IN VITRO ASSESSMENT OF MESENCHYMAL STROMAL CELL CHARACTERISTICS: IMPLICATIONS FOR THEIR CLINICAL USE
INTRODUCTION: Bone marrow (BM) stroma represents a source of progenitor stromal cells, termed mesenchymal stromal cells (MSCs), which are multipotent and can differentiate into cartilage, bone, and adipose tissue. Several questions have arisen regarding their long-term expansion and their safety before use.
OBJECTIVE: Our goal was to assess the long-term expansion and safety of MSCs in clinical practice.
METHODS: MSCs from BM of children with benign hematologic disorders and solid tumors without BM involvement were isolated and cultured for 10 consecutive passages (P). Immunophenotypic and functional characteristics, apoptosis, and the expression of cell cycle regulatory genes (p53, p16, and Rb) and signal transduction genes (H-Ras) involved in oncogenesis were assessed.
RESULTS: MSCs expressed mesenchymal-related surface antigens, >85% from P1. They had the ability to differentiate into osteocytes, adipocytes, and chondrocytes (reverse-transcription polymerase chain reaction). Colony forming units (fibroblast) ranged from 40.71 ± 4.3 at P1 to 15.5 ± 6.7 at P10. Their doubling time was 2.01 ± 0.14 days at P1 and 3.5 ± 1.19 days at P9. A low percentage of apoptotic cells was detected (7-amino-actinomycin D [7AAD]) at P2 until P10. MSCs were resistant to apoptosis under serum-deprivation conditions. The expression of the cell cycle genes studied was not statistically different compared with controls, and cells did not grow on soft agar.
CONCLUSIONS: MSCs isolated from BM of children retain their characteristics for a serial number of passages and survive under serum-deprivation conditions, a necessary process in a transplantation setting. The cells do not have oncogenic properties, as shown by normal expression levels of oncogenes and tumor suppressor genes, and no growth on soft agar. These findings enhance the use of MSCs in clinical applications.
Submitted by Helen Dimitriou
- Copyright © 2008 by the American Academy of Pediatrics