Pleiotrophin Is a Growth Factor for Hematopoietic Stem Cells and Induces Stem Cell Self-Renewal

The ability to undergo self-renewal is a defining feature of hematopoietic stem cells (HSCs) but the extrinsic signals which regulate HSC self-renewal remain unclear. We performed a genome-wide expression analysis on primary human brain ECs (HUBECs, n=10) which support the ex vivo expansion of HSCs in non-contact culture (Blood 100:4433–4439; Blood 105:576–583) and non-brain ECs which do not support HSC expansion (n=8) in order to identify soluble proteins overexpressed by the HSC-supportive HUBECs. We identified pleiotrophin (PTN), an 18 kD heparin binding growth factor, to be 32-fold overexpressed in HUBECs as compared to non-supportive EC lines. PTN has established activity in angiogenesis, embryogenesis, neuronal cell growth and tumorigenesis, but has no known function in hematopoiesis. We first tested whether secreted PTN was responsible for the amplification of HSCs that we have observed in co-cultures of HSCs with HUBECs via “loss of function” studies in which a blocking anti-PTN antibody was added to HUBEC cultures and HSC content was measured. Competitive repopulating unit (CRU) assays were performed in which limiting doses of donor CD45.1+ bone marrow (BM) 34−c-kit+sca-1+lin− (34-KSL) HSCs (10, 30 or 100 cells) or their progeny following 7 day non-contact culture with HUBECs + IgG or HUBECs + a blocking anti-PTN were transplanted into lethally irradiated CD45.2+ C57Bl6 mice. Mice transplanted with the progeny of 34-KSL cells cultured with HUBECs demonstrated 4–6 fold increased levels of donor-derived CD45.1+ multilineage repopulation at 8-, 12- and 24-weeks post-transplantation as compared to mice transplanted with input 34-KSL cells. In contrast, mice transplanted with the progeny of 34-KSL cells following culture with HUBECs + anti-PTN demonstrated significant reduction in donor CD45.1+ cell repopulation compared to mice transplanted with the progeny of HUBEC cultures and no difference in donor CD45.1+ cell engraftment compared to mice transplanted with input 34-KSL cells. CRU frequency within day 0 34-KSL cells was estimated to be 1 in 40 cells (95% Confidence Interval [CI]: 1/22-1/72), whereas the CRU estimate within the progeny of 34-KSL cells following HUBEC culture was 1 in 4 cells (CI: 1/2-1/9). The addition of anti-PTN to the HUBEC co-culture decreased the CRU estimate to 1 in 29 cells (CI: 1/16-1/52), suggesting that PTN signaling was responsible for the expansion of HSCs observed in HUBEC co-cultures. In order to confirm whether PTN is indeed a novel growth and self-renewal factor for HSCs, we next performed “gain of function” studies in which 34-KSL cells were placed in liquid suspension cultures with cytokines (thrombopoietin 50 ng/mL, SCF 120 ng/mL, flt-3 ligand 20 ng/mL) with and without the addition of increasing doses of recombinant murine PTN (10, 50 and 100 ng/mL) and total cell expansion and HSC content were compared. The addition of 100 ng/mL PTN to cytokine cultures caused a 20-fold increase in KSL cell content at day 7 compared to input (P<0.001), whereas a decline in KSL cells was observed with cytokine cultures alone (P<0.001), suggesting that PTN caused an expansion of stem/progenitor cells in vitro. Competitive repopulating assays were performed in which CD45.2+ recipient mice were lethally irradiated and transplanted with limiting doses (10, 30 and 100 cells) of CD45.1+ donor BM 34-KSL cells or their progeny following culture with cytokines alone or cytokines + 100 ng/mL PTN. CRU analysis at 4 weeks post-transplantation revealed that the CRU frequency within input 34-KSL cells was was 1 in 32 cells (CI: 1/18-1/57) and the CRU estimate within the progeny of 34-KSL cells cultured with cytokines alone was 1 in 69 (CI: 1/36-1/130). Conversely, the CRU estimate within the progeny of 34-KSL cells cultured with cytokines + PTN was 1 in 4 cells (CI: 1/2-1/10), indicating a 8-fold increase in short term repopulating cell content in response to PTN treatment. Longer term analysis will be performed in these mice to confirm whether PTN treatment induces the self-renewal and amplification of long-term repopulating HSCs in culture. Taken together, these data demonstrate that secreted PTN is primarily responsible for amplification of HSCs that we have observed in cultures of HSCs with ECs and the addition of PTN alone induces the expansion of phenotypic and functional HSCs in culture. PTN is therefore a novel soluble growth factor for HSCs and appears to play an important role in the extrinsic regulation of HSC self-renewal.