Expansion and Multipotencial Differentiation of Mesenchymal Stem Cells Isolated from Patients after High Dose Chemotherapy.

Background. High dose chemotherapy (HDCT) followed by autologous PBSC rescue has been increasingly used for the treatment of several human diseases. However, little is known on the extent of this therapy on the marrow mesenchymal stem cells (MSCs).

Aims. To evaluate the feasibility of expansion and multipotencial differentiation of MSCs isolated from patients after HDCT.

Patients and Methods. Twelve lymphoma’s patients (LP) free of disease in bone marrow (BM) were enrolled in the study. They were submitted to BEAM’s protocol with autologous PBSC rescue 28 to 1836 days before the sample collection. Six normal bone marrow donors (ND) were used as controls. The LP and ND median age were 37.5 (range 22–49) and 31.5 years old (range 23–42), respectively. MSCs were isolated by plastic adherence and expanded ex vivo by cultivation in flasks with α-MEM with 15% fetal bovine serum. Media was changed every 3–4 days. At 90% confluence, the cells were re-plated and expanded. The isolation efficiency, colony-forming unit-fibroblast (CFU-F) frequency, growth kinetics, phenotypic characteristics, cell cycle status, multi-lineage differentiation capacity as well as hematopoiesis-supportive function were determined and compared with those of ND-MSCs. This study protocol and the consent form were approved by the institution ethics committees.

Results. The results were analyzed by Mann-Whitney test and are expressed as median (range) to LP and ND, respectively. MSCs were successful isolated from all BM samples collected for this study. The cell population showed typical fibroblast-like morphology, appearing as an adherent, spindle shaped cell layer and growing to confluence after a few weeks of culture. The number of CFU-F found at 14 days of culture were 0.94 (0.00–3.75) and 1.25 (0.13–9.25) x10⁻⁵ nucleated cells (p = 0.4421). The doubling time between the 1^st and 2^nd passages was 80.66 (34.08–195.35) and 46.30 (36.36–270.59) hours (p = 0.1025). The cell clones proliferated extensively until 8.17 (1.81–28.27) and 18.11 (11.85–27.48) population doublings (p = 0.0668) in 71.50 (46–88) and 81 (57–103) cultivation days (p = 0.1505). Immunophenotypically, these cells were positive for the CD73, CD105, CD90, CD29, CD13, CD44, CD49e, CD54, HLA-class 1 and Stro-1 markers and negative for CD34, CD45, CD14, CD51/61, HLA-DR and KDR. Regarding the cell cycle status, 85.63 (63.19–92.17) and 82.41 (82.19–87.02) % were in G_O-G₁ phase (p = 1,000), while only 12.17 (3.33–36.81) and 10.67 (6.59–12.05) % were in S phase (p = 0,6828). All samples tested were capable of differentiating along adipogenic, osteogenic and chondrogenic lineages in vitro, demonstrated by morphology, cyto- and imunohistochemistry or RT-PCR reaction (PPARg and osteopontin genes expression). After co-culture with CD34⁺ cord blood cells for 1 and 4 weeks, no significant difference CD34⁺ expansion or colony-forming cells (BFU-E or CFU-GM) were observed between the CD34⁺ cells/LP-MSCs and CD34⁺ cells/ND-MSCs co-cultures with cytokines or not.

Interpretation and Conclusions. Our results demonstrate that is possible to cultivate and expand MSCs with multipotential differentiation capabilities and hematopoiesis-supportive function from patients after HDCT. Despite there were no significant differences in the median values between LP and ND, the comparative study indicates a possible damage in MSCs by HDCT.