Hemophilia A (HA) is a bleeding disorder characterized by spontaneous and prolonged hemorrhage. The disease is caused by mutations in the coagulation factor 8 gene (F8) leading to factor VIII (FVIII) deficiency. Since FVIII is primarily produced in endothelial cells (ECs) in a non-diseased human being, ECs hold great potential for development as a cell therapy for HA. We showed that HA patient-specific induced pluripotent stem cells (HA-iPSCs) could provide a renewable supply of ECs. The HA-iPSC-derived ECs were transduced with lentiviral vectors to stably express the functional B domain deleted F8 gene, the luciferase gene, and the enhanced green fluorescent protein gene (GFP). When transplanted intramuscularly into neonatal and adult immune deficient mice, the HA-iPSC-derived ECs were retained in the animals for at least 10-16 weeks and maintained their expression of FVIII, GFP, and the endothelial marker CD31, as demonstrated by bioluminescence imaging and immunostaining, respectively. When transplanted into HA mice, these transduced HA-iPSC-derived ECs significantly reduced blood loss in a tail-clip bleeding test and produced therapeutic plasma levels (11.2%-369.2%) of FVIII. Thus, our studies provide proof-of-concept that HA-iPSC-derived ECs can serve as a factory to deliver FVIII for the treatment of HA not only in adults but also in newborns.

Purpose

Augmentation of in utero myelomeningocele repair with human placental mesenchymal stromal cells seeded onto extracellular matrix (PMSC-ECM) improves motor outcomes in an ovine myelomeningocele model. This study evaluated the safety of PMSC-ECM application directly onto the fetal spinal cord in preparation for a clinical trial.

Methods

Laminectomy of L5-L6 with PMSC-ECM placement directly onto the spinal cord was performed in five fetal lambs at gestational age (GA) 100-106 days. Lambs and ewes were monitored for three months following delivery. Lambs underwent magnetic resonance imaging (MRI) of the brain and spine at birth and at three months. All organs from lambs and uteri from ewes underwent histologic evaluation. Lamb spinal cords and brains and ewe placentas were evaluated for persistence of PMSCs by polymerase chain reaction for presence of human DNA.

Results

MRIs demonstrated no evidence of abnormal tissue growth or spinal cord tethering. Histological analysis demonstrated no evidence of abnormal tissue growth or treatment related adverse effects. No human DNA was identified in evaluated tissues.

Conclusion

There was no evidence of abnormal tissue growth or PMSC persistence at three months following in utero application of PMSC-ECM to the spinal cord. This supports proceeding with clinical trials of PMSC-ECM for in utero myelomeningocele repair.

Level of evidence

N/A TYPE OF STUDY: Basic science.

Background

Myelomeningocele (MMC) is the congenital failure of neural tube closure in utero, for which the standard of care is prenatal surgical repair. We developed clinical-grade placental mesenchymal stromal cells seeded on a dural extracellular matrix (PMSC-ECM), which have been shown to improve motor outcomes in preclinical ovine models. To evaluate the long-term safety of this product prior to use in a clinical trial, we conducted safety testing in a murine model.

Methods

Clinical grade PMSCs obtained from donor human placentas were seeded onto a 6 mm diameter ECM at a density of 3 × 10⁵ cells/cm². Immunodeficient mice were randomized to receive either an ECM only or PMSC-ECM administered into a subcutaneous pocket. Mice were monitored for tumor formation until two study endpoints: 4 wk and 6 mo. Pathology and histology on all tissues was performed to evaluate for tumors. Quantitative polymerase chain reaction (qPCR) was performed to evaluate for the presence of human DNA, which would indicate persistence of PMSCs.

Results

Fifty-four mice were included; 13 received ECM only and 14 received PMSC-ECM in both the 4-wk and 6-mo groups. No mice had gross or microscopic evidence of tumor development. A nodular focus of mature fibrous connective tissue was identified at the subcutaneous implantation pocket in the majority of mice with no significant difference between ECM only and PMSC-ECM groups (P = 0.32 at 4 wk, P > 0.99 at 6 mo). Additionally, no human DNA was detected by qPCR in any mice at either time point.

