Engraftment Of Hematopoietic Stem/progenitor Cells Research Articles

Human serum albumin promotes self-renewal and expansion of umbilical cord blood CD34+ hematopoietic stem/progenitor cells.

We investigated the effect of human serum albumin (HSA) on human umbilical cord blood (UCB) CD34+ hematopoietic stem/progenitor cells (HSPCs) cultured in vitro and transplanted in vivo. Human umbilical cord blood mononuclear cells were obtained by density gradient centrifugation. CD34+ cells were then sorted by CD34 conjugated magnetic microbeads. The sorted cells were cultured with or without HSA for 8 days in vitro. After 8 days, all cells were harvested for flow phenotyping and colony formation cell (CFC) experiments. The cells were injected into immunodeficient mice (NOD/Shi-scid/IL2Rγnull, NOG) via intravenous injections. From 4 weeks post-transplantation, flow cytometry was used to calculate human cell chimerism in the peripheral blood (PB) every 2 weeks. Flow phenotyping of human cell chimerism in bone marrow and spleen was calculated 16 weeks post-transplantation. Compared to the control group, CD34+ cells cultured with HSA increased significantly in vitro. The long-term engraftment of HSPCs and the hematopoietic multilineage reconstruction capacity were preserved by HSA. Normal engraftment of human cells could be maintained via HSA treatment could maintain normal engraftment of human cells in recipient PB. Here, we found that HSA was beneficial to maintaining CD34+ cell expansion and short-term colony formation in vitro and optimizing multilineage reconstitution in immunodeficient mice in vivo.

Novel Evidence That NADPH Oxidase 2 (Nox-2) Enhances Trafficking of Hematopoietic Stem Progenitor Cells (HSPCs) By Involving ROS Mediated Activation of Nlrp3 Inflammasome

Background. NADPH oxidase 2 (Nox-2) is a superoxide-generating enzyme that forms reactive oxygen species (ROS) to regulate the redox state involved in the self-renewal of hematopoietic stem/progenitor cells (HSPCs). Moreover, ROS are released from Nox-2 expressed by cell surface and mitochondria membranes and are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates several aspects of HSPCs biology, including their trafficking, proliferation, and metabolism. Our previous research demonstrated a novel role of Nlrp3 inflammasome in the migration, mobilization, homing, and engraftment of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). The role of ROS was extensively studied in the proliferation of hematopoietic cells; however, its role in the migration of HSPCs is not very well understood. Aim of the study.Based on the role of Nox-2 in activating Nlrp3 inflammasome, we hypothesized that activation of Nox-2-ROS-Nlrp3 inflammasome axis in both HSPCs and BM microenvironment orchestrates trafficking of HSPCs.Materials and Methods. First, we studied in Transwells chemotaxis of Nox-2 deficient cells to bone marrow (BM) chemoattractants, including SDF-1, S1P, and eATP. Next, to shed more light on the role of Nox2 in the trafficking of HSPCs, we performed in Nox-2-KO animals G-CSF- and AMD3100-induced pharmacological mobilization. We also studied homing and engraftment of Nox-2-KO cells in BM of wild-type (WT) animals and homing and engraftment o WT BM cells in Nox-2-KO recipients. Nox-2-KO mice were also sublethal irradiated, and we followed the recovery of peripheral blood counts in these animals. Activation of Nlrp3 inflammasome in Nox-2-KO murine Sca-1+c-kit+lineage- HSPCs was evaluated by Glow assay. To ''bypass'' Nox-2-ROS activation in control experiments, nigericin has been employed as a direct activator of Nlrp3 inflammasome. Since membrane lipid rafts (MLRs) play a crucial role optimal migration of HSPCs, we evaluated the formation of membrane lipid rafts by employing confocal analysis in Nox-2-KO cells stimulated with SDF-1. We also studied the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in providing lipid components for MLRs assemble in activated Nox-2-KO cells. Results. We noticed that Nox-2-KO animals have a profound defect in the trafficking of HSPCs as evidenced by impaired chemotaxis to BM chemoattractants, decrease in pharmacological mobilization, and defect in homing and engraftment after transplantation. Nox-2-KO mice have also defect in recovery from sublethal irradiation. At the molecular level, HSPCs from Nox-2-KO mice displayed a defect in activation of Nlrp3 inflammasome and in cholesterol and sphingolipids synthesizing enzymes, which provide lipid components required for optimal assemble of MLRs. These defects were partially ameliorated by using direct activator of Nlrp3 inflammasome that is nigericin. Conclusions. We provide for the first time evidence that Nox-2-ROS-Nlrp3 inflammasome axis operates in normal HSPCs and is crucial for regulating trafficking and post-irradiation recovery of these cells. These effects are mediated by providing components for MLRs required for optimal migration and signaling in HSPCs

Novel Evidence That Intracellular Complement Network (complosome) Is Expressed in Hematopoietic Stem Cells (HSPCs) and Regulates Their Trafficking, Metabolism, and Proliferation in an Intracrine-Dependent Manner By Engaging Pattern Recognition Receptor Nlrp3 Inflammasome

