Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)–transfected human stromal cells

Stromal cells modulate TCL1 expression, interacting AP-1 components and TCL1-targeting micro-RNAs in chronic lymphocytic leukemia

“…At a concentration of 10 7 per ml in 20 ml, CLL cells were incubated in 75 cm 2 tissue culture flasks (Techno Plastic Products, Trasadingen, Switzerland). The bone marrow stroma cell (BMSC) lines KUSA-H1 (murine) and StromaNKtert (human) 23 were purchased from RIKEN (Ibaraki, Japan) and cultured as described. 12 CLL cell viability was studied by the analysis of mitochondrial transmembrane potential using 3,3 0 -dihexyloxacarbocyanine iodide (DioC6; Molecular Probes, Life Technologies, Grand Island, NY, USA) and by cell membrane permeability to propidium iodide (SigmaAldrich, St Louis, MO, USA), and measured by FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).…”

Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID–ß2m–/– repopulating cells.

“…Preparation of primary stromal cells was described previously. 23 Telomerized human stromal cells (hTERT stromal cells) that exhibited a prolonged life span with the same hematopoietic progenitor support as primary stromal cells were employed to establish gene-modified stromal cells. 23 Human stromal cells were cultured in long-term culture (LTC) medium containing minimum essential medium-␣ (Sigma Chemical, St Louis, MO), 12.5% horse serum (Gibco BRL, Rockville, MD), 12.5% fetal calf serum (Gibco BRL), 1 ϫ 10 Ϫ6 M hydrocortisone (Sigma Chemical), and 1 ϫ 10 Ϫ4 M ␤-mercaptoethanol (Sigma Chemical) at 37°C under 5% CO 2 in a humidified atmosphere.…”

“…To avoid the presence of stromal cells in the resulting cell suspensions, a 30-minute adhesion procedure was performed at 37°C as described previously. 23,26 The coculture was maintained by adding 5 mL fresh medium containing cytokines on day 28. This allowed us to observe the formation of cobblestone areas (CAs) from hematopoietic cells that transmigrated beneath the layer of stromal cells.…”

“…23,28 Aliquots of cells were cultured in quadruplicate with the use of 35-mm tissue culture dishes in 1 mL MethoCult GF H4434V (Stem Cell Technologies, Vancouver, Canada). After 14 days of culture in a humidified environment at 37°C under 5% CO 2 , colonies consisting of 50 or more cells were scored under a microscope.…”

Expansion of hematopoietic stem/progenitor cells

“…The telomere length of human bone marrow-derived CD34 1 CD38 lo cells is found to be shorter than the cells from fetal liver or umbilical cord blood, suggesting a loss of telomeric DNA during cell proliferation [53]. Then, in order to confer replicative immortality on human cells and obtain sufficient number of primary stromal cells, Kawano et al developed a new human stromal cell line that over-expressed the human telomerase catalytic subunit (hTERT) gene (so-called ''hTERT stromal cells'') [16]. The human progenitor cells cocultured with hTERT stromal cells were expanded more than 1,000-fold in the presence of SCF, TPO, and Flk-2/Flt-3 ligand in 7 weeks.…”