Mesenchymal stromal cell therapeutic potency is dependent upon viability, route of delivery, and immune match

Abstract

Culture-adapted bone marrow mesenchymal stromal cells (MSCs) deploy paracrine anti-inflammatory and tissue regenerative functionalities that can be harnessed as a living cell pharmaceutical product. Independent of clinical indication, a near majority of human clinical trials administer MSC IV, often with an allogeneic MSC cell product immediately after thawing from cryostorage. Despite hundreds of studies in a wide assortment of inflammatory, degenerative, and acute tissue injury syndromes, human clinical outcomes often fail to mirror promising rigorously conducted preclinical animal studies. Using a mouse model of toxic colitis, we demonstrate that replication fit MSCs harvested in log phase of growth have substantial impact on colitis clinical and pathologic endpoints when delivered subcutaneously or intraperitoneally, whereas the maximum tolerated IV bolus dosing failed to do so. We also demonstrate that heat-inactivated MSCs lose all therapeutic utility and the observation is mirrored by use of viable MSC administered immediately postthaw from cryostorage. Using luciferase transgenic MSC as donor cells, we demonstrate that transient in vivo engraftment is severely compromised when MSCs are dead or thawed and further demonstrate that MSC redosing is feasible in relapsing colitis, but only syngeneic MSCs lead to sustained improvement of clinical endpoints. These data support the notion that pharmaceutical potency of MSC requires viability and functional fitness. Reciprocally, IV administration of thawed MSC products may be biased against positive clinical outcomes for treatment of colitis and that extravascular administration of syngeneic, fit MSCs allows for effect in a recurrent therapy model.

Graphical abstract

Figure 1.

Metabolic fitness is indispensable for BM-MSCs’ therapeutic ability in DSS-induced colitis. (A) Trypan blue dye exclusion assay to measure the viability of fresh, postthawed or heat-inactivated BM-MSCs. (B) Schematic of the experimental setup for results presented in panels C-F. (G) Schematic of the experimental setup for results indicated in panels H-K (mice, N = 5 per test group). Development of colitis was examined by measuring body weight change relative to the initial body weight at day 0 (C,H), disease activity index (D,I), H&E staining of colon (E,J), and histological score (F,K). (E,J) Bars represent 100 μm. Statistical analysis was assessed by Student t test (E,J), 1-way ANOVA (Tukey test) (A), and 2-way ANOVA (Tukey test) for all other experiments. **P < .01, ***P < .001, ****P < .0001.

Figure 2.

BM-MSC delivery through IV route has no effect in colitis prevention. (A) Cartoon depicts the experimental schemes for results depicted in panels B-E. Mice (N = 5 per test group) received 4.0% (w/v) of DSS orally for 6 days. BM-MSCs were transferred IP, SC, or IV into the syngeneic mice at days 2 and 4. The development of colitis is examined by measuring body weight change relative to the initial body weight at day 0 (B), disease activity index (C), H&E staining (D), and histological score (E). Bars represent the mean ± SEM (N = 5 for all experiments). (D) Bars represent 100 μm. Statistical analysis was assessed by Student t test (E) and 2-way ANOVA (Tukey test) (B-C). **P < .01, ***P < .001, ****P < .0001.

Figure 3.

Dwell time of IP/SC delivered thawed and heat-inactivated BM-MSCs are compromised. (A) Longitudinal in vivo images of representative mice are shown at selected time points. Bioluminescence (BL) is symbolized by pseudocolored heat maps in which values in scale bars are photons/s/cm²/sr. BL was assessed in mice receiving fresh culture-rescued BM-MSC (A,E), cryopreserved BM-MSC (B,F), or HI-BM-MSC (C,G) via IP (A-C) or SC (E-G) on the indicated days of postcell injection. (D,H) BL was measured from all in vivo ROIs at each time point as total flux (photons per second). The graph shows averages ± SEM from 3 independent experiments, N = 5 mice per group per experiment. *P < .05, **P < .01, ****P < .0001 using Student t test. ns, not significant.

Figure 4.

Allogeneic BM-MSC are immune rejected by MHC-mismatched colitis mice. (A) Experimental schemes for results shown in panels B-E. Mice (N = 5 per test group) received 4.0% (w/v) of DSS orally for 6 days and normal drinking water afterward until day 15. For the second cycle of colitis induction, the same mice were administered DSS starting on day 16 and continued until day 22 following normal drinking water afterward until day 30. On days 2 and 4 of the first cycle, allogenic or syngenic fresh BM-MSCs (10 × 10⁶ cells) were delivered via IP (N = 5 per group) injection and the mice were treated with the second challenge of fresh MSC administration on days 18 and 20. After 30 days of complete cycle, all mice were sacrificed. The development of colitis is monitored by measuring body weight change relative to the initial body weight at day 0 (B), disease activity index (C), H&E staining (D), and histological score (E). BM-MSC transfusion into B6 mice served as positive control. Error bars represent the mean ± SEM (N = 5 for all experiments). (D) Bars represents 100 μm. Statistical analysis was performed by Student t test (E) and by 2-way ANOVA (Tukey test) (B-C). **P < .01, ***P < .001, ****P < .0001.