Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function

Abstract

The chemokine stromal-derived factor-1 (SDF-1) controls many aspects of stem cell function. Details of its regulation and sites of production are currently unknown. We report that in the bone marrow, SDF-1 is produced mainly by immature osteoblasts and endothelial cells. Conditioning with DNA-damaging agents (ionizing irradiation, cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and in CXCR4-dependent homing and repopulation by human stem cells transplanted into NOD/SCID mice. Our findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.

Figure 1

Anti–SDF-1 reactive immature BM osteoblasts lining the endosteum region (OB) and stromal cells (ST; arrows, ×200) (a), venules (arrow) (b), and arteriole (arrow) (c) in human BM sections (×1000). No peroxidase staining of control mAb was observed on identical tissue sections (data not shown).

Figure 2

(a) Expression of SDF-1 and CXCR4 by RT-PCR. Lane a: endothelial MBA-2.1 cells; lane b, adipocyte 14F1.1 cells; lane c, osteoblast MBA-15 cells; lane d, HUVECs; lane e, primary human stroma. Expression of GAPDH and SDF-1: primary HOBs (lanes 1–4), osteosarcomas MG-63 (lane 5), and SaOS-2 (lane 6). (b) Migration of CD34⁺ cells to CM: SDF-1 migration to 125 ng/ml SDF-1, MBA-15, HOB3, HOB4, MG-63, 14F1.1, MBA-2.1, HUVECs. αCXCR4 or T22 pretreatment of CD34⁺ before Transwell migration. The results are the mean of seven different experiments ± SE. (c) Engraftment by CD34 cells migrating (M) and nonmigrating (NM) to MBA15 CM. Human CD45⁺CD19⁺ cells (R1) in the BM of mice transplanted with cells migrating to CM of MBA-15.

Figure 3

Increased levels of SDF-1 after TBI. (a) SDF-1 mRNA expression in the BM of two NOD/SCID mice per time point. (b) Migration of human CD34⁺ cells to the NOD/SCID BM CM 4, 24, and 48 hours after TBI. Spontaneous migration (CTRL) and migration to SDF-1 (SDF-1). Results are mean ± SE from seven experiments. (c) Semisolid colony assay for progenitors migrating to NOD/SCID BM CM collected before or 48 hours after TBI, without or with anti-CXCR4 Ab pretreatment. (d) Engraftment by mononuclear cells (MNC), CD34⁺ cells or sorted CD34⁺/CD38^–/low cells transplanted immediately or 48 hours after TBI. Mean ± SE from nine experiments. P values determined using student’s t test. GEMM, granulocyte erythrocyte macrophage megakeryocyte; GM, granulocytes-macrophages; BFU-E, burst forming unit erythroid.

Figure 4

Increased levels of SDF-1 in the spleen of NOD/SCID after TBI. (a) SDF-1 mRNA in the BM and spleen from the same mouse per time point. (b) Semisolid colony assay for migrating progenitors to spleen CM collected before, 24 hours, or 48 hours after TBI with and without anti-CXCR4 Ab pretreatment.

Figure 5

Influence of DNA-damaging treatment on SDF-1 expression. (a) Semiquantitative RT-PCR for murine cell lines MBA-2.1, 14F1.1, and MBA-15, before and 48 hours after 300-cGy radiation. (b) ELISA for CM of astrocytes U373 and BM mesenchymal stromal cells MS5 48 hours after stimulation with IL-1β (Promega Corp.) or irradiation. (c) Semiquantitative RT-PCR for SDF-1 mRNA from BM of Cy- or 5-FU–treated BALB/c mice, murine MBA-15 osteoblasts (two mice or two tissue-culture plates per time point), human primary HOB3, and control levels of β-actin expression.

Figure 6

Increased homing of human CD45⁺ cells transplanted 48 hours after treatment with TBI, Cy, or 5-FU. (a) FACS plots, CD45⁺: CB MNC, human CB MNC before transplantation; CTRL, nontransplanted murine BM; NT, transplantation of human CB MNC into nontreated mice; Irrad. 0 h, transplanted immediately after TBI; Irrad. 48 h, transplanted 48 hours after TBI; 5-FU or Cy, transplanted 48 hours after treatment. (b) Total numbers of human CD45⁺ cells in the murine BM and spleen, 24 hours after transplantation of human CB MNC (2 × 10⁷/mouse) into nontreated (NT) or treated NOD/SCID mice. Results from two experiments, mean ± SE, are shown. Amounts of human cells were calculated for the total organ based on acquisition number of cells and cell numbers in the total organ. Numbers inside the chart represent P values calculated with Student’s t test. FL-1, anti-CD45 FITC; FL-2, internal fluorescence.