In Vivo Therapy for Prenatal Repair of the Ovine Fetal Myelomeningocele Model Using Human Umbilical Cord-Derived Mesenchymal Stem Cells: Proof of Principle

Major Points and Findings:

This is an animal study in unborn lambs. No humans were treated. A team at the University Medical Center Hamburg-Eppendorf in Germany created a spina bifida (myelomeningocele) defect in the spines of fetal lambs and then repaired it in the womb, as surgeons do in human fetal surgery. In some lambs they first placed human umbilical cord mesenchymal stem cells (MSCs) directly onto the exposed spinal cord. The cells came from Desacell, a ready-to-use product made from the umbilical cord tissue of a single healthy newborn. Earlier sheep studies had used cells seeded onto a patch, so this was the first report of applying a liquid cell suspension in this way.

The study showed that the cells could be applied in all five lambs intended to receive them. It could not show whether the cells helped. Only three of the seven repaired lambs were born alive (two treated, one untreated), all three had normal or near-normal movement scores, and the untreated lamb actually had the highest density of surviving large nerve cells. The authors themselves say the sample was too small for any comparison between groups.

Aim:

The main aim was to test whether a ready-to-use human umbilical cord MSC product (Desacell) could be applied locally to the spinal cord during fetal spina bifida repair in sheep. Secondary aims were to see whether the cells improved movement on the Sheep Locomotor Rating (SLR) scale, whether they increased the size of the spinal cord and its gray matter, and whether the density of large nerve cells related to movement.

Methods:

Pregnant ewes from a single breeder, all carrying lambs sired by the same ram, were used. The study was approved by the Hamburg state authority under the German Animal Welfare Act.

At about 75 days of pregnancy (a sheep pregnancy lasts about 145 days), surgeons opened the womb, removed the skin, back muscles and the bony arches of the spine from L1 to L5, and opened the membrane covering the spinal cord. This leaves the cord exposed to the amniotic fluid, mimicking spina bifida. About 25 days later the lambs had a second operation to repair the defect. Lambs were assigned two to one to repair with or without cells. In the cell group, scar tissue over the spinal cord was cleaned away and 1 mL of Desacell gel suspension containing 30 million cells was applied directly to the cord. All lambs then had a multilayer patch and skin closure. In twin pregnancies one twin was treated and the other served as a control or was left unoperated.

The cells came from one donor umbilical cord, met international MSC identity criteria and were tested for sterility, mycoplasma and endotoxin. They were thawed, washed, counted and shipped chilled (4 °C) to the operating site.

Lambs were born at term and filmed at 2 and 48 hours after birth. Reviewers who did not know which lambs had received cells scored movement on the 15-point SLR scale, where 15 is normal walking and 0 to 4 is severe impairment. The lambs were then put down and the lumbar spinal cord was cut into five to eight segments, stained and measured. A neuropathologist, also blinded, counted large motor nerve cells per square millimetre of gray matter. Before the study, the authors calculated that groups of three and six lambs would give only 25.5% power, meaning a real difference would most likely be missed.

Results:

Survival: Defects were created in 11 fetal lambs at an average of 76.9 days.

• Three were lost after the first operation. One ewe developed a wound breakdown with bowel obstruction and was put down, one fetus died after the ewe had a fever, and one died on the day of a wound complication.
• In one more lamb the fetus could not be reached surgically, so seven lambs were repaired, at a mean of 104.3 days. Five received cells and two did not.
• Four more fetuses died 11 to 17 days after repair: three of the five treated lambs and one of the two untreated lambs. One had a fungal abscess at the spine with widespread fungal infection. The cause of the other deaths was unclear.
• Three lambs were born alive at 144 to 147 days: lamb 1 (no cells) and lambs 3 and 4 (cells). The paper gives the overall live birth rate as 30%.

Movement (SLR scale, out of 15):

• Lamb 1 (no cells) scored 15 at 2 hours and 15 at 48 hours.
• Lamb 3 (cells) scored 13 at 2 hours and 15 at 48 hours.
• Lamb 4 (cells) scored 15 at 2 hours and 15 at 48 hours.

All three lambs therefore walked normally by 48 hours, whether or not they received cells, so the scores could not show any benefit.

Spinal cord tissue: No tumour was seen in any lamb, on the naked eye or under the microscope.

• Mean density of large nerve cells was 20.4 per mm² in the untreated lamb, against 15 and 13.1 per mm² in the two treated lambs. The authors note that this is the opposite of what earlier studies found.
• Mean gray matter area was larger in the treated lambs (274 and 218 units, against 171 in the untreated lamb), and the share of the cord made up of gray matter was 0.32 and 0.48, against 0.31. The authors suggest this “could possibly indicate better tissue preservation”.
• Areas were measured in image pixels, and no statistical tests comparing the lambs were reported.

Conclusions:

The authors conclude that applying a human umbilical cord MSC product during fetal spina bifida repair in sheep is feasible, and that larger studies with Desacell are needed to test movement, nerve protection and safety.

The study is a technical first step. Only one untreated and two treated lambs could be assessed, so the results cannot show whether the cells work. The authors list three main limitations: the high fetal death rate, which removed any chance of a meaningful comparison and may reflect surgical problems unrelated to the cells; no special staining (immunohistochemistry), so they could not confirm that the cells were still present in the repaired area; and too few survivors for the movement scores to mean anything. In addition, the untreated lamb walked normally, which suggests the defect in this model did not cause lasting weakness in these animals, and with one lamb per comparison the gray matter and nerve cell differences could easily be chance. Long-term outcomes such as bladder function or tethered cord were not studied, because the lambs were examined only up to 48 hours after birth.

Background Information:

Spina bifida affects about 1 in 2,000 pregnancies in Europe. The MOMS trial showed that repairing the defect before birth halves the need for a brain fluid shunt and doubles the chance of walking at 30 months, but 58% of children repaired before birth still cannot walk independently. Researchers hope that adding stem cells at the time of repair could further protect the exposed spinal cord.

The cell product, Desacell, is described as a ready-to-use product made using a patented isolation protocol. The paper does not name its manufacturer or the patent holder. The authors state that it has been tested in rodents and premature baboons, in adults with severe lung failure (CIRCA-19), in preterm babies at risk of chronic lung disease (HULC-1), and is being studied in adults with septic shock (UC-CISS II). The baboon study they cite, led by co-author Möbius, is titled “Prophylactic administration of mesenchymal stromal cells does not prevent arrested lung development in extremely premature-born non-human primates”. Two of the authors are from the Saxonian Center for Feto/Neonatal Health at the Technische Universität Dresden.

The study was funded by the German Research Foundation (DFG BA 7308/4-1). The authors state that they have no conflict of interest. The work is an animal experiment only, and the authors point to the ongoing CuRe trial, which uses placental cells on a patch, as the first human study of stem cells given during fetal spina bifida repair.