Safety Profile of Good Manufacturing Practice Manufactured Interferon γ-Primed Mesenchymal Stem/Stromal Cells for Clinical Trials

Abstract

Mesenchymal stem/stromal cells (MSCs) are widely studied by both academia and industry for a broad array of clinical indications. The collective body of data provides compelling evidence of the clinical safety of MSC therapy. However, generally accepted proof of therapeutic efficacy has not yet been reported. In an effort to generate a more effective therapeutic cell product, investigators are focused on modifying MSC processing protocols to enhance the intrinsic biologic activity. Here, we report a Good Manufacturing Practice-compliant two-step MSC manufacturing protocol to generate MSCs or interferon γ (IFNγ) primed MSCs which allows freshly expanded cells to be infused in patients on a predetermined schedule. This protocol eliminates the need to infuse cryopreserved, just thawed cells which may reduce the immune modulatory activity. Moreover, using (IFNγ) as a prototypic cytokine, we demonstrate the feasibility of priming the cells with any biologic agent. We then characterized MSCs and IFNγ primed MSCs prepared with our protocol, by karyotype, in vitro potential for malignant transformation, biodistribution, effect on engraftment of transplanted hematopoietic cells, and in vivo toxicity in immune deficient mice including a complete post-mortem examination. We found no evidence of toxicity attributable to the MSC or IFNγ primed MSCs. Our data suggest that the clinical risk of infusing MSCs or IFNγ primed MSCs produced by our two-step protocol is not greater than MSCs currently in practice. While actual proof of safety requires phase I clinical trials, our data support the use of either cell product in new clinical studies. Stem Cells Translational Medicine 2017;6:1868-1879.

Keywords: Good Manufacturing Practice (GMP); Interferon gamma; Mesenchymal stem cells; Mesenchymal stromal cells (MSC).

Figure 1

Schematic representation of the 2‐step GMP manufacturing process. (A): The isolation and primary expansion of MSCs through cryopreservation; (B): the thawing and secondary expansion of MSCs. The opportunity for cytokine priming, if appropriate, is shown. See text for details. Abbreviations: MNC, mononuclear cells; MSCs, mesenchymal stem/stromal cells.

Figure 2

Clustering of global transcriptional responses of MSCs following priming with IFNγ. (A): Principal Component Analysis (PCA) of differential gene expression of MSCs (open shapes) and γMSC (filled shapes) from each donor (same color). (B): Euclidean distance between samples to assess overall similarity among the samples. The darker blue indicates a shorter distance, more similar; lighter blue indicates greater distance, less similar. (C): Heat map analysis of RNA‐Seq gene expression data from MSCs and γMSC. The heatmap was generated using the normalized expression values for the 20 most upregulated and downregulated genes following interferon γ priming. Color code indicates relative expression; red highest, blue lowest. Abbreviation: MSCs, mesenchymal stem/stromal cells.

Figure 3

Immunophenotype of interferon γ primed Mesenchymal stem/stromal cell (MSCs). Flow cytometric analysis of select surface marker expression on γMSCs (solid line) and the isotype control (shaded peak).

Figure 4

Soft agar colony forming assay to detect malignant transformation. Representative photographs of triplicate agar plates for each condition are shown. The positive control was a human ES‐2 ovarian clear cell carcinoma cell line. The negative control was saline. Original magnification was ×46. Abbreviations: IFNγ, interferon γ; MSCs, mesenchymal stem/stromal cells.

Figure 5

Analysis of hematopoietic engraftment after cotransplantation of hematopoietic cells and MSCs or γMSCs. C57BL/6 mice were lethally irradiated and then transplanted with 2 × 10⁶ unfractionated GFP‐expressing C57BL/6 marrow cells and 250,000 GMP‐manufactured human MSCs or γMSCs (both clinical products) or 250,000 murine MSCs or γMSCs. (A): Kinetics of leukocyte recovery after transplantation for each cohort of mice. The control mice underwent hematopoietic cell transplantation, but did not receive MSC/γMSC cotransplantation. n.s., not significant. (B): The absolute lymphocyte count of each cohort assessed 21 days after transplantation. The values represent the mean ± standard deviation for each cohort, n = 5, *, p < .05 versus each cohort, one‐way ANOVA with Tukey's post hoc test. (C, top) Representative dot plot of flow cytometric analysis of unfractionated marrow harvested 21 days after transplantation revealing the percentage of donor (GFP‐expressing) marrow cells. (bottom) Similar analysis of the lin‐sca‐1+c‐kit+ (LSK) cells which demarcate the primitive stem/progenitors cells from marrow. Abbreviations: GFP, green fluorescent protein; FSC, forward scatter; LSK, lin‐sca‐1+c‐kit+; mMSCs, murine mesehcymal stromal cells; hMSCs human mesenchymal stromal cells; γ‐hMSCs, interferon gamma primed human mesenchymal stromal cells.

Figure 6

Post‐mortem examination. Ten weeks after NSG mice were infused with mesenchymal stem/stromal cells (MSCs)/γMSCs, animals were sacrificed and underwent complete post‐mortem examination. (A): Representative photomicrograph of a histologic section of lung tissue taken at ×40 original magnification. This section was taken from a mouse which received γMSCs. (B): Photomicrograph of a histologic section of spleen showing the osseous metaplasia (white arrows) taken at ×100 original magnification. This section was taken from one of the two MSC‐infused mice where this finding was observed.