Prenatal AAV9-GFP administration in fetal lambs results in transduction of female germ cells and maternal exposure to virus.

Prenatal somatic cell gene therapy (PSCGT) could potentially treat severe, early-onset genetic disorders such as spinal muscular atrophy (SMA) or muscular dystrophy. Given the approval of adeno-associated virus serotype 9 (AAV9) vectors in infants with SMA by the U.S. Food and Drug Administration, we tested the safety and biodistribution of AAV9-GFP (clinical-grade and dose) in fetal lambs to understand safety and efficacy after umbilical vein or intracranial injection on embryonic day 75 (E75) . Umbilical vein injection led to widespread biodistribution of vector genomes in all examined lamb tissues and in maternal uteruses at harvest (E96 or E140; term = E150). There was robust GFP expression in brain, spinal cord, dorsal root ganglia (DRGs), without DRG toxicity and excellent transduction of diaphragm and quadriceps muscles. However, we found evidence of systemic toxicity (fetal growth restriction) and maternal exposure to the viral vector (transient elevation of total bilirubin and a trend toward elevation in anti-AAV9 antibodies). There were no antibodies against GFP in ewes or lambs. Analysis of fetal gonads demonstrated GFP expression in female (but not male) germ cells, with low levels of integration-specific reads, without integration in select proto-oncogenes. These results suggest potential therapeutic benefit of AAV9 PSCGT for neuromuscular disorders, but warrant caution for exposure of female germ cells.

Recovering fetal signals transabdominally through interferometric near-infrared spectroscopy (iNIRS).

Noninvasive transabdominal fetal pulse oximetry can provide clinicians critical assessment of fetal health and potentially contribute to improved management of childbirth. Conventional pulse oximetry through continuous wave (CW) light has challenges measuring the signals from deep tissue and separating the weak fetal signal from the strong maternal signal. Here, we propose a new approach for transabdominal fetal pulse oximetry through interferometric near-infrared spectroscopy (iNIRS). This approach provides pathlengths of photons traversing the tissue, which facilitates the extraction of fetal signals by rejecting the very strong maternal signal from superficial layers. We use a multimode fiber combined with a mode-field converter at the detection arm to boost the signal of iNIRS. Together, we can detect signals from deep tissue (>∼1.6 cm in sheep abdomen and in human forearm) at merely 1.1 cm distance from the source. Using a pregnant sheep model, we experimentally measured and extracted the fetal heartbeat signals originating from deep tissue. This validated a key step towards transabdominal fetal pulse oximetry through iNIRS and set a foundation for further development of this method to measure the fetal oxygen saturation.

A bio-instructive parylene-based conformal coating suppresses thrombosis and intimal hyperplasia of implantable vascular devices.

Implantable vascular devices are widely used in clinical treatments for various vascular diseases. However, current approved clinical implantable vascular devices generally have high failure rates primarily due to their surface lacking inherent functional endothelium. Here, inspired by the pathological mechanisms of vascular device failure and physiological functions of native endothelium, we developed a new generation of bioactive parylene (poly(p-xylylene))-based conformal coating to address these challenges of the vascular devices. This coating used a polyethylene glycol (PEG) linker to introduce an endothelial progenitor cell (EPC) specific binding ligand LXW7 (cGRGDdvc) onto the vascular devices for preventing platelet adhesion and selectively capturing endogenous EPCs. Also, we confirmed the long-term stability and function of this coating in human serum. Using two vascular disease-related large animal models, a porcine carotid artery interposition model and a porcine carotid artery-jugular vein arteriovenous graft model, we demonstrated that this coating enabled rapid generation of self-renewable "living" endothelium on the blood contacting surface of the expanded polytetrafluoroethylene (ePTFE) grafts after implantation. We expect this easy-to-apply conformal coating will present a promising avenue to engineer surface properties of "off-the-shelf" implantable vascular devices for long-lasting performance in the clinical settings.

