Page 49 - Haematologica May 2020
P. 49

EGR1 regulation of human BMSC
    consequently, improved ex vivo expansion of hematopoiet- ic cells by up-regulating a panel of hematopoiesis-support- ing genes, including cytokines and cell surface expressed molecules such as CCL28 and VCAM1.
EGR1 knockdown induces bone marrow stromal cell proliferation mediated by elevated reactive oxygen species
EGR1 functions either as a cell cycle promoter or a cell
proliferation inhibitor depending on the cell type and the environment. We therefore investigated how EGR1 expression impacted BMSC proliferation. Our data showed that EGR1 knockdown BMSC exhibited higher percentages of dividing cells, shorter population doubling times, and enhanced colony-forming capacities (Figure 5A-C and Online Supplementary Figure S5A). On the other hand, CFU-F activity was virtually absent in EGR1 over- expressing cells and population doubling times were sub-
 A
BCD
EF
G
Figure 2. Increased EGR1 expression in mesenchymal stromal cells (MSC) promotes the ex vivo expansion of transplantable cord blood CD34+ cells. Five thousand
 cord blood CD34+ cells were co-cultured for four days on a feeder layer of 10,000 BMSC transfected with scramble control, shEGR1, green fluorescent protein control (GFP ctr) and EGR1 overexpression plasmids, respectively, in SFEM supplemented with 25 ng/mL of SCF, TPO and Flt3L (STF25). (A) Representative FACS profiles of co-culture generated cells are shown. The type of feeder cells is indicated on top of the respective FACS plot. (B-D) Fold change of total number of hematopoietic cells (B), CD34+ cells (C), and CD34+CD90+ cells (D) produced after four days in culture. Results are shown as fold change relative to the cell number of standard CD34+ culture (STF25) without stroma support. N=9-12. *P<0.05. (E-G) Analysis of human hematopoietic cell engraftment following intravenous transplantation of the culture equivalent (4 days culture in SFEM, STF25) of 50,000 input CD34+ cells into NSG mice. Human engraftment was assessed 8, 12 and 16 weeks after transplantation by flow cytometry using human-specific antibodies against CD45, CD15/CD33/CD66b and CD19. *P<0.05 between EGR1 OE and each of the other groups. (E) Percentage of human CD45+ cells in the peripheral blood of NSG mice at different time points after intravenous transplantation. Data represent the meanĀ±standard deviation (SD) of a total of 4-5 mice per time point except no stroma control, which shows the data for two mice. (F) Lineage distribution of human cells (meanĀ±SD, n= 2-5) and (G) representative FACS plots 16 weeks after transplantation.
  haematologica | 2020; 105(5)
1209
   





















































































   47   48   49   50   51