to deliver cells into the brain. In this context, intranasal cell administration has boomed over the past few years as an effective cell delivery method. However, comprehensive safety studies are required before translation to the clinic. Our study shed light on how intranasally administrated mesenchymal stem cells may be used to safely treat neurological disorders. Abstract Mesenchymal stem cell (MSC)-based therapy is a promising therapeutic approach in the management of several pathologies, including central nervous system diseases. Previously, we demonstrated the therapeutic potential of human adipose-derived MSCs for neurological sequelae of oncological radiotherapy using the intranasal route as a non-invasive delivery method. However, a comprehensive investigation of the safety of intranasal MSC treatment should be performed before clinical applications. Here, we cultured human MSCs in compliance with quality control standards and administrated repeated doses of cells into the nostrils of juvenile immunodeficient mice, mimicking the design of a subsequent clinical trial. Short- and long-term effects of cell administration were evaluated by in vivo and ex vivo studies. No serious adverse events were reported on mouse welfare, behavioral performances, and blood plasma analysis. Magnetic resonance study and histological analysis did not reveal tumor formation or other abnormalities in the examined organs of mice receiving MSCs. Biodistribution study reveals a progressive disappearance of transplanted cells that was further supported by an absent expression of human GAPDH gene in the major organs of transplanted mice. Our data indicate that the intranasal application of MSCs is a safe, simple and non-invasive strategy and encourage its use in future clinical trials.

Our safety study demonstrated that the intranasal delivery of MSCs is safe at 12 and 24 weeks after administration. In addition, animal welfare assessment, that was carried out daily over the whole study period, did not evidence any indicator of compromised health in MSC-transplanted mice at any time point (Supplemental Table 2), suggesting that cell therapy is also safe during the first weeks after transplantation. The lack of adverse events described in this study may be associated with the fact that MSCs are short-lived. Although we cannot fully conclude that cells disappear after week 4 post-transplantation ( Figure 6A), we suggest that MSCs had a limited durability after intranasal delivery, based on previous studies. In this regard, a report using immunocompetent mice demonstrated that mouse MSCs have a short survival time (i.e. 1-2 week) after intravenous infusion [3]. Therefore, as proposed by other authors, MSCs might pass on their effects to the host cells before dying via secretion of paracrine factors that will carry on the long-term beneficial effects [4][5][6]. Even so, we cannot discard the possibility that a small number of MSCs could survive longer after transplantations, being also responsible for the therapeutic effect. In this regard, RT-PCR determinations indicated that if this is the case, the number of cells surviving until week 24 post-transplantation is below the threshold of detection ( Figure 6F). Further studies should be performed to determine the survival time of transplanted cells in the donor and to identify potential safety issues shortly after cell therapy (e.g. at 7 weeks after administration).

Production and QC standards for MSCs
Prior MCS administration, QC standards were performed according to GMP requirements. The QC are briefly described as follow: Viability: Cell viability was measured by trypan Blue vital dye exclusion test (0,4%, Sigma Aldrich) according to standard procedure assays. Adequacy >90%.
Sterility test: Absence of microbial contamination of final product and culture supernatant was verified by direct inoculation of the sample into test media with Thioglycollate Broth Penase (VWR International Eurolab, S.L, Barcelona, Spain) to detect facultative anaerobes and aerobes, and test media with Tryptic Soya Broth Penase (VWR International Eurolab) for strict aerobes and fungi. Adequacy was reached when all cultures scored negative after a 14-day culture period.
Mycoplasma test: Absence of mycoplasma contamination was verified using the commercial kit Venor GeM (Minerva Biolabs GmbH, Germany). Adequacy: no contamination detected.
Endotoxin test: Endotoxin content was performed by chromogenic Limulus Amebocyte Lysate (LAL)-based kinetic method using Endosafe-PTS system (Charles River Laboratories; Barcelona, Spain), following the manufacturer's instructions. Cartridges with 0.05 -5.0 EU/mL sensitivity were used in this study. For the endotoxin testing, a test result was considered valid when the percentage of spike recovery was between 50% and 200% with a coefficient of variation less than 25%. Adequacy <5.0 EU/ml.
Karyotyping: Molecular karyotyping performed by Affymetrix Human SNP Array 6.0 and the CytoScan HD Array in the Genome Core Facility of CABIMER.
Cell differentiation: MSCs were differentiated into osteoblasts, adipocytes and chondrocytes using lineage specific induction media (Differentiation medium BulletKit TM ; Lonza, Basel, Switzerland), according to manufacturer´s instructions. Supplemental Figure S4. Evaluation of the effects of intranasally delivered MSCs in whole-brain irradiated mice. Animals in this study were assigned to six experimental groups: intact control mice (CTR group), mice receiving intranasal PBS (PBS group), mice receiving intranasal MSCs (MSC group), mice receiving intranasal positive control cancer cells (U87 group), mice receiving cranial radiation and intranasal PBS (XRT+PBS group) and mice receiving cranial radiation and intranasal MSC (XRT+MSC group). (A) Schema outlining treatments used in the survival study. Mice received a total dose of 10 Gy head-only XRT in 2 fractions (2 x 5 Gy), as previously described [2]. The day after, mice were treated with a weekly dose of MSCs for 4 consecutive weeks (5·10 5 of cells/dose). Image created with BioRender.com (B) Kaplan-Meier curve showing the percentage of survival mice. Survival curves were plotted using the Kaplan-Meier method, which include any animal found dead or euthanized. Note that all whole-brain irradiated mice exhibited shortened survival (i.e., XRT+PBS or XRT+MSC), as compared to non-irradiated mice (i.e., CTR, PBS, MSC, and U87 mice). Importantly, MSC treatment does not affect the time that mice survive after radiation exposure. The red line is not visible because it overlaps with the grey line. Log-rank test. n =9-17 per group. (C) Representative immunofluorescence images of the brain of CTR, XRT+PBS and XRT+MSC mice, using CD68 and Iba1 markers, the day after the 4th doses of MSCs (i.e. 4 weeks after radiation). Note that the XRT+MSC group exhibited lower neuroinflammation than XRT+PBS mice, as evidenced by reduced CD68 and Iba1 immunoreactivity. Scale bar, 200 µm. (D) Quantification of the number of CD68+ cells shown in C. n=4 per group. Data are represented as mean ± SEM. *p<0.005 compared to CTR group. One-way ANOVA.