Evaluation of a biodegradable polyurethane patch for repair of diaphragmatic hernia in a rat model: A pilot study.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) repair is an area of active research. Large defects requiring patches have a hernia recurrence rate of up to 50%. We designed a biodegradable polyurethane (PU)-based elastic patch that matches the mechanical properties of native diaphragm muscle. We compared the PU patch to a non-biodegradable Gore-Tex™ (polytetrafluoroethylene) patch. METHODS: The biodegradable polyurethane was synthesized from polycaprolactone, hexadiisocyanate and putrescine, and then processed into fibrous PU patches by electrospinning. Rats underwent 4 mm diaphragmatic hernia (DH) creation via laparotomy followed by immediate repair with Gore-Tex™ (n = 6) or PU (n = 6) patches. Six rats underwent sham laparotomy without DH creation/repair. Diaphragm function was evaluated by fluoroscopy at 1 and 4 weeks. At 4 weeks, animals underwent gross inspection for recurrence and histologic evaluation for inflammatory reaction to the patch materials. RESULTS: There were no hernia recurrences in either cohort. Gore-Tex™ had limited diaphragm rise compared to sham at 4 weeks (1.3 mm vs 2.9 mm, p = 0.003), but no difference was found between PU and sham (1.7 mm vs 2.9 mm, p = 0.09). There were no differences between PU and Gore-Tex™ at any time point. Both patches formed an inflammatory capsule, with similar thicknesses between cohorts on the abdominal (Gore-Tex™ 0.07 mm vs. PU 0.13 mm, p = 0.39) and thoracic (Gore-Tex™ 0.3 mm vs. PU 0.6 mm, p = 0.09) sides. CONCLUSION: The biodegradable PU patch allowed for similar diaphragmatic excursion compared to control animals. There were similar inflammatory responses to both patches. Further work is needed to evaluate long-term functional outcomes and further optimize the properties of the novel PU patch in vitro and in vivo. LEVEL OF EVIDENCE: Level II, Prospective Comparative Study.

A bioactive material with dual integrin-targeting ligands regulates specific endogenous cell adhesion and promotes vascularized bone regeneration in adult and fetal bone defects.

Significant progress has been made in designing bone materials capable of directing endogenous cells to promote vascularized bone regeneration. However, current strategies lack regulation of the specific endogenous cell populations for vascularized bone regeneration, thus leading to adverse tissue formation and decreased regenerative efficiency. Here, we engineered a biomaterial to regulate endogenous cell adhesion and promote vascularized bone regeneration. The biomaterial works by presenting two synthetic ligands, LLP2A and LXW7, explicitly targeting integrins α4ß1 and αvß3, respectively, expressed on the surfaces of the cells related to bone formation and vascularization, such as mesenchymal stem cells (MSCs), osteoblasts, endothelial progenitor cells (EPCs), and endothelial cells (ECs). In vitro, the LLP2A/LXW7 modified biomaterial improved the adhesion of MSCs, osteoblasts, EPCs, and ECs via integrin α4ß1 and αvß3, respectively. In an adult rat calvarial bone defect model, the LLP2A/LXW7 modified biomaterial enhanced bone formation and vascularization by synergistically regulating endogenous cells with osteogenic and angiogenic potentials, such as DLX5+ cells, osteocalcin+ cells, CD34+/CD45- cells and CD31+ cells. In a fetal sheep spinal bone defect model, the LLP2A/LXW7 modified biomaterial augmented bone formation and vascularization without any adverse effects. This innovative biomaterial offers an off-the-shelf, easy-to-use, and biologically safe product suitable for vascularized bone regeneration in both fetal and adult disease environments.

Early investigations into improving bowel and bladder function in fetal ovine myelomeningocele repair.

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.

Long-term safety evaluation of placental mesenchymal stromal cells for in utero repair of myelomeningocele in a novel ovine model.

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.

Efficacy of clinical-grade human placental mesenchymal stromal cells in fetal ovine myelomeningocele repair.

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.

Preliminary Evaluation of a Novel Fetal Guinea Pig Myelomeningocele Model.

