Abstract
Introduction:
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 6mm diameter ECM at a density of 3x105 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 weeks and 6 months. 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-week and 6-month 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 weeks, p>0.99 at 6 months). 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.
Keywords: Stromal cells, tumorigenicity, safety, cellular retention, myelomeningocele
Introduction
Myelomeningocele (MMC), the most severe form of spina bifida, is the congenital failure of the closure of the neural tube in utero. This results in progressive spinal cord damage via chemical and mechanical trauma from both amniotic fluid and the uterine wall. The intra-uterine spinal cord damage that occurs can then result in varying degrees of loss of lower limb motor function and can even result in complete paralysis for the child after birth. Historically MMC was treated with postnatal repair of the spinal cord defect, but prenatal repair became the standard of care after the landmark Management of Myelomeningocele Study (MOMS)(1). Prenatal surgery resulted in improvements in motor function, and when evaluated at 30 months, infants in the prenatal surgery group were more likely than those in the postnatal surgery group to be able to walk without orthotics or devices (44.8% vs. 23.9%, p=0.004)(2). These results demonstrated the potential for improvement in motor function, suggesting that the lost neurologic function of the damaged spinal cord could be restored or protected from further damage by operating in utero. However, there remains substantial room for improvement, as the majority of prenatally repaired children were still unable to walk independently at 30 months old (1).
We have developed a stromal cell product to be used to augment the prenatal surgery with the aim of further improving motor function. The development of this product was established after several years of investigating different treatment modalities, such as induced pluripotent stem cell-derived neural crest stem cells(3) and amniotic membrane patches(4). We have found that placental mesenchymal stromal cells (PMSCs) represent the best therapeutic candidate for in utero treatment of MMC due to their superior paracrine secretion and neuroprotective capabilities(5, 6). Furthermore, we found that PMSCs can be rapidly expanded and banked in viable cell banks and that PMSCs can be delivered on an extracellular matrix (ECM) while maintaining cell viability and protein secretion(7). We have previously demonstrated that PMSCs seeded on a dural extracellular matrix (PMSC-ECM) improves motor function when used in the in utero repair of MMC in a fetal ovine model(8). In order to augment the in utero repair of the MMC defect in human patients, we plan to use this PMSC-ECM product in a first-in-human clinical trial. However, the long-term safety of the PMSC-ECM product, including lack of tumorigenicity, has not been established as the surgical MMC defect creation in the ovine model makes long-term lamb survival difficult(9, 10). Evaluation of long-term safety of this product prior to use in human patients is necessary.
In order to support the clinical use of our PMSC-ECM product, manufactured under Current Good Manufacturing Practice (CGMP), this study aimed to evaluate the preclinical safety of our PMSC-ECM in an established tumorigenicity murine model. This specific murine model was designed to test our PMSC-ECM product in accordance with Food & Drug Administration (FDA) recommendations, as there is limited data regarding the safety of PMSCs administration by subcutaneous implantation, as most PMSC safety studies evaluate the safety after either intravenous or intramuscular injection. We hypothesized that the clinical grade PMSC-ECM product would not result in local or distant tumor formation in this immunodeficient mouse model. PMSCs have been demonstrated to exist transiently in tissues and to act via a paracrine manner, we therefore also hypothesized that PMSCs, measured by presence of human DNA, would not be detectable in the mouse tissue by the study endpoints.
Materials and Methods
Murine Model
The University of California Davis Institutional Animal Care and Use Committee (IACUC) approved all animal protocols, and all animal care was in compliance with the Guide for the Care and Use of Laboratory Animals. All facilities used during the study period were accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.
NOD/SCID/Gamma−/− (NSG) mice were used for this study, due to their immunodeficient status and propensity for tumor formation. The NOD/SCID/Gamma−/− (NSG) murine model is the long standing, standard model for a tumorigenicity study(11-13). These mice were randomized into one of two study groups: ECM only or PMSC-ECM. For each group there were two study endpoints with half of the mice being survived to 4 weeks and the other half to 6 months (Table 1) to evaluate for both short- and long-term tumorigenicity.
