Long-Term Safety of Urethral Injection of Stem Cell-Derived Muscle Progenitors

Objectives: There is growing interest in using pluripotent stem cells (PSC) and their derivatives for treatments of degenerative conditions. A major advantage of this stem cell source is that it allows for expansion of homogeneous cell doses sufficient for clinical applications. However, iPSCs are noted for their ability to form tumors in vivo. The process of differentiating iPSC into smooth muscle progenitor cells for urethral regeneration may reduce this risk. Long-term safety after clinical site transplantation has not been tested. In this preliminary study, we sought to examine engraftment, biodistribution, and safety of human iPSC-derived progenitors of smooth muscle cells (pSMC) after peri-urethral injection into the immune-deficient NSG mouse over 12 months.

Methods: Dermal fibroblasts from two women were genetically reprogrammed into iPCSs using a modified mRNA/miRNA reprogramming method through transduction of mRNA encoding Oct4, Klf4, Sox2, c-Myc, Lin28 and miR-302/367. These iPSC lines were then differentiated into pSMCs using our xeno-free protocol. Smooth muscle phenotype and function, and cell heterogeneity were confirmed by RT-qPCR, FACs, and single cell RNA sequencing and carbachol stimulation. pSMCs from each patient were injected peri-urethrally in three groups of NSG mice (n=10/group) with the following cell doses: (1) pSMCs-only (4 million), (2) pSMCs (4 million)+0.2%-1% parental iPSCs, and (3) pSMCs (4 million)+0.5x106 parental iPSCs. Controls were injected with saline. Throughout the 12-month study period, mice were assessed for clinical signs, body weight and food consumption, behavioral changes, and clinical pathology. Rats from each group were euthanized at different time points and organs including urethra, bladder, vagina, liver, spleen, kidney, and brain were harvested for RT-qPCR of the human Alu gene sequence to detect presence of human cells.

Results: By 6-12 months, human cells were present in tissues around the injection site (urethra, bladder, and vagina) and in the liver. Gross and histologic examination of the liver did not reveal solid tumors. No human cells were detected in the spleen, kidneys, or brain throughout the study. Benign uterine polyps and uterine prolapse were noted at 9-12 months. Throughout the 12-month period, there was no evidence of failure to thrive, teratoma formation or malignant masses in any of the groups, including saline controls and groups injected with pSMCs combined with a small amount of parental iPSCs to simulate residual iPSCs in the iPSC-derived pSMC population.

Conclusions: This 12-month study demonstrated that human iPSC-derived pSMCs can engraft in the peri-urethral region long term. The pSMCs can migrate to the liver without evidence of tumor formation or adverse clinical effect. The prolonged survival at the clinical site and the absence of malignancy support further translational research efforts utilizing pSMCs for treatment of stress incontinence.