INTRODUCTION: Translational models of myelomeningocele (MMC) are needed to test novel in utero interventions. An ideal animal model for MMC has locomotor function at birth and is low cost enough to allow for high throughput. The rat MMC model is limited by immature locomotor function at birth. The ovine MMC model is a costly surgical model. Guinea pigs are uniquely suited for an MMC model being a small animal model with locomotor function at birth. We aimed to develop a retinoic acid (RA) model of MMC in the guinea pig and to evaluate if pregnant guinea pigs could tolerate uterine manipulation. METHODS: Time-mated Dunkin Hartley guinea pig dams were dosed with 60 mg/kg of RA between gestation age (GA) 12 and 15 days in the development of an RA model. Fetuses were grossly evaluated for MMC lesions at Cesarean section after GA 31 days. Evaluation of the ability of pregnant guinea pig dams to tolerate uterine surgical intervention was performed by hysterotomy of a separated group of time-mated guinea pigs at GA 45, 50, and 55. RESULTS: Forty-two pregnant guinea pigs were dosed with RA, with a total of 189 fetuses. The fetal demise rate was 38% (n = 71). A total of 118 fetuses were viable, 83% (n = 98) were normal fetuses, 8% (n = 10) had a neural tube defect, and 8% (n = 10) had a hematoma or other anomalies. No fetuses developed an MMC defect. None of the fetuses that underwent hysterotomy survived to term. CONCLUSION: RA dosed at 60 mg/kg in guinea pigs between GA 12 and 15 did not result in MMC. Dunkin Hartley guinea pigs did not tolerate a hysterotomy near term in our surgical model. Further work is needed to determine if MMC can be induced in guinea pigs with alternate RA dosing.

Placental Mesenchymal Stromal Cells: Preclinical Safety Evaluation for Fetal Myelomeningocele Repair.

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.

A Novel Model of Fetal Spinal Cord Exposure Allowing for Long-Term Postnatal Survival.

BACKGROUND: The inherent morbidity associated with fetal ovine models of myelomeningocele (MMC) has created challenges for long-term survival of lambs. We aimed to develop a fetal ovine surgical spinal exposure model which could be used to evaluate long-term safety after direct spinal cord application of novel therapeutics for augmentation of in utero MMC repair. METHODS: At gestational age (GA) 100-106, fetal lambs underwent surgical intervention. Laminectomy of L5-L6 was performed, dura was removed, and an experimental product was directly applied to the spinal cord. Paraspinal muscles and skin were closed and the fetus was returned to the uterus. Lambs were delivered via cesarean section at GA 140-142. Lambs were survived for 3 months with regular evaluation of motor function by the sheep locomotor rating scale. Spinal angulation was evaluated by magnetic resonance imaging at 2 weeks and 3 months. RESULTS: Five fetal surgical intervention lambs and 6 control lambs who did not undergo surgical intervention were included. All lambs survived to the study endpoint of 3 months. No lambs had motor function abnormalities or increased spinal angulation. CONCLUSION: This model allows for long-term survival after fetal spinal cord exposure with product application directly onto the spinal cord.

Diagnosing Hirschsprung disease by detecting intestinal ganglion cells using label-free hyperspectral microscopy.

Hirschsprung disease (HD) is a congenital disorder in the distal colon that is characterized by the absence of nerve ganglion cells in the diseased tissue. The primary treatment for HD is surgical intervention with resection of the aganglionic bowel. The accurate identification of the aganglionic segment depends on the histologic evaluation of multiple biopsies to determine the absence of ganglion cells in the tissue, which can be a time-consuming procedure. We investigate the feasibility of using a combination of label-free optical modalities, second harmonic generation (SHG); two-photon excitation autofluorescence (2PAF); and Raman spectroscopy (RS), to accurately locate and identify ganglion cells in murine intestinal tissue without the use of exogenous labels or dyes. We show that the image contrast provided by SHG and 2PAF signals allows for the visualization of the overall tissue morphology and localization of regions that may contain ganglion cells, while RS provides detailed multiplexed molecular information that can be used to accurately identify specific ganglion cells. Support vector machine, principal component analysis and linear discriminant analysis classification models were applied to the hyperspectral Raman data and showed that ganglion cells can be identified with a classification accuracy higher than 95%. Our findings suggest that a near real-time intraoperative histology method can be developed using these three optical modalities together that can aid pathologists and surgeons in rapid, accurate identification of ganglion cells to guide surgical decisions with minimal human intervention.

Minimizing the risk of occupational Q fever exposure: A protocol for ensuring Coxiella burnetii-negative pregnant ewes are used for medical research.