Table 1:
Study Design and Group Numbers. ECM: extracellular matrix; PMSC-ECM: placental mesenchymal stromal cell seeded on extracellular matrix.
| Study Groups | # Males / # Females |
Sacrifice Time Point |
|---|---|---|
| ECM only | 6/7 | 4 Weeks |
| ECM only | 6/7 | 6 Months |
| PMSC-ECM | 7/7 | 4 Weeks |
| PMSC-ECM | 7/7 | 6 Months |
PMSC-ECM Preparation
Based on Food & Drug Administration (FDA) guidelines for the manufacturing of cellular therapy products, a manufacturing plan for donor eligibility and consent, generation of PMSCs under Current Good Manufacturing Practice (CGMP), and screening criteria of the banked PMSCs was established and Institutional Review Board approval was obtained (IRB #796298). All product manufacturing procedures were conducted in the GMP Facility under CGMP conditions at the University of California, Davis (UC Davis) by strictly following the approved Standard Operating Procedures (SOPs). The extracellular matrix (ECM) used was an FDA-approved clinical grade Biodesign® Dural Graft Extracellular Matrix (Cook Biotech Incorporated, Indiana, USA).
PMSCs used for this study were from one of our expanded product banks at passage 4. PMSCs in the cell banks were from consented donors that underwent rigorous screening and release testing as follows. Initial placental tissue donor infectious disease analysis consisted of Cytomegalovirus (CMV), Hepatitis B and C, Human Immunodeficiency Virus (HIV) 1&2, Human T-cell Lymphotropic Virus (HTLV) 1&2, West Nile Virus, Zika, Syphilis, Chlamydia, and Gonorrhea. Seed banks were tested with 14-day sterility testing, mycoplasma testing, and endotoxin testing. Product bank cells were also tested with 14-day sterility testing, mycoplasma testing, endotoxin testing, bovine virus testing, adventitious virus testing, human infectious virus testing. All infectious testing was negative for the PMSCs and product bank used for this study.
72 hours prior to the date of each surgery, one vial of the product bank was thawed and seeded in tissue-culture treated flasks at 2x104 cells/cm2 and was cultured for 48 hours at 37°C, 5% CO2. After 48 hours, the cells were lifted and seeded onto a 6mm ECM punch-out that had been preequilibrated for 24 hours in PMSC Growth Medium at 3x105 cells/cm2 and cultured for additional 24 hours. For the ECM only group, ECMs were prepared exactly the same as the PMSC-ECM aside from the seeding of cells onto the ECM. On the day of surgery and prior to releasing the product for use in mice, to verify the adherence of PMSCs to the ECM and PMSC viability, PMSC-ECMs were stained with CalceinAM, a live cell viability stain, and imaged (Figure 1). After confirming viability and adherence, the PMSC-ECMs and ECMs without PMSCs were delivered on ice with a temperature recorder to the Immune Deficient Mouse Core Facility at the UC Davis Institute for Regenerative Cures (IRC). The surgeons were blinded as to which product was applied to the mice.
Figure 1:
PMSC Viability and Adherence. Representative PMSC-ECM stained with CalceinAM and imaged to assess for PMSC (green) adherence and viability after seeding on the ECM.
Surgery and Route of Administration
All procedures were done using proper sterile technique to reduce the risk of infection. After induction of anesthesia using inhaled isoflurane at 2%, a 1-2 cm skin incision was made on the right upper back of the mouse. The subcutaneous route of administration and this particular location were chosen as the mice were unable to reach the incision, thus reducing the chance of inadvertent premature staple removal and compromising the product. Mice were randomized to receive an ECM only or PMSC-ECM. A small subcutaneous pocket was created and either an ECM only or a PMSC-ECM was placed in this pocket. The incision was then closed with skin staples which were removed 7-10 days post-operatively.
Monitoring
All animals were monitored for up to 4 weeks or 6 months following surgery. Animals underwent regular observation, body weight measurements, palpation for tumors, and measurements of tumor size (if applicable). The animals were observed daily for the first week and then twice weekly for the remainder of the study period. According to IACUC policy on humane endpoints we planned to euthanize any animals that appeared sick or a developed a palpable tumor > 1.5 cm.
The blinded coding scheme was shared with the quality assurance director and was kept confidential. Research personnel, surgeons, and laboratory staff were kept blinded to animal study groups.
Euthanasia & Data Collection
At the study endpoints, animals were humanely euthanized by CO2 asphyxiation. Immediately after euthanization, blood was drawn from animals by cardiac puncture. Laboratory tests including a complete blood count, liver function tests, and complete chemistry panel were performed to evaluate for any potential adverse effects from the PMSCs. Tissues were harvested immediately after the animals were euthanized and processed by laboratory personnel at the UC Davis Comparative Laboratory (CPL), all of whom were also blinded to the animal study groups. The following tissues were harvested and evaluated by histological analysis: bilateral adrenal glands, bilateral kidneys, bilateral gonads, spleen, liver, heart, brain, lungs.
