Page 47 - Haematologica May 2020

EGR1 regulation of human BMSC
EGR1 might also have a role in human primary non- hematopoietic bone marrow stromal cells.
We therefore investigated the role of EGR1 in human BMSC and found that EGR1 was highly expressed in steady-state primary BMSC compared to regenerating adult and fetal BMSC. Overexpression of EGR1 increased BMSC hematopoietic stroma support function and inhib- ited cell proliferation, whereas decreased EGR1 expres- sion caused the opposite effects. Our data thus indicate a key dual role of EGR1 in BMSC regulation, which has important implications for stroma repair and replacement approaches, and possibly also for the understanding of important developmental aspects of hematopoietic stro- ma formation.
Methods
Human bone marrow and cord blood cells
Human bone marrow (BM) cells were collected at the Hematology Department, University of Lund, Sweden, from consenting healthy donors by aspiration from the iliac crest. Cord blood (CB) samples were obtained from consenting donors at the maternity wards at Lund, Helsingborg and Malmö Hospitals, Sweden. BM aspirates from patients with acute myeloid leukemia (AML), BM controls, and fetal long bones were obtained with informed consent at the Erasmus Medical Center, the Netherlands, as described previously.8 The process- ing of BM mononuclear cells (BM-MNC), isolation and charac- terization of primary bone marrow stroma cells (BMSC), and generation of cultured stroma cells including EGR1 over-express- ing and knockout cells from sorted CD45–CD271+CD140a– BMSC, is provided in the Online Supplementary Appendix: experi- mental procedures. The use of human samples was approved by the corresponding Institutional Review Board of the University of Lund and the Erasmus Medical Center, respectively, in accor- dance with the Declaration of Helsinki.
Co-cultureofcordbloodCD34+ cellswithstromalcells FACS-sorted GFP+ stromal cells (EGR1 knockdown, EGR1 over-expressing cells, and corresponding controls) were plated as adherent feeder cells into 96-well plates at 10,000 cells per well and cultured in MSC medium for three days. Medium was then removed and 5,000 CB CD34+ cells were added in serum-free expansion medium (SFEM, STEMCELL Technologies) supple- mented with or without stem cell factor, thrombopoietin, and FLT-3 ligand (all at 25 ng/mL, STF25) (Online Supplementary Appendix). Expanded CB cells were harvested, counted and ana-
lyzed after four days of co-culture.
Conditioned MSC medium was prepared by replacing the
MSC expansion medium with serum-free expansion medium (SFEM) and collecting the conditioned media after four days.
Transwell cultures were performed using the HTS Transwell- 96 System (Corning, New York, NY, USA); 10,000 BMSC in standard MSC culture medium were plated in the transwell reservoir. After three days of culture, the MSC culture medium was removed and replaced with SFEM (STF25), and 5,000 CB CD34+ cells in SFEM (STF25) were loaded into the insert (mem- brane pore size: 0.4 mm). After four days of co-culture, cells were harvested, counted and analyzed.
To evaluate the role of VCAM1 and CCL28 on CD34 expansion, stromal cells were pretreated with 100 ng/mL VCAM1 or CCL28 blocking antibody and control IgG, respectively, at 37°C for two hours followed by co-culture with CD34+ cells in either standard SFEM (STF25) or cytokine-free conditions at 37°C for four days.
Other methods
The details of the other methods used in this study, i.e. gener- ation of cultured MSC, EGR1 knockdown and EGR1 over- expressing stroma cells, FACS sorting and analysis, CFU-F assays, in vitro differentiation assays, real-time polymerase chain reaction (PCR), in vivo HSC repopulation assay, CCL28 ELISA, Illumina array, RNA-seq and proteome analysis, as well as infor- mation on the deposition of gene expression and proteomics data, are all provided in the Online Supplementary Appendix: experimental procedures.
Results
EGR1 is highly expressed in primary bone marrow stromal cells
Our previous gene expression profiling data demon- strated that expression levels of EGR1 in primary CFU-F (colony-forming unit, fibroblast)-enriched lin–CD45– CD271+ BMSC were substantially higher compared to non-colony-forming cells (lin–CD45–CD271–).1 We there- fore proceeded to investigate EGR1 expression and func- tion in highly purified lin–CD45–CD271–CD140a (PDGFRα)– BMSC, which we have recently demonstrated as a (close to) pure population of putative BM stromal stem cells with high CFU-F frequency, typical in vitro and in vivo differentiation capacities, and potent hematopoiet- ic stroma function.1 Expression of EGR1 was 128.9±28.4- fold higher in lin–CD45–CD271+CD140a– BMSC com- pared to non-colony-forming cells (lin–CD45–CD271–CD140a–), and 2.8±0.6-fold higher compared to lin–CD45– CD271+CD140a+ stromal cells, which have only limited CFU-F activity (Figure 1A).1 In addition, EGR1 expression was significantly higher in steady-state adult BMSC (CD31–CD271+) in comparison to fetal BMSC, BMSC in regenerating marrow, and BM endothelial cells (CD31+CD9+) (Figure 1B). None of the other EGR transcription factor family members were expressed at comparable levels in BMSC or endothelial cells (Online Supplementary Figure S1A and B).
Upregulation of EGR1 increases BMSC cell hematopoietic stroma-support
Motivated by the selective high expression of EGR1 in hematopoiesis-supporting BMSC, we went on to investi- gate the functions of EGR1 employing gain-of-function and loss-of-function approaches.
Effective reduction of EGR1 expression in BMSC was induced by two (shEGR1-1 and shEGR1-4) of four tested EGR1-specific shRNA (Online Supplementary Figure S2A). EGR1 overexpression in BMSC by more than two-fold was realized using the lentiviral vector encoding full length human EGR1 (Online Supplementary Figure S2B).
BMSC cell hematopoietic stroma supporting function was evaluated by co-culturing CB CD34+ cells with EGR1 knockdown stromal cells, EGR1 over-expressing stromal cells and corresponding controls, respectively, as adherent feeder cells. Total numbers of hematopoietic cells increased when CD34+ cells were expanded on stroma feeder layers compared to cultures without stroma sup- port, but no differences were observed between the dif- ferent stromal cell groups (Figure 2A and B). However, EGR1 knockdown feeder cells failed to effectively sup- port the CD34+ phenotype while the EGR1 over-express- ing stromal cells efficiently maintained this population
haematologica | 2020; 105(5)
1207