Q fever is a worldwide zoonosis caused by Coxiella burnetii that can lead to abortion, endocarditis, and death in humans. Researchers utilizing parturient domestic ruminants, including sheep, have an increased risk of occupational exposure. This study evaluated the effectiveness of our screening protocol in eliminating C. burnetii-positive sheep from our facility. From August 2010 to May 2018, all ewes (N = 306) and select lambs (N = 272; ovis aries) were screened twice for C. burnetii utilizing a serum Phase I and Phase II antibody immunofluorescence assay (IFA). The first screen was performed by the vendor prior to breeding, and the second screen was performed on arrival to the research facility. Ewes that were positive on arrival screening were quarantined and retested using repeat IFA serology, enzyme-linked immunosorbent assay, buffy coat polymerase chain reaction (PCR), and amniotic fluid PCR. The overall individual seroprevalence of C. burnetii in the flocks tested by the vendor was 14.2%. Ewes with negative Phase I and Phase II IFA results were selected for transport to the research facility. Upon arrival to the facility, two (0.7%) ewes had positive Phase I IFA results. Repeat testing demonstrated seropositivity in one of these two ewes, though amniotic fluid PCR was negative in both. The repeat seropositive ewe was euthanized prior to use in a research protocol. No Q fever was reported among husbandry, laboratory or veterinary staff during the study period. Serologic testing for C. burnetii with IFA prior to transport and following arrival to a research facility limits potential exposure to research staff.

Design and In Vivo Evaluation of a Non-Invasive Transabdominal Fetal Pulse Oximeter.

OBJECTIVE: Current intrapartum fetal monitoring technology is unable to provide physicians with an objective metric of fetal well-being, leading to degraded patient outcomes and increased litigation costs. Fetal oxygen saturation (SpO2) is a more suitable measure of fetal distress, but the inaccessibility of the fetus prior to birth makes this impossible to capture through current means. In this paper, we present a fully non-invasive, transabdominal fetal oximetry (TFO) system that provides in utero measures of fetal SpO2. METHODS: TFO is performed by placing a reflectance-mode optode on the maternal abdomen and sending photons into the body to investigate the underlying fetal tissue. The proposed TFO system design consists of a multi-detector optode, an embedded optode control system, and custom user-interface software. To evaluate the developed TFO system, we utilized an in utero hypoxic fetal lamb model and performed controlled desaturation experiments while capturing gold standard arterial blood gases (SaO2). RESULTS: Various degrees of fetal hypoxia were induced with true SaO2 values ranging between 10.5% and 66%. The non-invasive TFO system was able to accurately measure these fetal SpO2 values, supported by a root mean-squared error of 6.37% and strong measures of agreement with the gold standard. CONCLUSION: The results support the efficacy of the presented TFO system to non-invasively measure a wide-range of fetal SpO2 values and identify critical levels of fetal hypoxia. SIGNIFICANCE: TFO has the potential to improve fetal outcomes by providing obstetricians with a non-invasive measure of fetal oxygen saturation prior to delivery.

Surviving Lambs with Myelomeningocele Repaired in utero with Placental Mesenchymal Stromal Cells for 6 Months: A Pilot Study.

BACKGROUND: Fetal repair of myelomeningocele (MMC) with placental mesenchymal stromal cells (PMSCs) rescues ambulation in the ovine model up to 48 h postnatally. Outcomes past 48 h are unknown as MMC lambs have not been survived past this timepoint. OBJECTIVE: We aimed to survive lambs for 6 months following the fetal repair of MMC with PMSCs. METHODS: Fetal MMC lambs were repaired with PMSCs. Lambs received either no additional treatment or postnatal bracing and physical therapy (B/PT). Motor function was assessed with the sheep locomotor rating (SLR). Lambs with an SLR of 15 at birth were survived for 6 months or until a decline in SLR less than 15, whichever came first. All lambs underwent a perimortem MRI. RESULTS: The lambs with no postnatal treatment (n = 2) had SLR declines to 7 and 13 at 29 and 65 days, respectively, and were euthanized. These lambs had a spinal angulation of 57° and 47°, respectively. The B/PT lamb (n = 1) survived for 6 months with a sustained SLR of 15 and a lumbar angulation of 42°. CONCLUSION: Postnatal physical therapy and bracing counteracted the inherent morbidity of the absent paraspinal muscles in the ovine MMC model allowing for survival and maintenance of rescued motor function of the prenatally treated lamb up to 6 months.