To evaluate for the persistence of human PMSCs in the mice, the presence or absence of human DNA was evaluated by quantitative polymerase chain reaction (qPCR) using human-specific primers and probe, detecting the human endogenous retrovirus gene hERV3. The primers were first validated using human placenta DNA as positive control using 10-fold dilutions of DNA. Mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous quality control for DNA extraction. Commercially available GAPDH specific primers were used (Taqman (FAM/MGB probes) assay number: Mm99999915_g1). The cycling conditions were 2 minutes at 50°C, 10 minutes at 95°C, 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Fluorescent signals were collected during the annealing temperature and quantification cycle (Cq) values extracted with a threshold of 0.1 and baseline values of 3-12. qPCR testing was done on tissue from the implantation site and from the lung. The lungs were chosen as an additional site of qPCR evaluation because intravenously injected MSCs localize predominately to lung tissue (14-16). For interpretation, the detection limit of our assay was 1pg hDNA that has a Ct value of 29 cycles from the standard curve. There was also a pre-amplification of 15 cycles, followed by an additional 40 cycles. Therefore, any Ct value of greater than 38 can be strongly interpreted as negative. In addition, all qPCR samples were run in triplicates per the standard protocol. We thus regarded values as a false positive if only one of three Ct values was positive, as if human DNA was present it would be expected that there be detectable Ct values for all three triplicate runs, as is standard in interpretation of qPCR data.
Statistical Analysis
Continuous variables are reported as the median and interquartile range (IQR). Categorical variables are reported as frequencies and percentages. For continuous outcomes, bivariate associations were discerned using the Wilcoxon-Mann-Whitney test. All tests were 2-sided and used significance level of p=0.05. Statistical analysis was completed using GraphPad Prism (Version 8, La Jolla, CA, USA).
Results
In total, 54 mice underwent surgery; 26 received an ECM only and 28 received a PMSC-ECM. The final group numbers for the 4-week time point were: ECM only (6 males, 7 females), PMSC-ECM (7 males, 7 females). The final group numbers for the 6-month time point were: ECM only (6 males, 7 females), PMSC-ECM (7 males, 7 females) (Table 1).
There were no adverse effects observed in any of the groups at any point during monitoring up to 4 weeks or 6 months. No changes in weight, grooming, breathing, eating, or behavior were observed in the course of the 4-week or 6-month monitoring periods. No animal required early euthanasia according to the IACUC guidelines. There was no gross tumor formation identified in any animal.
For all animals at both time points, there were no unexpected abnormalities noted in blood samples (Tables 2 & 3). When comparing the median blood sample values between ECM only and PMSC-ECM animals, the only significant difference at the 4-week time point was glucose values. The PMSC-ECM median glucose value was significantly higher than the ECM only median glucose, however both values were within the normal reference range. At the 6-month time point, there were significant differences in albumin, blood urea nitrogen (BUN), and chloride. For both chloride and BUN, despite there being a difference between the two groups, values in both groups were within the normal reference ranges. For albumin, the ECM only group had a higher median of 4.1 compared to 3.9 in the PMSC-ECM group, which was just above the upper limit of normal (4.0).
Table 2:
4-week time point blood results summary. IQR: interquartile range; WBC: white blood cell; ECM: extracellular matrix; PMSC-ECM: placental mesenchymal stromal cell seeded on extracellular matrix. P value reflects comparison of the medians between groups. *Statistically significant.
