Phuong L. Doan

Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells

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.

Endothelial progenitor cell infusion induces hematopoietic stem cell reconstitution in vivo

Hematopoietic stem cells (HSCs) reside in association with bone marrow (BM) sinusoidal vessels in vivo, but the function of BM endothelial cells (ECs) in regulating hematopoiesis is unclear. We hypothesized that hematopoietic regeneration following injury is regulated by BM ECs. BALB/c mice were treated with total body irradiation (TBI) and then infused with C57Bl6-derived endothelial progenitor cells (EPCs) to augment endogenous BM EC activity. TBI caused pronounced disruption of the BM vasculature, BM hypocellularity, ablation of HSCs, and pancytopenia in control mice, whereas irradiated, EPC-treated mice displayed accelerated recovery of BM sinusoidal vessels, BM cellularity, peripheral blood white blood cells (WBCs), neutrophils, and platelets, and a 4.4-fold increase in BM HSCs. Systemic administration of anti-VE-cadherin antibody significantly delayed hematologic recovery in both EPC-treated mice and irradiated, non-EPC-treated mice compared with irradiated controls. These data demonstrate that allogeneic EPC infusions can augment hematopoiesis and suggest a relationship between BM microvascular recovery and hematopoietic reconstitution in vivo.

The vascular niche: home for normal and malignant hematopoietic stem cells

Hematopoietic stem cells (HSCs) are uniquely capable of self-renewal and provision of all of the mature elements of the blood and immune system throughout the lifetime of an individual. HSC self-renewal is regulated by both intrinsic mechanisms and extrinsic signals mediated via specialized microenvironments or ‘niches’ wherein HSCs reside. HSCs have been shown to reside in close association with bone marrow (BM) osteoblasts in the endosteal niche and also in proximity to BM sinusoidal vessels. An unresolved question surrounds whether the endosteal and vascular niches provide synchronous or redundant regulation of HSC fate or whether these niches provide wholly unique regulatory functions. Furthermore, while some aspects of the mechanisms through which osteoblasts regulate HSC fate have been defined, the mechanisms through which the vascular niche regulates HSC fate remain obscure. Here, we summarize the anatomic and functional basis supporting the concept of an HSC vascular niche as well as the precise function of endothelial cells, perivascular cells and stromal cells within the niche in regulating HSC fate. Lastly, we will highlight the role of the vascular niche in regulating leukemic stem cell fate in vivo.

Epidermal growth factor regulates hematopoietic regeneration after radiation injury.

The mechanisms that regulate hematopoietic stem cell (HSC) regeneration after myelosuppressive injury are not well understood. We identified epidermal growth factor (EGF) to be highly enriched in the bone marrow serum of mice bearing deletion of Bak and Bax in TIE2-expressing cells in Tie2Cre; Bak1−/−; Baxflox/– mice. These mice showed radioprotection of the HSC pool and 100% survival after a lethal dose of total-body irradiation (TBI). Bone marrow HSCs from wild-type mice expressed functional EGF receptor (EGFR), and systemic administration of EGF promoted the recovery of the HSC pool in vivo and improved the survival of mice after TBI. Conversely, administration of erlotinib, an EGFR antagonist, decreased both HSC regeneration and the survival of mice after TBI. Mice with EGFR deficiency in VAV-expressing hematopoietic cells also had delayed recovery of bone marrow stem and progenitor cells after TBI. Mechanistically, EGF reduced radiation-induced apoptosis of HSCs and mediated this effect through repression of the proapoptotic protein PUMA. Our findings show that EGFR signaling regulates HSC regeneration after myelosuppressive injury.

Inhibition of Aldehyde Dehydrogenase Expands Hematopoietic Stem Cells with Radioprotective Capacity

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However, the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34−c‐kit+Sca‐1+lineage− (34−KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34−KSL cells. Treatment of bone marrow (BM) 34−KSL cells with DEAB caused a fourfold increase in 4‐week competitive repopulating units, verifying the amplification of short‐term HSCs (ST‐HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment, confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB‐mediated expansion of ST‐HSCs in culture, suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST‐HSCs for transplantation purposes. STEM CELLS 2010;28:523–534

Tie2(+) bone marrow endothelial cells regulate hematopoietic stem cell regeneration following radiation injury.

