Preclinical safety & toxicity evaluation of pooled, allogeneic human bone marrow-derived mesenchymal stromal cells

Abstract

Background & objectives: Administration of ex vivo-expanded human bone marrow-derived mesenchymal stromal cells (hBMMSC) obtained from single donors has shown therapeutic benefits in both preclinical and clinical studies. In this study, the safety, toxicity and biodistribution profiles of a pooled hBMMSC population, produced from three healthy donors were assessed in rodent and non-rodents.

Methods: The pooled hBMMSC population was characterized by their expression of various cell surface markers, differentiation potential and immunomodulatory activity. To establish in vivo safety of the pooled cells, these were administered by various injection routes into rodents and non-rodents to determine overall toxicity, biodistribution and tumorigenic potential in a series of preclinical studies.

Results: Single injections of hBMMSC at various doses through intravenous or intramuscular routes did not cause toxicity in rats and rabbits. In addition, repeat administration of hBMMSC was also well tolerated by rats, and no prenatal toxicity was observed by multiple administration in the same animal species. Ex vivo-expanded and cryopreserved hBMMSCs did not induce tumour formation in severe combined immunodeficient (SCID) mice.

Interpretation & conclusions: Our results showed that the pooled hBMMSC population was non-toxic, non-teratogenic and non-tumorigenic in animals. Further studies need to be done to find out if it can be safely administered in human patients.

Fig. 1

Multilineage differentiation of pooled human bone marrow-derived mesenchymal stromal cells (hBMMSCs). Adipogenic differentiation was detected by oil droplet formation stained with Oil O red staining (D - differentiated), Osteogenic differentiation was confirmed by the stained mineralized matrix with alizarin red staining (E - differentiated), chondrogenic differentiation was confirmed through the presence of stained glycosaminoglycan by Alcian blue staining (F - differentiated). Undifferentiated control cultures of adipogenic, osteogenic and chondrogenic differentiation are shown in (A-C), respectively.

Fig. 2

Cytokine levels in the serum samples of rats (sex pooled) following single intravenous injection of pooled human bone marrow-derived mesenchymal stromal cells (BMMSCs) or vehicle. Serum cytokine levels were measured before cell injection (basal) at day 0 (A), day 7 (B) and day 90 (C) after cell injection. Human bone marrow-derived mesenchymal stromal cells were injected at low (12.6 × 106 cells/kg b.w.), medium (63 × 106 cells/kg b.w.) and high dose (126 × 106 cells/kg b.w.). No significant differences in the levels of cytokines were observed between cell- and vehicle-treated animals. Vertical bars represent standard error (n=6). IL-1β, interleukin-1 beta; IFNγ, interferon gamma; TNFα, tumour necrosis factor alpha.

Fig. 3

Pooled human bone marrow-derived mesenchymal stromal cell (hBMMSC) population was non-tumorigenic in severe combined immunodeficient (SCID) mice. Tumour volumes were measured twice weekly in both male (A) and female (B) mice. Progressive tumour growth was detected visually at the site of cell injection in the positive control animals (D) and not detected in either vehicle- or human bone marrow-derived mesenchymal stromal cell-treated animals (C, E & F). Neoplastic cell growth with central area of necrosis (arrows) was observed in all (10/10) positive control animals (H) and absent in vehicle- or hBMMSCs-treated animals (G, I & J). Magnification, ×4 (G-J).

Fig. 4

In vivo biodistribution profile of CM-DiI-labelled pooled human bone marrow-derived mesenchymal stromal cell (hBMMSC) at different time points (A-E & F-J) showing the distribution profile of hBMMSCs administered through intravenous route in nude rats and nude mice, respectively, and K-O showed biodistribution profile in nude mice through intramuscular route.