| Lab Value | Group | Median | IQR | Normal Range | p value |
|---|---|---|---|---|---|
| WBC (K/μl) | ECM Only | 2.4 | 1.5-3.6 | 5.1-14.7 | 0.36 |
| PMSC-ECM | 1.6 | 1.2-5.0 | |||
| Hemoglobin (g/dL) | ECM Only | 14.0 | 13.3-14.8 | 11.7-16.2 | 0.93 |
| PMSC-ECM | 14.1 | 13.1-14.9 | |||
| Hematocrit % | ECM Only | 50.0 | 46.2-51.9 | 38.3-54.0 | 0.77 |
| PMSC-ECM | 58.5 | 46.6-51.6 | |||
| Platelets (K/μL) | ECM Only | 1036.0 | 891.5-1476.0 | 574-1079 | 0.76 |
| PMSC-ECM | 1062.0 | 705.3-1491.0 | |||
| Alanine Transaminase U/L | ECM Only | 39.0 | 23.5-67.9 | 0-403 | 0.69 |
| PMSC-ECM | 33.4 | 26.5-46.4 | |||
| Albumin g/dL | ECM Only | 3.7 | 3.5-4.0 | 2.9-4.0 | 0.69 |
| PMSC-ECM | 3.8 | 3.5-3.9 | |||
| Alkaline Phosphatase U/L | ECM Only | 105.3 | 95.2-152.7 | 49-172 | 0.38 |
| PMSC-ECM | 114.4 | 89.4-132.5 | |||
| Amylase U/L | ECM Only | 3702.0 | 3165.0-4215.0 | 2463-6660 | 0.22 |
| PMSC-ECM | 3242.0 | 2903.0-3940.0 | |||
| Aspartate Transaminase U/L | ECM Only | 126.1 | 63.8-258.6 | 0-552 | 0.76 |
| PMSC-ECM | 112.5 | 80.5-171.2 | |||
| Blood Urea Nitrogen mg/dL | ECM Only | 17.9 | 15.8-19.4 | 15.2-34.7 | 0.95 |
| PMSC-ECM | 17.5 | 15.5-20.0 | |||
| Calcium mg/dL | ECM Only | 11.4 | 11.0-12.0 | 9.6-11-5 | 0.19 |
| PMSC-ECM | 11.7 | 11.1-12.3 | |||
| Creatinine mg/dL | ECM Only | 0.1 | 0.1-0.1 | 0.0-0.3 | 0.23 |
| PMSC-ECM | 0.1 | 0.1-0.1 | |||
| Glucose mg/dL | ECM Only | 206.5 | 183.8-235.0 | 130-254 | 0.006* |
| PMSC-ECM | 241.1 | 222.0-267.2 | |||
| Phosphorus mg/dL | ECM Only | 11.7 | 11.3-12.9 | 7.5-10.7 | 0.51 |
| PMSC-ECM | 12.3 | 11.5-12.7 | |||
| Total Bilirubin mg/dL | ECM Only | 0.2 | 0.1-0.2 | 0.0-0.2 | 0.45 |
| PMSC-ECM | 0.2 | 0.1-0.2 | |||
| Total Protein g/dL | ECM Only | 5.1 | 4.7-5.6 | 4.7-6.1 | 0.65 |
| PMSC-ECM | 5.2 | 5.0-5.7 | |||
| Chloride mmol/L | ECM Only | 111.9 | 110.7-114.1 | 105-118 | 0.95 |
| PMSC-ECM | 111.9 | 111.0-113.7 | |||
| Potassium mmol/L | ECM Only | 11.1 | 10.2-12.0 | 6.9-10.0 | 0.90 |
| PMSC-ECM | 11.2 | 10.1-12.0 | |||
| Sodium mmol/L | ECM Only | 154.0 | 151.0-156.0 | 150-160 | 0.30 |
| PMSC-ECM | 153.0 | 151.8-154.3 |
Table 3:
6-month time point blood results summary. IQR: interquartile range; WBC: white blood cell. ECM: extracellular matrix; PMSC-ECM: placental mesenchymal stromal cell seeded on extracellular matrix. P value reflects comparison of the medians between groups. *Statistically significant.
