Safety and efficacy of intravenous infusion of allogeneic cryopreserved mesenchymal stem cells for treatment of chronic kidney disease in cats: results of three sequential pilot studies

Abstract

Introduction: Administration of mesenchymal stem cells (MSCs) has been shown to improve renal function in rodent models of chronic kidney disease (CKD), in part by reducing intrarenal inflammation and suppressing fibrosis. CKD in cats is characterized by tubulointerstitial inflammation and fibrosis, and thus treatment with MSCs might improve renal function and urinary markers of inflammation in this disease. Therefore, a series of pilot studies was conducted to assess the safety and efficacy of intravenous administration of allogeneic adipose-derived MSCs (aMSCs) in cats with naturally occurring CKD.

Methods: Cats enrolled in these studies received an intravenous infusion of allogeneic aMSCs every 2 weeks collected from healthy, young, specific pathogen-free cats. Cats in pilot study 1 (six cats) received 2 × 106 cryopreserved aMSCs per infusion, cats in pilot study 2 (five cats) received 4 × 106 cryopreserved aMSCs per infusion, and cats in pilot study 3 (five cats) received 4 × 106 aMSCs cultured from cryopreserved adipose. Serum biochemistry, complete blood count, urinalysis, urine protein, glomerular filtration rate, and urinary cytokine concentrations were monitored during the treatment period. Changes in clinical parameters were compared statistically by means of repeated measures analysis of variance (ANOVA) followed by Bonferroni's correction.

Results: Cats in pilot study 1 had few adverse effects from the aMSC infusions and there was a statistically significant decrease in serum creatinine concentrations during the study period, however the degree of decrease seems unlikely to be clinically relevant. Adverse effects of the aMSC infusion in cats in pilot study 2 included vomiting (2/5 cats) during infusion and increased respiratory rate and effort (4/5 cats). Cats in pilot study 3 did not experience any adverse side effects. Serum creatinine concentrations and glomerular filtration rates did not change significantly in cats in pilot studies 2 and 3.

Conclusions: Administration of cryopreserved aMSCs was associated with significant adverse effects and no discernible clinically relevant improvement in renal functional parameters. Administration of aMSCs cultured from cryopreserved adipose was not associated with adverse effects, but was also not associated with improvement in renal functional parameters.

Figure 1

Expression of cell surface markers by cryopreserved feline adipose-derived mesenchymal stem cells (aMSCs). Feline aMSCs expanded from cryopreserved adipose were passaged three times in culture, then collected by trypsinization and immunostained for assessment of cell surface marker expression by flow cytometry, as described in Methods. Feline aMSCs expressed high surface levels of CD90 (A) and CD44 (B) but did not express major histocompatibility complex (MHC) class II (C) or CD4 (D). Isotype controls are represented in red and unstained MSCs are represented in blue. Similar results were obtained with aMSCs from three donor cats (E).

Figure 2

Trilineage differentiation of feline adipose-derived mesenchymal stem cells (aMSCs) after cryopreservation as cells or adipose. aMSCs taken directly from cryopreservation (A-C) and cultured from cryopreserved adipose (D-F) were capable of trilineage differentiation. (A,D) aMSCs formed intracellular lipid vacuoles when incubated in adipocytic differentiation media for 21 days. (B,E) aMSCs stained positive for calcium with alizarin red following differentiation into osteocytic phenotype after 21 days of incubation in differentiation media. (C,F) Cryosection of pellets of cartilage matrix (stained with toluidine blue) formed by aMSCs when exposed to chondrocytic differentiation media for 21 days.

Figure 3

Assessment of renal function in adipose-derived mesenchymal stem cell (aMSC)-treated cats. (A) Serum creatinine values for cats in pilot study 1, who received three doses of 2 × 10⁶ cryopreserved aMSCs intravenously 2 weeks apart. A statistically significant decrease in creatinine is seen (P = 0.01), however the degree of decrease may not be clinically significant. (B) Serum creatinine values for cats in pilot study 2, who received three doses of 4 × 10⁶ cryopreserved aMSCs intravenously 2 weeks apart. No significant difference in creatinine was detected. (C) Serum creatinine values for cats in pilot study 3, who received three doses of 4 × 10⁶ aMSCs cultured from cryopreserved adipose intravenously 2 weeks apart. No significant difference in creatinine was detected. (D) Estimated glomerular filtration rate (GFR) by iohexol clearance results at 0 and 8 weeks for five cats in pilot study 2 that received 4 × 10⁶ cryopreserved aMSCs intravenously every 2 weeks for three treatments. Control chronic kidney disease cats had GFR performed at 0 and 8 weeks only. (E) GFR determined by nuclear scintigraphy results at 0 and 8 weeks for five cats in pilot study 3 that received 4 × 10⁶ aMSCs cultured from cryopreserved adipose intravenously every 2 weeks for three treatments.

Figure 4

Urinary cytokine levels in mesenchymal stem cell (MSC)-treated cats. Pilot study 1 (A,B): cats received 2 × 10⁶ cryopreserved aMSCs intravenously every other week for three injections. (A) Monocyte chemoattractant protein 1 (MCP-1):urine creatinine ratio throughout the study. There was a statistically significant decrease in MCP-1 (P = 0.0001). (B) Interleukin 8 (IL-8):urine creatinine ratio throughout the study. There was a statistically significant decrease in IL-8 (P = 0.01). Pilot study 2 (C-E): cats received 4 × 10⁶ cryopreserved aMSCs intravenously every other week for three injections. Cytokine:urine creatinine ratio data throughout the study for (C) MCP-1, (D) IL-8, (E) transforming growth factor (TGF)-β1 and (F) vascular endothelial growth factor (VEGF). There was no statistically significant change in urinary cytokines in pilot study 2. Pilot study 3 (G-I): cats received 4 × 10⁶ aMSCs cultured from cryopreserved adipose intravenously every other week for three injections. Cytokine:urine creatinine ratio data throughout the study for (G) IL-8, (H) TGF-β1 and (I) VEGF. There was no statistically significant change in urinary cytokines in pilot study 3.