In vivo safety profile and biodistribution of GMP-manufactured human skin-derived ABCB5-positive mesenchymal stromal cells for use in clinical trials

Abstract

Background aims: Human dermal ABCB5-expressing mesenchymal stromal cells (ABCB5⁺ MSCs) represent a promising candidate for stem cell-based therapy of various currently uncurable diseases in several fields of regenerative medicine. We have developed and validated a method to isolate, from human skin samples, and expand ABCB5⁺ MSCs that meet the guideline criteria of the International Society for Cellular Therapy. We are able to process these cells into a Good Manufacturing Practice-conforming, MSC-based advanced-therapy medicinal product.

Methods: To support the development of ABCB5⁺ MSCs for potential therapeutic topical, intramuscular and intravenous administration, we have tested our product in a series of Good Laboratory Practice-compliant nonclinical in-vivo studies addressing all relevant aspects of biosafety, including potential long-term persistence and proliferation, distribution to nontarget tissues, differentiation into undesired cell types, ectopic tissue formation, tumor formation and local tissue reaction.

Results: (i) Subcutaneous application of 1 × 10⁷ ABCB5⁺ MSCs/animal and intravenous application of 2 × 10⁶ ABCB5⁺ MSCs/animal, respectively, to immunocompromised mice did not result in safety-relevant biodistribution, persistence or proliferation of the cells; (ii) three monthly subcutaneous injections of ABCB5⁺ MSCs at doses ranging from 1 × 10⁵ to 1 × 10⁷ cells/animal and three biweekly intravenous injections of 2 × 10⁶ ABCB5⁺ MSCs/animal, respectively, to immunocompromised mice were nontoxic and revealed no tumorigenic potential; and (iii) intramuscular injection of 5 × 10⁶ ABCB5⁺ MSCs/animal to immunocompromised mice was locally well tolerated.

Discussion: The present preclinical in vivo data demonstrate the local and systemic safety and tolerability of a novel advanced-therapy medicinal product based on human skin-derived ABCB5⁺ MSCs.

Keywords: ABCB5; GMP; MSC; biodistribution; persistence; safety; stem cells; stromal cells; toxicity; tumorigenicity.

Figure 1

Concentration of human-origin cells as detected by qPCR in injection-site tissues (skin and skeletal muscle) and in the lung of NSG mice at 8 (black bars), 29 (light gray bars) and 92 days (medium gray bars) after intravenous injection of 2 × 10⁶ ABCB5⁺ MSCs. Shown are means of 10 animals; error bars indicate SD; **P < 0.01, non-paired one-way analysis of variance followed by Tukey’s multiple comparison test. A separate control group (not shown), comprising the mice injected with cell-free vehicle and sacrificed 92 days thereafter, did not reveal any detectable human-origin cells except for muscle tissue of one animal, which was considered an artifact (see Results).

Figure 2

Histological evaluation of the persistence of human cells in the lungs of NSG mice treated with three bi-weekly intravenous injections of 2 × 10⁶ ABCB5⁺ MSCs/mouse. Mice were sacrificed at 13 weeks after the first MSC injection. Tissues were stained with anti-mitochondrial antibody (AMA) (brown) as a marker of human-origin cells. (Left) Skin at the injection site of a mice injected subcutaneously with 1.08 × 10⁶ HeLa cells served as positive control. (Middle and Right) Representative microphotographs of lung sections of MSC-treated mice in which AMA staining was exceptionally rare. Scale bars: 50 μm.

Figure 3

Histopathological evaluation of local reactions in thigh muscle tissue of NOG mice to intramuscular injection of 200 μL HRG solution without (left) and with (right) 5 × 10⁶ ABCB5⁺ MSCs/mouse, split over four injection sites. (Left) Mice were sacrificed at 7 days after cell injection. Representative microphotograph of a thigh muscle section of a control animal, treated with vehicle only, showing slight focal myofiber degeneration (arrows) and regenerative myoblasts (arrowheads). (Right) Representative microphotograph of a thigh muscle section of an MSC-treated mouse, showing focal accumulation of mesenchymal cells with minimal fibrotic aspect. H&E staining; magnification 20 × (left), 40 × (right); scale bars: 20 μm.