| Lab Value | Group | Median | IQR | Normal Range | p value |
|---|---|---|---|---|---|
| WBC (K/μl) | ECM Only | 1.3 | 1.2-2.0 | 5.1-14.7 | 0.91 |
| PMSC-ECM | 1.3 | 1.2-2.3 | |||
| Hemoglobin (g/dL) | ECM Only | 13.1 | 12.6-13.2 | 11.7-16.2 | 0.21 |
| PMSC-ECM | 13.4 | 12.7-13.9 | |||
| Hematocrit % | ECM Only | 25.8 | 42.1-47.1 | 38.3-54.0 | 0.66 |
| PMSC-ECM | 46.3 | 42.5-48.2 | |||
| Platelets (K/μL) | ECM Only | 1218.0 | 1038.0-1422.0 | 574-1079 | 0.46 |
| PMSC-ECM | 1142.0 | 984.3-1338.0 | |||
| Alanine Transaminase U/L | ECM Only | 39.2 | 25.5-104.4 | 0-403 | 0.98 |
| PMSC-ECM | 38.7 | 23.1-627.0 | |||
| Albumin g/dL | ECM Only | 4.1 | 4.0-4.2 | 2.9-4.0 | 0.03* |
| PMSC-ECM | 3.9 | 3.8-4.0 | |||
| Alkaline Phosphatase U/L | ECM Only | 82.5 | 62.3-102.0 | 49-172 | 0.35 |
| PMSC-ECM | 74.4 | 61.1-87.6 | |||
| Amylase U/L | ECM Only | 3766.0 | 3524.0-4047.0 | 2463-6660 | 0.52 |
| PMSC-ECM | 3659.0 | 3338.0-3934.0 | |||
| Aspartate Transaminase U/L | ECM Only | 96.8 | 68.1-193.0 | 0-552 | >0.990 |
| PMSC-ECM | 110.2 | 55.5-887.8 | |||
| Blood Urea Nitrogen mg/dL | ECM Only | 25.0 | 22.5-26.1 | 15.2-34.7 | 0.02* |
| PMSC-ECM | 20.4 | 18.5-23.6 | |||
| Calcium mg/dL | ECM Only | 11.6 | 11.4-12.0 | 9.6-11-5 | 0.39 |
| PMSC-ECM | 11.6 | 11.2-11.8 | |||
| Creatinine mg/dL | ECM Only | 0.2 | 0.2-0.2 | 0.0-0.3 | 0.06 |
| PMSC-ECM | 0.2 | 0.2-0.2 | |||
| Glucose mg/dL | ECM Only | 219.9 | 206.7-248.0 | 130-254 | 0.90 |
| PMSC-ECM | 219.9 | 192.7-294.9 | |||
| Phosphorus mg/dL | ECM Only | 11.1 | 10.5-12.5 | 7.5-10.7 | 0.09 |
| PMSC-ECM | 12.3 | 11.7-12.6 | |||
| Total Bilirubin mg/dL | ECM Only | 0.1 | 0.1-0.2 | 0.0-0.2 | 0.73 |
| PMSC-ECM | 0.1 | 0.1-0.2 | |||
| Total Protein g/dL | ECM Only | 5.6 | 5.5-5.7 | 4.7-6.1 | 0.33 |
| PMSC-ECM | 5.5 | 5.3-5.7 | |||
| Chloride mmol/L | ECM Only | 112.5 | 112.0-114.0 | 105-118 | 0.01* |
| PMSC-ECM | 111.5 | 111.0-112.5 | |||
| Potassium mmol/L | ECM Only | 11.9 | 10.7-12.7 | 6.9-10.0 | 0.97 |
| PMSC-ECM | 11.6 | 11.3-12.1 | |||
| Sodium mmol/L | ECM Only | 152.0 | 150.0-154.5 | 150-160 | 0.88 |
| PMSC-ECM | 152.0 | 148.5-155.8 |
No mice in either time point group had gross or histological evidence of tumor development on pathologic evaluation in any examined tissues. Furthermore, there were no specific histologic findings suggesting treatment-related effects (Supplementary Figures 1 & 2). Regarding the histologic evaluation specifically of the subcutaneous implantation pocket site, a nodular focus of mature fibrous connective tissue was identified in the majority of mice both the ECM only group (n=12) and PMSC-ECM group (n=9) at the 4-week time point, with no difference between the groups (p=0.32). Similarly, this was seen in the majority of mice at the 6-month time point in the ECM only group (n=12) and PMSC-ECM group (n=12), also with no difference between groups (p>0.99). There were no inflammatory changes identified at the subcutaneous pocket site for any of the mice.
Due to the inadvertent formalin fixation of tissues from 11 animals, there are only qPCR results for 16 of the 4-week time point animals. Overall, there were no mice with the presence of human DNA remaining (Tables 4 & 5, Supplementary Tables 1 & 2). More specifically, one female mouse at the 4-week time point in the ECM only group had one detectable Ct value of 39 at the implantation site. As discussed in the methods, this value being greater than 38, it being present in only one triplicate run, and the fact that it is in a mouse in which no human DNA was implanted make this a false positive result. In the ECM only 6-month time point group, there was 1 male mouse that had a Ct value of 38.24 in the lung tissue in one triplicate run, which was also considered a false positive for the same aforementioned reasons. In the PMSC-ECM 6-month time point group, there was 1 male mouse with a single Ct value of 39 at the implantation site and 1 male mouse with 2 of the 3 triplicate Ct values of 38.19 and 38.17. In addition, there was 1 female mouse with a single Ct value of 38.82 at the implantation site and 1 female mouse with 2 of the 3 triplicate Ct values of 38.66 and 38.32. With regards to the lung tissue, there was 1 male mouse with 1 Ct value of 39.12. For these PMSC-ECM animals, since these were not positive in all three triplicate runs and all Ct values were greater than 38, these were all considered false positives. Therefore, overall our results indicate that there were no mice with the presence of human DNA remaining by either 4 weeks or 6 months.