Hematopoietic stem cells (HSCs) reside in proximity to bone marrow endothelial cells (BM ECs) and maintenance of the HSC pool is dependent upon EC‐mediated c‐kit signaling. Here, we used genetic models to determine whether radioprotection of BM ECs could facilitate hematopoietic regeneration following radiation‐induced myelosuppression. We developed mice bearing deletion of the proapoptotic proteins, BAK and BAX, in Tie2+ ECs and HSCs (Tie2Bak/BaxFl/− mice) and compared their hematopoietic recovery following total body irradiation (TBI) with mice which retained Bax in Tie2+ cells. Mice bearing deletion of Bak and Bax in Tie2+ cells demonstrated protection of BM HSCs, preserved BM vasculature, and 100% survival following lethal dose TBI. In contrast, mice that retained Bax expression in Tie2+ cells demonstrated depletion of BM HSCs, disrupted BM vasculature, and 10% survival post‐TBI. In a complementary study, VEcadherinBak/BaxFl/− mice, which lack Bak and Bax in VEcadherin+ ECs, also demonstrated increased recovery of BM stem/progenitor cells following TBI compared to mice which retained Bax in VEcadherin+ ECs. Importantly, chimeric mice that lacked Bak and Bax in HSCs but retained Bak and Bax in BM ECs displayed significantly decreased HSC content and survival following TBI compared to mice lacking Bak and Bax in both HSCs and BM ECs. These data suggest that the hematopoietic response to ionizing radiation is dependent upon HSC‐autonomous responses but is regulated by BM EC‐mediated mechanisms. Therefore, BM ECs may be therapeutically targeted as a means to augment hematopoietic reconstitution following myelosuppression. STEM CELLS2013;31:327–337

Diagnosis of Partial Body Radiation Exposure in Mice Using Peripheral Blood Gene Expression Profiles

In the event of a terrorist-mediated attack in the United States using radiological or improvised nuclear weapons, it is expected that hundreds of thousands of people could be exposed to life-threatening levels of ionizing radiation. We have recently shown that genome-wide expression analysis of the peripheral blood (PB) can generate gene expression profiles that can predict radiation exposure and distinguish the dose level of exposure following total body irradiation (TBI). However, in the event a radiation-mass casualty scenario, many victims will have heterogeneous exposure due to partial shielding and it is unknown whether PB gene expression profiles would be useful in predicting the status of partially irradiated individuals. Here, we identified gene expression profiles in the PB that were characteristic of anterior hemibody-, posterior hemibody- and single limb-irradiation at 0.5 Gy, 2 Gy and 10 Gy in C57Bl6 mice. These PB signatures predicted the radiation status of partially irradiated mice with a high level of accuracy (range 79–100%) compared to non-irradiated mice. Interestingly, PB signatures of partial body irradiation were poorly predictive of radiation status by site of injury (range 16–43%), suggesting that the PB molecular response to partial body irradiation was anatomic site specific. Importantly, PB gene signatures generated from TBI-treated mice failed completely to predict the radiation status of partially irradiated animals or non-irradiated controls. These data demonstrate that partial body irradiation, even to a single limb, generates a characteristic PB signature of radiation injury and thus may necessitate the use of multiple signatures, both partial body and total body, to accurately assess the status of an individual exposed to radiation.

