Unfractionated Marrow Mononuclear Cells Research Articles

Day +60 WT1 assessment on CD34 selected bone marrow better predicts relapse and mortality after allogeneic stem cell transplantation in acute myeloid leukemia patients.

The aim of this study was to evaluate the role of WT1 expression after allogeneic stem cell transplantation (alloHSCT) in patients with acute myeloid leukemia (AML). We studied WT1 expression in bone marrow cells from 50 patients in complete remission on day +60 after transplant. WT1 was assessed on unfractionated bone marrow mononuclear cells (MNC) and on CD34+ selected cells (CD34+). A ROC curve analysis identified 800 WT1 copies on CD34+ selected cells, as the best cut-off predicting relapse (AUC 0.842, p=0.0006, 85.7% sensitivity and 81.6% specificity) and 100 copies in MNC (AUC 0.819, p=0.007, 83.3% sensitivity and 88.2% specificity). Using the 800 WT1 copy cut off in CD34+ cells, the 2 year cumulative incidence of relapse was 12% vs 38% (p=0.005), and 2 year survival 88% vs 55% (p=0.02). Using the 100 WT1 copy cut off in unfractionated MNC, the 2 year cumulative incidence of relapse 13% vs 44% (p=0.01) and the 2 year survival 88% vs 55% (p=0.08). In a multivariate Cox analysis WT1 expression in CD34 cells proved to highly predictive of relapse (p=0.004); also WT1 expression on unfractionated cells predicted relapse (p=0.03). In conclusion, day-60 WT1 expression after allogeneic HSCT is a significant predictor of relapse, particularly when tested on CD34+ selected bone marrow cells.

Cell-Based Cardiac Repair

Received February 28, 2005; revision received April 25, 2005; accepted April 29, 2005. It has now been more than a decade since the first experiments were performed using cell transplantation for the prevention and treatment of heart failure.1–3 Although the biomedical community was initially somewhat skeptical of this approach, a large body of experimental evidence was amassed showing that injected cells could create new tissue and improve function of the failing heart. This evidence, coupled with the recognized limitations of heart failure treatments and the intuitively appealing concept of “regenerative medicine,” has contributed to a crescendo of activity in cell-based cardiac repair. Given the flurry of clinical trials that are currently under way, we think it is timely to review progress over the past 10 years and provide a critical assessment of where the field stands and where it appears to be headed. Cell-based cardiac repair began with studies of skeletal myoblasts derived from skeletal muscle satellite cells.1–3 Myoblasts were the initial choice because of their availability from autologous or syngeneic sources, their ability to proliferate, and their ability to withstand ischemia better than many cell types. Although it was originally hoped that these cells would transdifferentiate into cardiomyocytes, it is now clear that myoblasts remain stubbornly committed to form mature skeletal muscle in the heart3–5 (with the exception of rare cell fusion events at the graft–host interface6). Skeletal muscle is one of the few cell types in the body that does not normally express gap junction proteins, and hence, structural and physiological studies indicate that skeletal muscle cells do not form electromechanical junctions with cardiomyocytes when engrafted into the heart.7,8 Despite this, numerous studies have shown beneficial effects of skeletal myoblast grafting into the infarcted heart in rodents and large animals.8–13 Cardiomyocytes would …

Ex Vivo Expansion of Autologous Bone Marrow CD34+ Cells With Porcine Microvascular Endothelial Cells Results in a Graft Capable of Rescuing Lethally Irradiated Baboons

Hematopoietic stem cell (HSC) self-renewal in vitro has been reported to result in a diminished proliferative capacity or acquisition of a homing defect that might compromise marrow repopulation. Our group has demonstrated that human HSC expanded ex vivo in the presence of porcine microvascular endothelial cells (PMVEC) retain the capacity to competitively repopulate human bone fragments implanted in severe combined immunodeficiency (SCID) mice. To further test the marrow repopulating capacity of expanded stem cells, our laboratory has established a myeloablative, fractionated total body irradiation conditioning protocol for autologous marrow transplantation in baboons. A control animal, which received no transplant, as well as two animals, which received a suboptimal number of marrow mononuclear cells, died 37, 43, and 59 days postirradiation, respectively. Immunomagnetically selected CD34(+) marrow cells from two baboons were placed in PMVEC coculture with exogenous human cytokines. After 10 days of expansion, the grafts represented a 14-fold to 22-fold increase in cell number, a 4-fold to 5-fold expansion of CD34(+) cells, a 3-fold to 4-fold increase of colony-forming unit-granulocyte-macrophage (CFU-GM), and a 12-fold to 17-fold increase of cobblestone area-forming cells (CAFC) over input. Both baboons became transfusion independent by day 23 posttransplant and achieved absolute neutrophil count (ANC) >500/microL by day 25 +/- 1 and platelets >20,000/microL by day 29 +/- 2. This hematopoietic recovery was delayed in comparison to two animals that received either a graft consisting of freshly isolated, unexpanded CD34(+) cells or 175 x 10(6)/kg unfractionated marrow mononuclear cells. Analysis of the proliferative status of cells in PMVEC expansion cultures demonstrated that by 10 days, 99.8% of CD34(+) cells present in the cultures had undergone cycling, and that the population of cells expressing a CD34(+) CD38(-) phenotype in the cultures was also the result of active cell division. These data indicate that isolated bone marrow CD34(+) cells may undergo cell division during ex vivo expansion in the presence of endothelial cells to provide a graft capable of rescuing a myeloablated autologous host.