Conclusions

Subcutaneous implantation of the PMSC-ECM product did not result in tumor formation and we found no evidence that PMSCs persisted. These results support the safety of the PMSC-ECM product for use in a Phase 1/2a human clinical trial evaluating fetal MMC repair augmented with PMSC-ECM.

Background

While fetal repair of myelomeningocele (MMC) revolutionized management, many children are still unable to walk independently. Preclinical studies demonstrated that research-grade placental mesenchymal stromal cells (PMSCs) prevent paralysis in fetal ovine MMC, however this had not been replicated with clinical-grade cells that could be used in an upcoming human clinical trial. We tested clinical-grade PMSCs seeded on an extracellular matrix (PMSC-ECM) in the gold standard fetal ovine model of MMC.

Methods

Thirty-five ovine fetuses underwent MMC defect creation at a median of 76 days gestational age, and defect repair at 101 days gestational age with application of clinical-grade PMSC-ECM (3 × 10⁵ cells/cm², n = 12 fetuses), research-grade PMSC-ECM (3 × 10⁵ cells/cm², three cell lines with n = 6 (Group 1), n = 6 (Group 2), and n = 3 (Group 3) fetuses, respectively) or ECM without PMSCs (n = 8 fetuses). Three normal lambs underwent no surgical interventions. The primary outcome was motor function measured by the Sheep Locomotor Rating scale (SLR, range 0: complete paralysis to 15: normal ambulation) at 24 h of life. Correlation of lumbar spine large neuron density with SLR was evaluated.

Results

Clinical-grade PMSC-ECM lambs had significantly better motor function than ECM-only lambs (SLR 14.5 vs. 6.5, p = 0.04) and were similar to normal lambs (14.5 vs. 15, p = 0.2) and research-grade PMSC-ECM lambs (Group 1: 14.5 vs. 15, p = 0.63; Group 2: 14.5 vs. 14.5, p = 0.86; Group 3: 14.5 vs. 15, p = 0.50). Lumbar spine large neuron density was strongly correlated with motor function (r = 0.753, p<0.001).

Conclusions

Clinical-grade placental mesenchymal stromal cells seeded on an extracellular matrix rescued ambulation in a fetal ovine myelomeningocele model. Lumbar spine large neuron density correlated with motor function, suggesting a neuroprotective effect of the PMSC-ECM in prevention of paralysis. A first-in-human clinical trial of PMSCs in human fetal myelomeningocele repair is underway.

Introduction

Fetal myelomeningocele (MMC) repair improves lower extremity motor function. We have previously demonstrated that augmentation of fetal MMC repair with placental mesenchymal stromal cells (PMSCs) seeded on extracellular matrix (PMSC-ECM) further improves motor function in the ovine model. However, little progress has been made in improving bowel and bladder function, with many patients suffering from neurogenic bowel and bladder. We hypothesized that fetal MMC repair with PMSC-ECM would also improve bowel and bladder function.

Methods

MMC defects were surgically created in twelve ovine fetuses at median gestational age (GA) 73 days, followed by defect repair at GA101 with PMSC-ECM. Fetuses were delivered at GA141. Primary bladder function outcomes were voiding posture and void volumes. Primary bowel function outcome was anorectal manometry findings including resting anal pressure and presence of rectoanal inhibitory reflex (RAIR). Secondary outcomes were anorectal and bladder detrusor muscle thickness. PMSC-ECM lambs were compared to normal lambs (n = 3).

Results

Eighty percent of PMSC-ECM lambs displayed normal voiding posture compared to 100% of normal lambs (p = 1). Void volumes were similar (PMSC-ECM 6.1 ml/kg vs. normal 8.8 ml/kg, p = 0.4). Resting mean anal pressures were similar between cohorts (27.0 mmHg PMSC-ECM vs. normal 23.5 mmHg, p = 0.57). RAIR was present in 3/5 PMSC-ECM lambs that underwent anorectal manometry and all normal lambs (p = 0.46). Thicknesses of anal sphincter complex, rectal wall muscles, and bladder detrusor muscles were similar between cohorts.

Conclusion

Ovine fetal MMC repair augmented with PMSC-ECM results in near-normal bowel and bladder function. Further work is needed to evaluate these outcomes in human patients.