The Reduction of In Vitro Radiation‐Induced Fas‐Related Apoptosis in CD34⁺Progenitor Cells by SCF, FLT‐3 Ligand, TPO, and IL‐3 in Combination Resulted in CD34⁺Cell Proliferation and Differentiation

Abstract

Recovery from radiation-induced (RI) bone marrow aplasia depends on appropriate cytokine support. The early effects of exogenous cytokines at the hematopoietic stem and progenitor cell (HSPC) level following irradiation are still largely unknown, especially those of survival factors such as stem cell factor (SCF) and Flt-3 ligand (FL). This study was aimed at A) clarifying Fas/Fas-Ligand (Fas-L) implication in RI apoptosis of CD34 + cells and B) assessing the capacity of a combination of cytokines to mitigate RI apoptosis in HSPCs in vitro. We showed that most of in vitro gamma-irradiated CD34 + HSPCs incubated in a medium devoid of cytokines underwent progressive apoptosis-related changes from 6 h (i.e., decreased CD34 antigen expression, Annexin V binding); then Fas/Fas-L coexpression occurred from 10 h on. A strong DNA fragmentation, as assessed by TUNEL assay and propidium iodide staining, was observed at 24 h. Within a 2.5-to 6-Gy dose range, the RI apoptotic process finally led to 97% CD34 + cell death within 48 h with a complete loss of functionality. Unirradiated cells incubated in the same conditions displayed a significantly reduced apoptotic pattern. The early addition of a combination of SCF, FL, thrombopoietin, and interleukin 3 (4F) after cell irradiation prevented 15% (2.5 Gy) and 12% (4 Gy) of HSPCs, respectively, from RI apoptosis, whereas these cytokines used as single factors were inefficient. Furthermore, irradiated HSPCs (2.5 Gy) incubated with 4F in a serum-free culture system for seven days proliferated, giving rise to an increase in the number of total cells (× 5.6-fold) and CD34 + cells (× 4.2-fold) and to megakaryocytic and granulomonocytic precursors. These results show that the prevention of apoptosis in in vitro irradiated HSPCs depends on an early combination cytokine support. These data suggest that the early therapeutic administration of anti-apoptotic cytokines may be critical for preserving functional HSPCs from in vivo radiation damage.

Mesenchymal stem cells rescue CD34+ cells from radiation-induced apoptosis and sustain hematopoietic reconstitution after coculture and cografting in lethally irradiated baboons: is autologous stem cell therapy in nuclear accident settings hype or reality?

“…This homogeneous dose induces the cell death of 95% of HSPCs 6 and mimics the damage undergone by underexposed BM areas after heterogeneous TBI in the range of 8-10 Gy. Cultures were then performed for 7 days (75 cm 2 flasks; Corning Costar, Dutscher, Issy-les-Moulineaux, France) using a serum-free medium (CTM-H00 containing human albumin, Mabio-RTM, Tourcoing, France) and a combination of four cytokines, Stem Cell Factor þ FLT-3 ligand þ Thrombopoietin þ Interleukin-3 (4F; 50 ng/ml each).…”

“…2 This problem is mainly due to the 'apoptotic burst' that occurs during the first hours following TBI. 6 Culturing HSPCs could allow significant amplification of the HSPC pool but the efficacy of cytokines may be reduced in cases of damaged cells. Moreover, the hematopoietic potential of rescued cells is suspected to be impaired.…”

“…We have previously shown that the homogeneous dose of 2.5 Gy induces the death of 95% of HSPCs and mimics the damage undergone by underexposed BM areas after heterogeneous TBI in the range of 8-10 Gy. 6 This model allowed us to explore major aspects of ACT, circumventing the difficulty of the collection phase.…”

Le syndrome hématopoïétique induit par une irradiation accidentelle : évaluation et traitements possibles

“…La seconde hypothèse est qu'il est possible d'amplifier in vitro et de façon efficace des cellules hématopoïétiques irradiées. Ceci a été vérifié par plusieurs groupes, soit à partir de CSH enrichies (Drouet et al, 1999), soit…”

Flt-3 and Its Ligand Are Expressed in Neural Crest–Derived Tumors and Promote Survival and Proliferation of Their Cell Lines

“…Flt-3 is expressed on human hematopoietic progenitors, immature thymocytes, monocytes, acute myelogenous leukemia (AML) and B cell precursors acute lymphoblastic leukemia (ALL), mouse fetal liver, and various adult mouse tissues, such as brain and cerebellum (Birg et al 1992;Drexler et al 1999;. Its ligand, flt-3 ligand (FL), is a cytokine that promotes the proliferation, survival, and differentiation of hematopoietic progenitors (Broxmeyer et al, 1995;Hannum et al, 1994;Lyman et al, 1994;Rasko et al, 1995;Vanu et al, 1999;Zeigler et al, 1994) in synergy with other growth factors, namely SCF, IL-3, IL-6, IL-11, IL-12, and GM-CSF (Drouet et al, 1999;Jacobsen et al, 1995). In association with G-CSF, FL mobilizes stem and progenitor cells to peripheral blood (Molineux et al, 1997;Papayannopoulou et al, 1997).…”