Validation of a Novel Transabdominal Fetal Oximeter in a Hypoxic Fetal Lamb Model.

Current intrapartum fetal oxygen saturation (SaO2) monitoring methodologies are limited, mostly consisting of fetal heart rate monitoring which is a poor predictor of fetal hypoxia. A newly developed transabdominal fetal oximeter (TFO) may be able to determine fetal SaO2 non-invasively. This study is to validate a novel TFO in determining fetal SaO2 in a hypoxic fetal lamb model. Fetal hypoxia was induced in at-term pregnant ewe by placing an aortic occlusion balloon infrarenally and inflating it in a stepwise fashion to decrease blood flow to the uterine artery. The inflation was held at each step for 10 min, and fetal arterial blood gases (ABGs) were intermittently recorded from the fetal carotid artery. The balloon catheter was deflated when fetal SaO2 fell below 15%, and the fetus was recovered. A total of three desaturation experiments were performed. The average fetal SpO2 reported by the TFO was derived at each hypoxic level and correlated with the ABG measures. Fetal SaO2 from the ABGs ranged from 10.5 to 66%. The TFO SpO2 correlated with the ABG fetal SaO2 (r-squared = 0.856) with no significant differences (p > 0.5). The fetal SpO2 measurements from TFO were significantly different than the maternal SpO2 (p < 0.01), which suggests that the transcutaneous measurements are penetrating through the maternal abdomen sufficiently and are expressing the underlying fetal tissue physiology. The recently developed TFO system was able to non-invasively report the fetal SpO2, which showed strong correlation with ABG measures and showed no significant differences.

Rapid endothelialization of small diameter vascular grafts by a bioactive integrin-binding ligand specifically targeting endothelial progenitor cells and endothelial cells.

Establishing and maintaining a healthy endothelium on vascular and intravascular devices is crucial for the prevention of thrombosis and stenosis. Generating a biofunctional surface on vascular devices to recruit endothelial progenitor cells (EPCs) and endothelial cells (ECs) has proven efficient in promoting in situ endothelialization. However, molecules conventionally used for EPC/EC capturing generally lack structural stability, capturing specificity, and biological functionalities, which have limited their applications. Discovery of effective, specific, and structurally stable EPC/EC capturing ligands is desperately needed. Using the high-throughput One-Bead One-Compound combinatorial library screening technology, we recently identified a disulfide cyclic octa-peptide LXW7 (cGRGDdvc), which possesses strong binding affinity and functionality to EPCs/ECs, weak binding to platelets, and no binding to inflammatory cells. Because LXW7 is cyclic and 4 out of the 8 amino acids are unnatural D-amino acids, LXW7 is highly proteolytically stable. In this study, we applied LXW7 to modify small diameter vascular grafts using a Click chemistry approach. In vitro studies demonstrated that LXW7-modified grafts significantly improved EPC attachment, proliferation and endothelial differentiation and suppressed platelet attachment. In a rat carotid artery bypass model, LXW7 modification of the small diameter vascular grafts significantly promoted EPC/EC recruitment and rapidly achieved endothelialization. At 6 weeks after implantation, LXW7-modified grafts retained a high patency of 83%, while the untreated grafts had a low patency of 17%. Our results demonstrate that LXW7 is a potent EPC/EC capturing and platelet suppressing ligand and LXW7-modified vascular grafts rapidly generate a healthy and stable endothelial interface between the graft surface and the circulation to reduce thrombosis and improve patency. STATEMENT OF SIGNIFICANCE: In this study, One-Bead One-Compound (OBOC) technology has been applied for the first time in discovering bioactive ligands for tissue regeneration applications. Current molecules used to modify artificial vascular grafts generally lack EPC/EC capturing specificity, biological functionalities and structural stability. Using OBOC technology, we identified LXW7, a constitutionally stable disulfide cyclic octa-peptide with strong binding affinity and biological functionality to EPCs/ECs, very weak binding to platelets and no binding to inflammatory cells. These characteristics are crucial for promoting rapid endothelialization to prevent thrombosis and improve patency of vascular grafts. LXW7 coating technology could be applied to a wide range of vascular and intravascular devices, including grafts, stents, cardiac valves, and catheters, where a "living" endothelium and healthy blood interface are needed.

A Decade of Experience with the Ovine Model of Myelomeningocele: Risk Factors for Fetal Loss.

