Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells

Abstract

Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC numbers in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34(+)CDCD38(-)Lin(-) cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration.

Figure 1

Pleiotrophin is overexpressed by HUBECs, and treatment with pleiotrophin induces the expansion of phenotypic HSCs in culture. (a) Unsupervised hierarchical cluster analysis of 1,335 genes upregulated in HUBECs (the region of the heat map indicated by the red bar) compared to nonbrain endothelial cells (ECs) (red, increased expression; green, decreased expression). (b) Left, scatter plot of pleiotrophin gene expression determined by microarray analysis in HUBECs versus nonbrain ECs (mean 25.1 ± 7.4 versus 1.0 ± 0.3, n = 6–8 samples per group, P = 0.001). Horizontal lines represent mean pleiotrophin expression in each group. Middle, pleiotrophin expression determined by quantitative RT-PCR in HUBECs versus coronary and pulmonary artery ECs (means ± s.e.m., n = 2 or 3 samples per group, HUBECs 1 versus coronary, P = 0.004; HUBECs 1 versus pulmonary, P = 0.004). Right, pleiotrophin concentrations determined by ELISA of HUBEC-conditioned medium (CM) compared to nonbrain EC–conditioned medium (means ± s.e.m., n = 3 samples per group, * P = 0.04). (c) Left, a representative high-power field microscopic image of RPTP-β/ζ staining in mouse bone marrow mononuclear cells versus isotype control (n=3 mice). Right, flow cytometric analysis of RPTP-β/ζ expression on mouse bone marrow KSL cells (n=1). (d) The total number of bone marrow cells, the percentage of KSL cells and the number of KSL cells after 7 day culture of highly purified CD34⁻KSL cells (97.3% ± 2.1%) with cytokines (TSF) with or without pleiotrophin (PTN). Left, data are means ± s.d., n = 3 experiments per group, * P = 0.01, ** P = 0.006 versus TSF. Middle, data are means ± s.d., n = 3, * P = 0.04, ** P = 0.004 versus TSF. Right, data are means ± s.d., n = 3 experiments per group,* P = 0.005, ** P = 0.006 versus TSF. All comparisons were one-tailed t tests. (e) Bone marrow CD34⁻KSL cells were placed in culture with TSF alone or TSF plus pleiotrophin for 7 days and representative FACS plots show the presence of side population cells in the progeny of TSF alone and TSF plus pleiotrophin cultures (n=2 experiments; percentage of cells that efflux the Hoechst 33342 dye are shown in the gates; UV1-A = Hoechst Red and UV2-A represents Hoechst Blue staining).

