Abstract
Introduction: Recently, co-transplantion of mesenchy-mal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been shown to alleviate complications such as GVHD and speeding recovery of HSCs. This in vivo finding suggests that coculture of MSCs and HSCs may enhance their growth potentials in vitro. As the large-scale expansion of HSCs has been achieved by NASA’s suspension culture system, we further examined the effects of this suspension culture system (rotary bio-reactor) on MSCs’ proliferation and differentiation potentials in vitro.
Methods: Mononuclear cell fractions (MNCs) of human bone marrow aspirates (n=6, ages 46–81) were collected by density gradient centrifugation. The cells were inoculated into bioreactor (RCCS, Synthecon Inc., Texas, USA) at the concentration 1x106 cells/ml, in MyelocultTM medium supplemented with 50ng/ml SCF, 20ng/ ml rhIL-3 and rhIL-6 (10ng/ml SCF, 2ng/ml IL-3 and IL-6 after the first feeding) and 10-6 M hydrocortisone for 8 days. The medium was fully exchanged after 3 days and 20% daily thereafter. Total cell numbers in the bioreactor were counted daily using hemacytometer. Cells from day 1, 4, and 8 cultures were subjected to tri-color flow cytometry examination using CD34, CD44, and Stro-1 antibodies. By the end of 8 day culture, the output cells were resuspended in DMEM medium with 10% FBS and cultured in T75 flasks at 1x105 cells/cm2 for further 3 weeks. Upon harvest, half of the attached MSCs were prepared for western blotting assay using various antibodies. The other half was further cultured for 13–28 days in osteogenic, chondrogenic, and adipogenic induction medium respectively. Cell differentiation results were examined by histology staining, immunohistochemistry (ICC) and transmission electron microscope (TEM) examinations.
Results: After 8-day culture in bioreactor, flow-cytometric analysis confirmed that two cell populations, CD34+CD44+ (HSCs) and Stro-1+CD44+ (MSCs), increased 8-fold and 29-fold respectively, when compared to the values of the MNCs prior to bioreactor treatment. Cell counting revealed that the total cell expansion over 8 days was 9-fold above the number of the input MNCs. Western blotting data confirmed that bioreactor-expanded MSCs population remained in their early-stage with the expression of primitive MSCs markers such as CD105 (endoglin, SH-2) and Vimentin, whereas no expression of differentiation markers including osteocalcin (osteogenesis), Type II collegen (chondrogenesis) and C/EBPα (adipogenesis). Upon differentiation induction, the bioreactor-expanded MSCs were capable of differentiating into osteocytes, chondrocytes, and adipocytes as evidenced by histology staining, ICC and TEM examinations.
Discussion: Our study has shown that the percentage of MSCs (Stro-1+CD44+) increased 29 folds in the bone marrow derived MNCs after they have been cultured with Myelocult¢â medium in bioreactor for 8 days. The suspension culture system did not affect the subsequent in vitro proliferation and differentiation potentials of MSCs. Current study indicates that rotary bioreactor may be used to rapidly expand the numbers of traditionally attachment-dependent MSCs from bone marrow-derived MNCs, which may be very useful in clinical tissue engineering applications.
Correspondence should be addressed to Mr Carlos Wigderowitz, Honorary Secretary BORS, University Dept of Orthopaedic & Trauma Surgery, Ninewells Hospital & Medical School, Dundee DD1 9SY.
None of the authors have received anything of value from a commercial or other party related directly or indirectly to the subject of the presentation