INTRODUCTION: The ovine model is the gold standard large animal model of myelomeningocele (MMC); however, it has a high rate of fetal loss. We reviewed our experience with the model to determine risk factors for fetal loss. METHODS: We performed a retrospective review from 2009 to 2018 to identify operative factors associated with fetal loss (early fetal demise, abortion, or stillbirth). Operative risk factors included gestational age at operation, operative time, reduction of multiple gestations, amount of replaced amniotic fluid, ambient temperature, and method of delivery. RESULTS: MMC defects were created in 232 lambs with an overall survival rate of 43%. Of the 128 fetuses that died, 53 (42%) had demise prior to repair, 61 (48%) aborted, and 14 (11%) were stillborn. Selective reduction of multiple gestations in the same uterine horn was associated with increased fetal demise (OR 3.03 [95% CI 1.29-7.05], p = 0.01). Later gestational age at MMC repair and Cesarean delivery were associated with decreased abortion/stillbirth (OR 0.90 [95% CI 0.83-0.90], p = 0.03, and OR 0.37 [95% CI 0.16-0.31], p = 0.02), respectively. CONCLUSION: Avoiding selective reduction, repairing MMC later in gestation, and performing Cesarean delivery decreases the rate of fetal loss in the ovine MMC model.

In utero treatment of myelomeningocele with placental mesenchymal stromal cells - Selection of an optimal cell line in preparation for clinical trials.

BACKGROUND: We determined whether in vitro potency assays inform which placental mesenchymal stromal cell (PMSC) lines produce high rates of ambulation following in utero treatment of myelomeningocele in an ovine model. METHODS: PMSC lines were created following explant culture of three early-gestation human placentas. In vitro neuroprotection was assessed with a neuronal apoptosis model. In vivo, myelomeningocele defects were created in 28 fetuses and repaired with PMSCs at 3 × 105 cells/cm2 of scaffold from Line A (n = 6), Line B (n = 7) and Line C (n = 5) and compared to no PMSCs (n = 10). Ambulation was scored as ≥13 on the Sheep Locomotor Rating Scale. RESULTS: In vitro, Line A and B had higher neuroprotective capability than no PMSCs (1.7 and 1.8 respectively vs 1, p = 0.02, ANOVA). In vivo, Line A and B had higher large neuron densities than no PMSCs (25.2 and 27.9 respectively vs 4.8, p = 0.03, ANOVA). Line C did not have higher neuroprotection or larger neuron density than no PMSCs. In vivo, Line A and B had ambulation rates of 83% and 71%, respectively, compared to 60% with Line C and 20% with no PMSCs. CONCLUSION: The in vitro neuroprotection assay will facilitate selection of optimal PMSC lines for clinical use. LEVEL OF EVIDENCE: n/a. TYPE OF STUDY: Basic science.

Placental Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Myelin Regeneration in an Animal Model of Multiple Sclerosis.

Mesenchymal stem/stromal cells (MSCs) display potent immunomodulatory and regenerative capabilities through the secretion of bioactive factors, such as proteins, cytokines, chemokines as well as the release of extracellular vesicles (EVs). These functional properties of MSCs make them ideal candidates for the treatment of degenerative and inflammatory diseases, including multiple sclerosis (MS). MS is a heterogenous disease that is typically characterized by inflammation, demyelination, gliosis and axonal loss. In the current study, an induced experimental autoimmune encephalomyelitis (EAE) murine model of MS was utilized. At peak disease onset, animals were treated with saline, placenta-derived MSCs (PMSCs), as well as low and high doses of PMSC-EVs. Animals treated with PMSCs and high-dose PMSC-EVs displayed improved motor function outcomes as compared to animals treated with saline. Symptom improvement by PMSCs and PMSC-EVs led to reduced DNA damage in oligodendroglia populations and increased myelination within the spinal cord of treated mice. In vitro data demonstrate that PMSC-EVs promote myelin regeneration by inducing endogenous oligodendrocyte precursor cells to differentiate into mature myelinating oligodendrocytes. These findings support that PMSCs' mechanism of action is mediated by the secretion of EVs. Therefore, PMSC-derived EVs are a feasible alternative to cellular based therapies for MS, as demonstrated in an animal model of the disease.