Table 4:
4-week time point qPCR data summary. ND=not detected; ECM: extracellular matrix; PMSC-ECM: placental mesenchymal stromal cell seeded on extracellular matrix. All samples run in triplicate. Eleven of the ECM only animals were not able to be analyzed secondary to inadvertent formalin fixation of the tissue, therefore the total ECM only animals is 16.
| Tissue Location (sex) | ECM Only Value (sample size) |
PMSC-ECM Value (sample size) |
|---|---|---|
| Implantation Site (Male) | ND/ND/ND (n=4) | ND/ND/ND (n=4) |
| Implantation Site (Female) | ND/ND/ND (n=3) 39/ND/ND (n=1) |
ND/ND/ND (n=4) |
| Lung (Male) | ND/ND/ND (n=4) | ND/ND/ND (n=4) |
| Lung (Female) | ND/ND/ND (n=4) | ND/ND/ND (n=4) |
Table 5:
6-month time point qPCR data summary. ND=not detected; ECM: extracellular matrix; PMSC-ECM: placental mesenchymal stromal cell seeded on extracellular matrix. All samples run in triplicate.
| Tissue Location (sex) | ECM Only Value (sample size) |
PMSC-ECM Value (sample size) |
|---|---|---|
| Implantation Site (Male) | ND/ND/ND (n=6) | ND/ND/ND (n=5) 38.19/38.71/ND (n=1) 39/ND/ND (n=1) |
| Implantation Site (Female) | ND/ND/ND (n=7) | ND/ND/ND (n=5) 38.66/38.32/ND (n=1) 38.82/ND/ND (n=1) |
| Lung (Male) | ND/ND/ND (n=5) 38.24/ND/ND (n=1) |
ND/ND/ND (n=6) 39.12/ND/ND (n=1) |
| Lung (Female) | ND/ND/ND (n=7) | ND/ND/ND (n=7) |
Discussion
This study evaluates the tumorigenicity and safety of the use of GMP-manufactured clinical-grade PMSC-ECM product in the gold standard immunodeficient murine model. In total 54 mice underwent surgical subcutaneous implantation of either ECM only or PMSC-ECM and were survived to 4 weeks or 6 months. No mice had gross abnormalities in their blood samples or had gross or microscopic evidence of tumor development. qPCR results showed that no mice had human DNA detected at either 4 weeks or 6 months at either the implantation site or lung tissue, which is in line with previous MSC studies which have found the cells to be short-lived, as they primarily function through local paracrine secretion at the site of implantation(17-19), and are not expected to persist in the tissues. These results support the safety of the PMSC-ECM product for use in a Phase 1/2a human clinical trial of fetal MMC repair.
With an abundance of research on stem cell therapy, including their use in clinical trials, the safety of stem cell products is an important focus. Prior studies have evaluated the toxicity and tumorgenicity of human adipose tissue-derived MSCs, bone marrow derived MSCs (BM-MSCs), and umbilical cord MSCs. There is substantial literature supporting the safety of MSCs derived from tissues other than the placenta as therapeutic agents. Choi et al found that transplantation of human adipose tissue-derived MSCs into rat femurs found no significant changes in hematological values or histopathological findings(20). Ra et al found that intravenous injection of human adipose tissue-derived MSCs into immunodeficient mice resulted in no evidence of tumor development(21). Melnik et al implanted BM-MSCs into a subcutaneous pocket in immunodeficient mice and found that after 42 days there was no macro- or microscopic evidence of tumor formation (22). Rengasamy et al injected BM-MSC through both intravenous and intramuscular routes in rats and rabbits and found that these injections did not cause toxicity after 14 or 90 days. In addition, BM-MSC were implanted subcutaneously and injected intramuscularly in immunodeficient mice and after 6 months there was no evidence of tumor formation(23). BM-MSCs have additionally been tested for safety in human patients. Centeno et al treated human patients with degenerative disc disease with BM-MSCs via percutaneous intradiscal injection in a preliminary safety study and there were no adverse effects, including tumor formation(24). Yang et al implanted umbilical cord MSCs under the spinal cord arachnoid in rats, which resulted in no immune system toxicity or organ dysfunction, and no tumor formation(25). Despite all of this favorable research on different types of MSCs, this is the first study regarding the safety and tumorigenicity of placental MSCs. One of the key outcomes of this study was to evaluate for tumor formation and adverse effects of subcutaneous implantation of PMSCs during both a short-term (4-week) and long-term (6-month) period of time. Our results showed that no mice that had gross or pathologic evidence of tumor development or specific histologic findings that suggested treatment-related adverse effects. These findings are promising regarding the safe use of PMSCs clinically for the treatment of a wide array of indications, including MMC.