Figure 2

Treatment with pleiotrophin induces the expansion of mouse short- and long-term HSCs. (a) Scatter plots showing the percentages of total CD45.1⁺ donor cells and donor-derived B220⁺ (B lymphoid), Mac-1⁺/Gr-1⁺ (myeloid) and Thy1.2⁺ (T cell) populations in the peripheral blood (PB) of mice transplanted with 10 bone marrow CD34⁻KSL cells or their progeny after culture (n = 8–10 mice per group; means ± s.d). Horizontal lines represent the mean engraftment levels for each group. PB, peripheral blood. (b) Representative flow cytometric analysis of peripheral blood donor-derived (CD45.1⁺) multilineage engraftment at 12 weeks after transplantation in mice transplanted with ten bone marrow CD34⁻KSL cells versus mice transplanted with the progeny of ten bone marrow CD34⁻KSL cells after culture with TSF and 100 ng ml⁻¹ pleiotrophin. The percentage of cells in each quadrant is indicated (representative of analysis of 8–10 mice per group) (c) Poisson statistical analysis after limiting-dilution analysis; plots were obtained to allow estimation of CRU content within each condition (n = 8–10 mice transplanted at each dose per condition; n = 75 mice total). The plot shows the percentage of recipient mice containing less than 1% CD45.1⁺ cells in the peripheral blood at 12 weeks after transplantation versus the number of cells injected per mouse. BM, bone marrow. (d) The number of CD45.1⁺ donor-derived cells in the peripheral blood of mice transplanted with day 0 CD34⁻KSL cells or with pleiotrophin plus TSF-treated or TSF-alone treated CD34⁻KSL cells at 4, 8, 12 and 24 weeks after transplantation (means ± s.e.m., n = 6–10 per group, * P = 0.006, * P = 0.002, * P = 0.006, P = 0.05; ^ P = 0.005, ^ P = 0.002, ^ P = 0.007, P = 0.05,) (e) Donor-derived CD45.1⁺ cell engraftment 12 weeks after transplantation in secondary recipient mice transplanted with bone marrow isolated from primary mice transplanted with day 0 CD34⁻KSL cells or with TSF plus pleiotrophin or TSF-alone treated CD34⁻KSL cells (means ± s.e.m., n = 5 or 6 per group, P = 0.003 and 0.02 for TSF plus pleiotrophin group versus day 0 CD34-KSL group and TSF alone group, respectively; Mann-Whitney test). Horizontal bars represent mean levels of CD45.1⁺ cell engraftment in the peripheral blood. (f) Representative FACS analysis of CD45.1⁺ cell engraftment and B220⁺, Mac-1⁺/Gr-1⁺ and Thy 1.2⁺ engraftment at 12 weeks after transplantation in secondary recipient mice transplanted with bone marrow from primary mice transplanted with day 0 CD34⁻KSL cells or with TSF plus pleiotrophin treated CD34⁻KSL cells (representative of n= 5–6 mice per group). (g) The mean levels of donor CD45.1⁺ cells in the bone marrow (BM) of CD45.2⁺ recipient mice at 24 h after transplantation of CD45.1⁺ bone marrow Sca-1⁺Lin⁻ cells (4 × 10⁴) or their progeny after 7 d of culture with TSF or TSF plus pleiotrophin (n = 3–5 per group, means ± s.d.).

Figure 3

Treatment with pleiotrophin induces the expansion of human HSCs. (a) Total number of cells, percentage of CD34⁺CD38⁻Lin⁻ cells and number of CD34⁺CD38⁻Lin⁻ cells after culture of highly purified human cord blood CD34⁺CD38⁻Lin⁻ cells (98.6% ± 0.4%, Input = day 0, 3×10³ cells/culture) with pleiotrophin (either 100 or 500 ng⁻¹ ml) plus TSF or TSF alone (means ± s.d., n = 3 per group, * P = 0.02 versus TSF, ** P = 0.04 versus TSF, ^ P = 0.04 versus TSF). (b) The number of CFCs obtained from 1×10³ day 0 cord blood CD34⁺CD38⁻Lin⁻ cells or after culturing of these cells with pleiotrophin plus TSF or TSF alone (means ± s.d., n = 3 per group). * P = 0.0004 and 0.01 versus day 0 and TSF group, respectively (CFU-GM=colony forming unit - granulocyte monocyte; BFU-E=burst forming unit-erythroid; CFU-GEMM=colony forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte). (c) Mean levels of human CD45⁺ cell engraftment in the peripheral blood of NOD-SCID mice at 4 weeks in mice transplanted with cord blood CD34⁺CD38⁻Lin⁻ cells (1,000 cells) or their progeny after 7 d of culture with TSF or TSF plus pleiotrophin (means ± s.d., n = 10–13 per group; * P = 0.04 and 0.05 versus day 0 CD34⁺CD38⁻Lin⁻ and TSF groups, respectively). (d) A scatter plot showing the levels of human CD45⁺ cell engraftment in the bone marrow of NOD-SCID mice 8 weeks after transplantation with 500 or 2,500 cord blood CD34⁺CD38⁻Lin⁻ cells or their progeny after culture with TSF or TSF plus pleiotrophin (n = 7–13 mice per group). Horizontal bars represent the mean levels of human CD45⁺ cell engraftment in the bone marrow in each group. 500 cell dose: CD34⁺CD38⁻Lin⁻ group = 0.3% ± 0.3% human CD45⁺ cells, TSF = 1.9% ± 0.9% human CD45⁺ cells, TSF plus pleiotrophin = 2.6% ± 1.0 human CD45⁺ cells; 2,500 cell dose: CD34⁺CD38⁻Lin⁻ group = 3.1% ± 1.9% human CD45⁺ cells, TSF = 17% ± 6.4% human CD45⁺ cells, TSF plus pleiotrophin = 21% ± 8.0% human CD45⁺ cells. (e) FACS plots of representative human CD45⁺ hematopoietic cell engraftment (top) and multilineage differentiation (bottom) in NOD-SCID mice at 8 weeks after transplantation with 2,500 cord blood (CB) CD34⁺CD38⁻Lin⁻ cells or their progeny after culture with TSF or TSF plus pleiotrophin (representative of 7–13 mice per group).

