Stem Cell Preparation & Analysis

Director: Diane Krause
Associate Director: Bernard Forget

Services: Myeloid cell line preparation

Introduction

Cell lines that can be induced to differentiate down multiple lineages in vitro have been enormously useful in studies of hematopoiesis because they allow for analysis of uniform populations of large numbers of cells at different stages of differentiation. Uniform populations of primary cells are often not available in sufficient quantities for in depth analyses of gene expression and/or chromatin immunoprecipitation.

As a service to individual investigators, the YCEMH will produce murine myeloid cell lines using one of the two potential retroviral approaches.

EML and EPRO Cells

Primary BM cells are transduced with a retrovirus that encodes a dominant negative form of the retinoic acid receptor, which, when the cells are selected in the presence of SCF or GM-CSF, results in cells referred to as EML and EPRO cells, respectively. This approach, developed by Dr. Schickwann Tsai, who is now at the University of Utah, results in cell lines that can be induced to differentiate into different cell types with the addition of supraphysiological levels of all-trans retinoic acid (ATRA) plus specific growth factors 10, 11.

Primary immortalized cell lines

Primary BM cells are transduced with a retrovirus that encodes the resistance gene to puromycin, but does not encode any genes that independently induce cell immortalization or a block in differentiation. When primary cells are infected with this retrovirus and selected for growth in the presence of puromycin, the rare cell lines that develop are clonal expansions of cells in which the retrovirus has inserted stably into a region of the genomic DNA such that a gene promoting cell growth and/or a block in differentiation is activated or repressed.

Quality Control

For every cell line created, we will characterize the purity, sterility, growth rate, and function. The purity will be assessed by morphology of cytospin preparation and by FACS analysis. Acceptable criteria for the EML-type cell lines will be a uniform blast-like morphology for at least 80% of the SCF-dependent undifferentiated cells in any particularly line, and acceptable differentiation will include differentiation of at least 80% of the cells into early (band) or late neutrophils after induction. For the EPRO cell lines, they need to have a uniform promyelocyte-like morphology for at least 80% of the GM-CSF-dependent undifferentiated cells, and acceptable differentiation will include differentiation of at least 80% of the cells into early (band) or late neutrophils after induction.

Product Specification Sheet

Users will receive a Product Specification Sheet for each cell line supplied that describes not only the methodology used to create the line, but also the optimal medium for growing and differentiating the cells, the growth rate and optimal range of cell concentrations for growing the cells, and the % differentiation over time after induction. Sterility will be assessed by microbiological culture plus a commercial test for mycoplasma. Cell lines will not be released if they are mycoplasma positive, and, obviously, if there is any gross bacterial or other contamination.

For additional information or to order this service, please contact Stephanie Halene at stephanie.halene@yale.edu. Charges will vary based on the amount of time required to obtain the cell lines requested.