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S. Romero Marquez et al.
In the bone marrow, the main function of the microenvi- ronment, or niche, is to limit recruitment of HSC into the cell cycle during stress responses. However, to expand, HSC must divide and retain their LTR ability. In vivo, this process is tightly regulated, so that not all LTR-HSC are recruited and then depleted, but a significant portion of these cells return to quiescence and, thus, self-renew. Since the role of the bone marrow niche is to limit the cell cycle of HSC activation,3 it is likely that extrinsic factors govern the retention of stem cell properties of HSC during their cell cycle progression. However, the exact factors mediating these processes have, to date, not been identified.
To identify possible factors involved in the maintenance and expansion of HSC, we generated murine stromal cell lines from different tissues of the mid-gestation mouse embryo.4,5 Two of these, UG26-1B6 and EL08-1D2, support the maintenance of both murine HSC and human cord blood CD34+ cells.4,5 Interestingly, we found that UG26-1B6 stromal cells support the maintenance of HSC in a non-con- tact setting,6,7 and we showed that repopulating HSC could be maintained in conditioned medium (CM) from this cell line.1 In the latter study, we further identified nerve growth factor (NGF) and collagen 1 (Col1) to be the most effective substitute for UG26-1B6-CM. The main effect of this CM seemed to be to improve survival of phenotypic HSC to almost 97% of the input cells during culture, allowing for almost complete recruitment of cells into cell division.1 In addition, the CM supported symmetrical cell division of HSC with LTR ability, which is a requirement for expansion of these cells.
In comparisons aimed at defining stem cell-maintaining factors from different stromal cells, factors characterizing the developmental origin of stromal cells should be distin- guished from factors required for their HSC-maintaining function.8 A problem with this approach is that for optimal filtering for functional secreted HSC-supportive factors, multiple sources of stromal cells secreting such factors to support HSC self-renewal without the requirement of direct stroma-contact should be available. To date, only two such cell lines have been described.1,6,7 Here, we describe mouse embryonic fibroblasts (MEF) as an additional source of mid-gestation stromal cells to maintain phenotypic HSC with repopulating activity. CM from MEF (MEF-CM) has previously been used to maintain both murine and human embryonic stem cells.9 Interestingly, MEF-CM also facili- tates the differentiation of embryonic stem cells into the hematopoietic lineage.10 Whether and how MEF-CM affects adult HSC and their repopulating activity under serum-free conditions remain to be established.
We here show that MEF-CM maintains the self-renewal of murine HSC with repopulating ability in single cell cul- tures. MEF represent a robust and easily accessible stromal cell source to study the mechanisms of the maintenance of HSC by niche factors and identify further factors support- ing expansion of HSC with repopulating ability.
Methods
Mice
Eight- to 10-week old C57BL/6.J (B6; CD45.2), B6.SJL- Ptprca.Pep3b/BoyJ (Ly5.1; CD45.1), 129S2/SvPasCrl (129; CD45.2) mice were obtained from Charles River Laboratories (Lyon, France). All experiments were approved by the Government of Upper Bavaria. Animals were housed in micro-isolators under spe-
cific pathogen-free conditions, according to Federation of Laboratory Animal Science Associations and institutional recom- mendations.
Primary cells and mouse embryonic fibroblasts
MEF were generated from embryos (E11.5 or E13.5) using a generic protocol11 without trypsin digestion of the embryonic tis- sue. The dissected embryos were cultured in MEF medium (see Online Supplementary Methods) at 37°C and passaged four times every 3 to 4 days.
Generation of conditioned medium
CM was prepared essentially as described previously.1 In brief, 70,000/cm2 MEF (passage 4) were cultured to near confluence in MEF medium (for details see Online Supplementary Methods). Twelve hours later the cells were γ-irradiated (30 Gy X-Ray; Gulmay Type RS225), washed twice with Dulbecco phosphate- buffered saline (DPBS; Gibco) and cultured for 72 h with serum- free medium (SFM; StemSpan, Stemcell Technologies).1
Flow cytometry analyses
Surface antigens were stained with antibodies from either Invitrogen- or eBioscience-ThermoFisher (Online Supplementary Table S1). Flow cytometry was performed on a CyAn ADP Lx P8; cell sorting was performed using an Astrios high speed sorter (both from Beckman-Coulter). Data were analyzed with FlowJo software (TreeStar).
Cell sorting and single cell cultures
For single cell cultures, single murine CD34- CD48- CD150+ Lineage- SCA1+ KIT+ (LSK) cells (CD34- SLAM cells) were sorted directly into a 96-well round-bottomed plate on an Astrios high speed cell sorter (Beckman Coulter). Single cells were deposited with an efficiency of 90%. These CD34- SLAM cells comprise both CD34- and CD34low populations, as these have been reported to contain similar frequencies of long-term repopulating cells.12 These plates were preloaded with 100 mL of filtered (0.20 mm, Sartorius) CM supplemented with murine stem cell factor (SCF: 100 ng/mL) and murine interleukin-11 (IL-11; 20 ng/mL) (two growth factors; 2GF), both from R&D Systems (BioTechne). Additional cultures were further supplemented with Col1 (300 μg/mL; BioVendor) and human NGF (250 ng/mL; R&D Systems- BioTechne) (four growth factors; 4GF). Every 24 h, the number of cells per well was enumerated using a light microscope. After 5 days, cells that had divided at least once were harvested, pooled, stained with antibodies, and analyzed by flow cytometry. In some experiments, colony-forming ability of pools from divided cells was assessed using standard assays (M3434; Stemcell Technologies).
In vivo transplantation assay
An in vivo repopulation assay using competitive transplantation
was performed as described previously.13,14 For this purpose, 20 wells showing at least one cell division of CD34- SLAM cells from 129xLy5.1 F1 (129Ly5.1; CD45.1xCD45.2) donors were harvested, pooled, and transplanted into lethally irradiated 129xB6 F1 (129B6; CD45.2) recipient mice, together with 1x105 bone marrow cells and 5x105 spleen cells from 129B6 mice. Donor cell engraftment was analyzed every 5, 10 and 16 weeks in the peripheral blood and 16 weeks after transplantation in the bone marrow cells, as describedpreviously.13,14
Apoptosis assay
Apoptosis was determined in 48 h cultures of LSK cells in 12- well plates, prefilled with 2 mL SFM or MEF-CM with the growth
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