Figure 4

Pleiotrophin mediates bone marrow progenitor cell expansion via activation of PI3K and Notch signaling The total number of cells, percentage of KSL cells and number of KSL cells in untreated bone marrow CD34⁻KSL cells (5×10²) or after treatment of these cells with TSF plus pleiotrophin or TSF alone. TSF plus pleiotrophin treatment was with or without addition of LY294002 (LY29), a PI3K inhibitor, or 30 μM γ-secretase inhibitor (γ-sec), a Notch signaling inhibitor. TSF plus pleiotrophin caused a significant increase in percentage KSL cells and total KSL cells compared to culture with TSF alone (means ± s.d., n = 3 per group, * P = 0.04 and 0.02 for comparison with TSF alone; the addition of LY294002 caused a significant decrease in total cells, percentage KSL and KSL cells, respectively (^ P = 0.002, 0.03, and 0.002 for comparison with TSF plus pleiotrophin); the addition of γ secretase inhibitor caused a significant decrease in percentage KSL and KSL cells compared to TSF plus pleiotrophin ** P = 0.01 and 0.002).

Figure 5

Pleiotrophin induces bone marrow stem and progenitor cell regeneration in vivo. Bone marrow stem and progenitor cell content and function are shown for mice irradiated with 700 cGy total body irradiation and subsequently treated intraperitoneally for 7 d with pleiotrophin, G-CSF or saline (day 4, 7 and 14 = days post-irradiation). (a) Total number of bone marrow cells (means ± s.d., n = 10, * P = 0.02 versus saline, ^ P = 0.04 versus G-CSF). (b) Percentage KSL cells (means ± s.d., n = 5 per group; day 4, * P = 0.04 and ** P = 0.003 versus saline; day 7, * P = 0.03 and ^ P = 0.004 versus saline- and G-CSF groups, respectively; day 14, * P = 0.04 and ^ P = 0.02 versus saline- and G-CSF-treated groups, respectively). (c) Bone marrow CFCs (n = 3 per group, means ± s.d.; * P = 0.03, 0.004, and 0.006 for comparison between G-CSF and saline groups at days 4, 7 and 14, respectively; * P = 0.004 and 0.04 for comparison between pleiotrophin and saline groups at days 7 and 14, respectively; ^ P = 0.04, 0.04 and 0.02 for comparison between pleiotrophin and G-CSF groups at days 4, 7 and 14, respectively. (d) Bone marrow LTC-ICs (n = 4–6 per group, means ± s.d.; * P = 0.02 and P = 0.02 versus saline group at days 7 and 14, respectively; ^ P = 0.03 and P = 0.02 versus G-CSF group at days 7 and 14, respectively). (e) Competitive repopulation assays, performed with 5 × 10⁵ CD45.1⁺ bone marrow cells collected at day 7 from mice that had been irradiated with 700 cGy and subsequently treated with pleiotrophin or saline for 7 days. Bone marrow was collected at weeks 4, 8 and 12 after transplantation, and donor CD45.1⁺ cell engraftment was measured in the peripheral blood of recipient CD45.2⁺ mice. (n = 9 per group, means ± s.d. * P = 0.001, 0.04, and 0.04 at weeks 4, 8 and 12, respectively).