Abstract
Introduction
Mesenchymal stem cells (MSC) are now in the limelight of stem cell researchers. The growing number of preclinical studies gives feedback for using MSC in the various fields of medicine. Their immunomodulatory function gives them a scientific rationale to be used in Graft-versus-Host Disease (GvHD) treatment. The MSC can be isolated from bone marrow (BM), adipose tissue, cord blood (CB) or umbilical cord (UC). However, BM harvest puts a donor at risk of procedure complication. In the contrary, Wharton Jelly (WJ) - derived MSC can be collected safely, easily and they are reach in MSC, which makes them more preferable source than CB.
We would like to present the first in Europe international cooperation that led to the clinical application of WJ-derived MSC in patients with GvHD. In the present publication we describe the results of collection, transport, culture, investigation, cryopreservation and the first examples of clinical usage of MSC derived from more than 500 UC collected by our group of stem cells banks (www.famicord.eu).
Methods
WJ-derived MSC were obtained from third party unrelated donors after natural deliveries as well as caesarian sections. They were collected to the sterile vessel containing 0,9% natrium chloratum and 1% antibiotic and transported in the temperature of 18-24°C. After an isolation by mechanical dissection of cord’s blood vessels, they underwent culture in the 37°C in the atmosphere of 5% CO2 in the air with human MSC growth medium as well as supplement containing fetal bovine serum and antibiotic. They were enumerated and their viability was evaluated. Then the cells were cryopreserved in the presence of DMSO and placed in the vapour phase of liquid nitrogen in <150°C. The repeated cell counting, viability test, flow cytometric immunophenotyping, and functional in vitro differentiation assays were performed from the thawed reference samples.
Results
Low contamination level (less than 2%) of the UC tissue collected after both natural deliveries and caesarian section was reported. We have not noticed any differences in growth, cell number and morphology in the primary cultures of tissue fragments from placental, central and baby side of the cord. The first adherent cells with fibroblast-like morphology were well-distinguishable within a week after the initiation of the cell culture. The immunophenotype remained stable (CD45-/CD34-/CD19-/CD14-/HLA-DR-/CD73+/CD90+/CD105+) during the whole period of culture (with extreme limit of 15 passages). MSC were capable of differentiation into adipogenic, chondrogenic and osteogenic cells. The WJ-derived MSC have been applied to the ten patients with steroid-refractory GvHD always after approval of bioethical committee. Three patients were diagnosed with chronic form and 7 with acute one. Five children had multiple infusions, up to 4 doses with 1-2 week intervals. No adverse effects were described during infusions apart from low grade fever in 1 adult patient.
Conclusion
The results described above demonstrate a repeatable method to obtain an adequate number of cells for the clinical use. The international cooperation between Polish, Hungarian, Romanian and Spanish stem cell banks, enabled us to use WJ-derived MSC in the setting of GvHD. No serious adverse effects were described. Third party donor WJ-derived MSCs are safe and effective treatment of GvHD, however further studies are needed.
Disclosures:
No relevant conflicts of interest to declare.
A Review of Clinical Applications and Side Effects of Methotrexate in Ophthalmology