In addition to the safety profile of MSCs, an understanding of their in vivo cellular fate and distribution are important for translation to human clinical use. There are numerous studies evaluating BM-MSCs that have demonstrated that when systemically delivered, the utility of BM-MSCs were limited initially by entrapment of cells mainly in the lungs(16, 26, 27). Regarding MSC homing and half-life, Lee et al showed that after injecting human multipotent MSCs intravenously into mice, most of the cells were trapped in the lungs, with only trace amounts in other tissues, and furthermore that the cells in the lungs disappeared with a half-life of about 24 hours(17). Hara et al injected BM-MSCs into rats and found that cells injected into the femoral artery were detectable for less than 3 days in the lung tissue and after intramuscular injections were detectable only for about 7 days(28). Casiraghi et al injected BM-MSCs into either the portal vein or tail vein of mice and found that after 7 and 21 days, cells were almost undetectable in all tissues(29). While there have been studies evaluating the homing and half-life of other types of MSCs, the data regarding these characteristics for PMSCs is not as well characterized. There is one study that evaluated PMSCs specifically. Ramot et al showed that after intramuscular injection in a mouse model, the cells were confined to the injection site muscle and there was no human DNA detected in any of the other tissues evaluated, including the blood and femur bone marrow. Furthermore, the concentration of human DNA was highest one day after injection and then decreased over a three month period(30). In our study, persistent human DNA, representing human PMSCs in this model, could raise concerns about the potential for continued or future proliferation of cells which could result in cell-related adverse effects or tumor formation in this murine model. However, we demonstrated that there was no evidence that human DNA remained at the implantation site or in the lungs as early as 4 weeks after implantation, suggesting that any cell-related adverse effects are extremely unlikely, as the cells were no longer present by 4 weeks.
The absence of tumor formation and the absence of any remaining human DNA in this immunodeficient murine model support the overall safety of the PMSC-ECM product for use in a Phase 1/2a clinical trial for human patients. Together these safety data will allow us to proceed with a first-in-human clinical trial for fetal patients with MMC using our PMSC-ECM product to augment fetal MMC repair.
Conclusions
This study evaluated tumorigenicity of PMSCs seeded on Cook Biodesign® Dural Graft Extracellular Matrix (PMSC-ECM) in an established immunodeficient NOD/SCID/Gamma−/− (NSG) murine model. In this study we evaluated for tumor formation at two different time points. There was no gross tumor formation in any group and histological evaluation and blood samples were all within normal limits. No mice had human DNA remaining at either 4 weeks or 6 months, therefore the likelihood of future tumor formation or cellular therapy-related complications is low. These safety results in a murine model support initiation of a Phase 1/2a clinical trial for human patients with MMC using our PMSC-ECM product to augment fetal MMC repair.
Supplementary Material
Highlights.
Placental Mesenchymal Stromal Cells seeded on a clinical grade extracellular matrix and implanted in an immunodeficient murine model did not result in any tumor formation.
There was no evidence that Placental Mesenchymal Stromal Cells remained at the implantation site or in lung tissue at 6 months.
Acknowledgements
The authors would like to acknowledge Dr. Jan Nolta, PhD, William Gruenloh, the staff at both the UC Davis Clinical Pathology Laboratory and the Institute for Regenerative Cures for their expertise and guidance in the conduct of these experiments.
Funding Sources/Disclosures:
This work was funded by the California Institute of Regenerative Medicine late stage preclinical CLIN1 grant (CLIN1-11404).
The project described was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant number UL1 TR001860 for author CMT. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
The remaining authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article.
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