Support of hMSCs transduced with TPO/FL genes to expansion of umbilical cord CD34+ cells in indirect co-culture

Abstract

A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34+ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34+ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions.

Cord Blood—An Alternative Source for Bone Regeneration

“…To prolong the proliferation rate but also to prevent from increased cellular aging in vitro, a co-culture system of cord blood MSC with human platelet lysates and / endothelial cells, mimicking the physiological in vivo conditions may be useful [124]. Some other data also suggest a clinical potential for using USSCs in a dose dependent manner to facilitate homing and engraftment for cord blood transplant recipients [125][126][127][128]. As published previously we found an increased bone healing of a critical size bone defect in a nude rat model after xenotransplantation of human CB-MSC [64].…”

Neural Network Analysis of Ex-vivo Expansion of Hematopoietic Stem Cells

“…The network input and output data were obtained from literature 1,2,[4][5][6][7][8][9][10][11][12][14][15][16][17][18][19][20][21][22][23][24][25]27,[29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][46][47][48][49] for evaluation and prediction. Altogether 341 data were collected and summarized in Table 1.…”

Expansion of human hematopoietic stem cells for transplantation: trends and perspectives

“…However, CD34 + and total CFU did not show a significant difference. Expanded cells also showed a bigger increase in the LTC-IC and similar engraftment to uncultured cells in the NOD/SCID murine model (Xie et al 2006). The feeder layer systems have achieved expansion, but Leaving aside the 3D support, Madlambayan et al designed a static bioprocess consisting of two gas permeable culture bags separated by a magnetic system to eliminate undesired cells from the culture (Madlambayan et al 2006).…”