Page 42 - 2019_04-Haematologica-web

E.-M. Demmerath et al.
protecting HSPCs from damage and preventing their cell death or by fostering subsequent hematopoietic regenera- tion.3-10 In contrast, only limited preclinical data on sub- stances protective for the human hematopoietic system in vivo are available.11,12
The aim of this study was to identify substances that protect human HSPCs from irradiation-induced apoptosis in vivo and to delineate their effects on the BCL-2 protein family. BCL-2 proteins are the master regulators of the intrinsic apoptosis pathway and have either pro- or anti- apoptotic function. Anti-apoptotic BCL-2 proteins (i.e. BCL-2, BCL-XL, MCL-1 and A1/BFL) protect cells from apoptotic stimuli by binding and inactivating their pro- apoptotic antagonists. The pro-apoptotic family members can be subdivided into the downstream ‘effector’ proteins, BAK and BAX, and the BH3-only proteins (e.g. BIM, PUMA, BMF, BAD and others) that act upstream as cell stress sensors. Upon activation, BH3-only proteins acti- vate BAX and BAK either directly or indirectly through inhibition of the anti-apoptotic BCL-2 proteins. BAX/BAK activation leads to outer mitochondrial membrane perme- abilization, caspase activation and cell death.13 Radiotherapy as well as most conventional chemothera- peutic drugs converge at the level of BCL-2 proteins and engage the intrinsic apoptosis pathway.2
A particularly attractive candidate for our study was the epidermal growth factor (EGF) that was recently described to prevent irradiation-induced apoptosis of murine HSPCs in vivo.10 Mechanistically, EGF receptor was up-regulated on bone marrow HSPCs subjected to irradiation, and binding of EGF resulted in suppression of p53-mediated transcriptional activation of the pro-apoptotic BCL-2 pro- tein PUMA,10 the p53 target responsible for most DNA damage-induced apoptosis in hematopoietic cells.14,15
A second candidate was prostaglandin E2 (PGE2), which was shown to have multiple beneficial effects on both murine and human HSPCs, including increased survival, self-renewal and homing, together resulting in increased long-term repopulation potential.5,16,17 In a mouse model of sublethal total body irradiation (TBI), treatment with the long-acting PGE2 analog, 16,16-dimethyl-PGE2 (dmPGE2), resulted in increased HSPC survival and accel- erated hematopoietic regeneration.9 The increase in apop- tosis resistance was explained by upregulation of BCL-2 and BCL-XL and reduced expression of BAX.17
Finally, the cytokines FLT3L, stem cell factor (SCF) and thrombopoietin (TPO) have protective effects on murine and human HSPCs and drive their proliferation in vitro.3 Together, these cytokines are frequently used for culture and ex vivo expansion of human CD34+ cells. We have shown earlier that their pro-survival activity can be attrib- uted to reduced transcription of BIM and BMF mRNA.18
None of these molecules have been tested yet for possi- ble protective effects on human hematopoiesis in vivo. We focused on EGF, PGE2, FLT3L and TPO but excluded SCF from our studies since it had been shown earlier to induce extensive proliferation and premature exhaustion in murine HSPCs.19 We analyzed the effects of these sub- stances on human HSPCs subjected to cell stress in vitro and, in addition, developed a xenograft model to analyze stress resistance and regeneration of human hematopoiesis in vivo.
Despite promising data obtained in the above-described mouse model, EGF was not able to protect human HSPCs in vitro nor to promote hematopoietic regeneration follow-
ing sublethal irradiation in vivo. PGE2 significantly reduced in vitro apoptotic susceptibility of human HSPCs to taxol and etoposide. This could, however, be ascribed to reduced proliferation rather than to a change in BCL-2 protein regulation. Accordingly, PGE2 did not accelerate regeneration of the human hematopoietic system in vivo. Repeated treatment of sublethally irradiated xenograft mice with the combination of FLT3L and TPO also result- ed in only minor beneficial effects during human hematopoietic regeneration.
Methods
Cell isolation and culture
Human umbilical cord blood was obtained after caesarean birth. Informed consent was obtained from the parents and the study was approved by the local ethics committee. CD34+ cells were enriched by MACS-technology (Miltenyi), cell purity was generally more than 90%. Purified cells were frozen in CryoStor CS10 (Stem Cell Technologies), stored in liquid nitrogen and used at later time points. Thawed cells were cultured in serum-free medium supplemented with 10% ES-FBS (Invitrogen), human TPO (50 ng/mL, Immunotools), FLT3L, SCF, IL3 (100 ng/mL each, Immunotools), human EGF (hEGF) (20 or 200 ng/mL Immunotools), PGE2 (10, 25 or 50 mM, Sigma-Aldrich) and/or cytotoxic drugs (etoposide, taxol, tunicamycin; Sigma-Aldrich). Alternatively, thawed cells were used for xenotransplantation.
Xenotransplantation
All experiments were performed according to the guidelines of the German "Tierversuchsgesetz" and approved by the local com- mittee (RP Freiburg/Germany). Rag2-/-γc-/- mice were irradiated at five weeks of age with 3 Gy and 6-8 hours (h) later they were injected intravenously into the retrobulbar venous plexus with 3x105 human CD34+ cells. Four weeks later, animals were irradiat- ed again. Subsequently, xenograft mice were treated once daily intraperitoneally (i.p.) with human EGF (0.5 mg/g body weight), murine EGF (0.5 μg/g), human dmPGE2 (2 mg/g), human FLT3L (40 ng/g), human TPO (40 ng/g), combinations thereof, or respective carrier solutions (Figure 1). At indicated time points, mice were sacrificed for analysis. Alternatively, mice were treated once daily for seven days with etoposide (20 mg/k, i.p.), and the anti-apop- totic substances were given simultaneously.
Proliferation, apoptosis and colony formation assays
Cell cycle status and proliferation were determined by double staining for Ki-67 (BioLegend) and DAPI (Sigma-Aldrich) or incu- bation with CFSE (1 mM; Sigma-Aldrich). Apoptosis was deter- mined by combined staining with 7-AAD and Annexin-V. Specific apoptosis triggered by stress was calculated as follows: (induced apoptosis – spontaneous apoptosis)/(100 – spontaneous apopto- sis). For colony forming assays, 150,000 human CD45+ cells isolat- ed from murine bone marrow (BM) were plated for 11 days on a semi-solid medium containing insulin, transferrin, human SCF, IL-3, IL-6, EPO, G-CSF and GM-CSF (SF H4436 MethoCult).
Flow cytometric analysis
Single cell suspensions of hematopoietic organs were surface- stained with antibodies conjugated with FITC, PE, APC, PerCP/Cy5.5, PE/Cy7 or biotin. Antibodies for murine markers: anti-CD45 (30-F11). Antibodies for human markers: H13 and 2D1, anti-CD45; AC136, anti-CD34. Biotinylated antibodies were detected using streptavidin-APC. Flow cytometric analysis was performed using a FACS-Fortessa (BD).
670
haematologica | 2019; 104(4)