Related Links References Publications-Human Publications-Mouse

background
The long-term culture system, developed first for mouse marrow in the late 1970’s,¹ and then successfully adapted for human cells,² establishes in vitro the essential cell types involved in hematopoiesis. When initiated with a relatively high density of marrow cells (>10⁶ cells/mL), long-term cultures are characterized by the formation of an adherent stromal layer of mesenchymal cells including endothelial cells, fibroblasts and adipocytes. Primitive hematopoietic cells associated with this stromal layer typically generate myeloid clonogenic progenitors and mature granulocytes for many weeks, provided that appropriate medium and supplements, incubation conditions, and feeding schedules are used. To this end, StemCell Technologies has developed the unique long-term culture medium MyeloCult^® . This medium promotes the formation of human or mouse primary stromal layers and allows the proliferation and differentiation of primitive hematopoietic progenitors.

long-term culture-initiating cell assay (LTC-IC)
The unique features of the long-term culture system have allowed the development of the long-term culture-initiating cell (LTC-IC) assay³ to detect and quantitate primitive hematopoietic cells which share phenotypic and functional properties with mouse and human in vivo repopulating cells.^4,27 In human long-term cultures, colony-forming cells (CFC) detected after >5 weeks represent the progeny of LTC-IC since CFC present in the input cell suspension have undergone terminal differentiation by this time. Quantitation of LTC-IC in a test cell suspension requires culturing the cells on a supportive feeder layer of irradiated marrow cells or suitable human or mouse fibroblast cell lines.^28,29 Limiting dilution analysis is used to determine the frequency of LTC-IC as well as the average number of CFC produced per LTC-IC. Once the average number of CFC per LTC-IC is established, the LTC-IC content of a sample can be determined by a bulk culture LTC-IC assay, provided that the same source of test cells (e.g. bone marrow, cord blood, mobilized peripheral blood) is used and the assay conditions are identical. The LTC-IC content is then calculated by dividing the total output of CFC by the average number of CFC produced per LTC-IC.³

applications
Quantitate Frequency of LTC-IC and Study their Phenotypic and Functional Properties. Primitive hematopoietic progenitors capable of initiating and sustaining myelopoiesis for several weeks in long-term culture have been called long-term culture-initiating cells (LTC-IC). The frequency of LTC-IC in humans³ or mouse^4,5 samples can be determined using the LTC-IC assay. Experiments using MyeloCult^® and related products show that LTC-IC are a heterogeneous population of cells which can differ in phenotype,^6,7 cell cycling characteristics⁸ and expansion potential.^9-11
Facilitate Gene Transfer. Culture in MyeloCult^® facilitates retroviral gene transfer into primitive hematopoietic progenitors and expansion of these transfected cells.^12,13
Expand Multi-Potential Hematopoietic Cells In Vitro. Human colony-forming cells (CFC) and LTC-IC have been expanded using MyeloCult^® in stirred suspension cultures.^14,15 Mouse totipotent hematopoietic cells expanded in static long-term cultures with MyeloCult^® can sustain lymphomyelopoiesis in irradiated recipients.¹²
Evaluate Factors Regulating Myelopoiesis. The role of stroma-derived factors (positive and/or negative regulators^16-18 and adhesion molecules¹⁹) in the regulation of myelopoiesis can be evaluated in long-term cultures using MyeloCult^®.
Examine Differences Between Normal and Malignant Cells. MyeloCult^® has been used to culture LTC-IC from patients with CML,^20-22 AML²³ and aplastic anemia.²⁴
Study Differentiation of CD34⁺ Cells into Natural Killer (NK) Cells. A subpopulation of CD34⁺ cord blood cells can be induced to differentiate into NK cells upon culture in MyeloCult^® in the presence of IL-2 and IL-7 or Stem Cell Factor (SCF) and IL-15.²⁵
Culture NK Cell Lines. MyeloCult^® is used for the culture of the human NK-92 cell line.²⁶

quality control
MyeloCult^® long-term culture reagents are prepared from components that have been selected after extensive screening for their ability to optimally support the growth of long-term hematopoietic cultures. All sera used in MyeloCult^® products have been extensively screened and selected for their ability to establish productive stroma-supported human and mouse myelopoietic cultures or mouse B-lymphoid (Whitlock-Witte) cultures.


FOR RESEARCH USE ONLY