Pleiotrophin mediates hematopoietic regeneration via activation of RAS

Hematopoietic stem cells (HSCs) are highly susceptible to ionizing radiation-mediated death via induction of ROS, DNA double-strand breaks, and apoptotic pathways. The development of therapeutics capable of mitigating ionizing radiation-induced hematopoietic toxicity could benefit both victims of acute radiation sickness and patients undergoing hematopoietic cell transplantation. Unfortunately, therapies capable of accelerating hematopoietic reconstitution following lethal radiation exposure have remained elusive. Here, we found that systemic administration of pleiotrophin (PTN), a protein that is secreted by BM-derived endothelial cells, substantially increased the survival of mice following radiation exposure and after myeloablative BM transplantation. In both models, PTN increased survival by accelerating the recovery of BM hematopoietic stem and progenitor cells in vivo. PTN treatment promoted HSC regeneration via activation of the RAS pathway in mice that expressed protein tyrosine phosphatase receptor-zeta (PTPRZ), whereas PTN treatment did not induce RAS signaling in PTPRZ-deficient mice, suggesting that PTN-mediated activation of RAS was dependent upon signaling through PTPRZ. PTN strongly inhibited HSC cycling following irradiation, whereas RAS inhibition abrogated PTN-mediated induction of HSC quiescence, blocked PTN-mediated recovery of hematopoietic stem and progenitor cells, and abolished PTN-mediated survival of irradiated mice. These studies demonstrate the therapeutic potential of PTN to improve survival after myeloablation and suggest that PTN-mediated hematopoietic regeneration occurs in a RAS-dependent manner.

Pharmacological Manipulation of the RAR/RXR Signaling Pathway Maintains the Repopulating Capacity of Hematopoietic Stem Cells in Culture

The retinoid X receptor (RXR) contributes to the regulation of diverse biological pathways via its role as a heterodimeric partner of several nuclear receptors. However, RXR has no established role in the regulation of hematopoietic stem cell (HSC) fate. In this study, we sought to determine whether direct modulation of RXR signaling could impact human HSC self-renewal or differentiation. Treatment of human CD34(+)CD38(-)lin(-) cells with LG1506, a selective RXR modulator, inhibited the differentiation of HSCs in culture and maintained long-term repopulating HSCs in culture that were otherwise lost in response to cytokine treatment. Further studies revealed that LG1506 had a distinct mechanism of action in that it facilitated the recruitment of corepressors to the retinoic acid receptor (RAR)/RXR complex at target gene promoters, suggesting that this molecule was functioning as an inverse agonist in the context of this heterodimer. Interestingly, using combinatorial peptide phage display, we identified unique surfaces presented on RXR when occupied by LG1506 and demonstrated that other modulators that exhibited these properties functioned similarly at both a mechanistic and biological level. These data indicate that the RAR/RXR heterodimer is a critical regulator of human HSC differentiation, and pharmacological modulation of RXR signaling prevents the loss of human HSCs that otherwise occurs in short-term culture.

Dickkopf-1 promotes hematopoietic regeneration via direct and niche-mediated mechanisms

The role of osteolineage cells in regulating hematopoietic stem cell (HSC) regeneration following myelosuppression is not well understood. Here we show that deletion of the pro-apoptotic genes Bak and Bax in osterix (Osx, also known as Sp7 transcription factor 7)-expressing cells in mice promotes HSC regeneration and hematopoietic radioprotection following total body irradiation. These mice showed increased bone marrow (BM) levels of the protein dickkopf-1 (Dkk1), which was produced in Osx-expressing BM cells. Treatment of irradiated HSCs with Dkk1 in vitro increased the recovery of both long-term repopulating HSCs and progenitor cells, and systemic administration of Dkk1 to irradiated mice increased hematopoietic recovery and improved survival. Conversely, inducible deletion of one allele of Dkk1 in Osx-expressing cells in adult mice inhibited the recovery of BM stem and progenitor cells and of complete blood counts following irradiation. Dkk1 promoted hematopoietic regeneration via both direct effects on HSCs, in which treatment with Dkk1 decreased the levels of mitochondrial reactive oxygen species and suppressed senescence, and indirect effects on BM endothelial cells, in which treatment with Dkk1 induced epidermal growth factor (EGF) secretion. Accordingly, blockade of the EGF receptor partially abrogated Dkk1-mediated hematopoietic recovery. These data identify Dkk1 as a regulator of hematopoietic regeneration and demonstrate paracrine cross-talk between BM osteolineage cells and endothelial cells in regulating hematopoietic reconstitution following injury.