Background. Complement cascade (ComC) proteins, including C3 and C5 components, are synthesized in the liver; however, recent reports indicate their expression in lymphocytes as well (Immunity 2020;52(3):513-527). Accordingly, intracellular ComC proteins have been reported to be cleaved to active C3a and C5a mediators that interact with corresponding receptors in an intracrine-depended manner. This novel regulatory loop operating in lymphocytes has been named ''complosome''. Aim of the study.Because hematopoiesis and lymphopoiesis have a common stem cell origin and similar mechanisms are involved in regulating the biology of both lineages, we asked if complosome is also expressed and functional in normal murine hematopoietic stem progenitor cells (HSPCs). We also address a challenging question if intracellular ComC mediators could activate pattern recognition receptor Nlrp3 inflammasome that, as we reported, regulates trafficking, proliferation and metabolism of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). Materials and Methods. First, we phenotyped purified murine and human HSPCs for expression of major complement elements, including C3, C5, C3aR, C5aR, and cathepsin L that cleaves intracellular C3 to C3a. Next, we focused on a crucial distal executive part of ComC, protein C5, and evaluated in C5-KO and C5aR-KO mice the number of HSPCs and their clonogeneic potential. We also studied hematopoietic recovery of these animals after sublethal irradiation (650 cGy). Activation of Nlrp3 inflammasome in C5a-KO and C5aR-KO in response to hematopoietic growth factors and cytokines was evaluated by immunofluorescence Glow assay. We also studied homing and engraftment of C5-KO and C5aR-KO mice-derived HSPCs transplanted into normal WT recipients. Finally, we performed a metabolomic analysis of C5-KO, C5aR-KO, and control WT mice in steady-state conditions and after stimulation with selected hematopoietic growth factors and cytokines. Results. We show for the first time that purified murine and human HSPCs express complosome elements, including C3, C5, C3aR, C5aR, and cathepsin L. Next, C5-KO and C5aR-KO mice also have a lower number of HSPCs and clonogenic progenitors in bone marrow (BM), and display defect of hematopoietic recovery from sublethal irradiation. Furthermore, BM cells isolated from these animals had impaired homing and engraftment in normal WT recipients, supported by the observation that HSPCs from complosome deficient mice had a defect in migration to stromal-derived factor-1 (SDF-1), which correlated with decreased activation of Nlrp3 inflammasome. This defect could be alleviated by direct activation of Nlrp3 inflammasome by nigericin. Finally, our metabolic studies revealed a defect in glucose, amino acids, and lipid metabolism in C5-KO and C5aR-KO mice which correlates with complosome deficiency and Nlrp3 inflammasome activation. Conclusions. We provide for the first time evidence that complosome is expressed and functional in normal HSPCs and by engaging Nlrp3 inflammasome regulates metabolism, proliferation, and migration of HSPCs. Thus, our data sheds new intriguing light on innate immunity as a regulator of hematopoiesis that involves intracellular activation of Nlrp3 inflammasome. We currently focus on Nlrp3 inflammasome-released alarmins released from the cells in response to complosome-Nlrp3 inflammasome activation that accounts for observed phenomena. These studies are essential to develop more efficient ex vivo expansion strategies of HSPCs and to understand better intrinsic mechanisms that regulate metabolism of these cells.

Novel evidence that the P2X1 purinergic receptor-Nlrp3 inflammasome axis orchestrates optimal trafficking of hematopoietic stem progenitors cells.

Our previous research demonstrated P2X purinergic receptors as important extracellular adenosine triphosphate (eATP) sensing receptors promoting the trafficking of hematopoietic stem progenitor cells (HSPCs). Accordingly, mice deficient in expression of P2X4 and P2X7 receptors turned out to mobilize poorly HSPCs. Similarly, defective expression of these receptors on transplanted HSPCs or in the bone marrow (BM) microenvironment of graft recipient mice led to defective homing, engraftment, and delayed hematopoietic reconstitution. This correlated with decreased activation of intracellular pattern recognition receptor Nlrp3 inflammasome. The P2X receptor family consists of seven purinergic receptors (P2X1-7) and we noticed that in addition to P2X4 and P2X7, HSPCs also highly express rapidly signaling the P2X1 receptor. Therefore, we asked if P2X1 receptor is also involved in HSPCs trafficking. We employed in vitro and in vivo murine models to study the role of P2X1 receptor blocked on HSPCs or bone marrow microenvironment cells by specific small molecular inhibitor NF499. First, we performed in vitro cell migration assays of bone marrow mononuclear cells (BMMNCs) isolated from normal mice that were exposed to NF499 and compared them to unexposed control cells. Next, in experiments in vivo we mobilized mice exposed to NF499 with G-CSF or AMD3100 and compared mobilization to control unexposed animals. Flow cytometry was employed to identify cell populations and clonogenic assays to enumerate the number of mobilized clonogenic progenitors. Similarly, in homing and engraftment experiments BMMNCs or recipient mice were exposed to NF499 and we evaluated homing and engraftment of transplanted cells by enumerating the number of cells labeled with fluorochromes in recipient mice BM and by evaluating the number of clonogenic progenitors in BM and spleen 24 hours and 12 days after transplantation. We also evaluated the potential involvement of Nlrp3 inflammasome in P2X1 receptor-mediated HSPCs trafficking. We report that the functional P2X1 receptor is highly expressed on murine and human HSPCs. We could demonstrate that the P2X1 receptor promotes the trafficking of murine cells in Nlrp3 inflammasome-dependent manner. Mice after exposure to P2X1 receptor inhibitor poorly mobilized HSPCs from the bone marrow into the peripheral blood. Mice transplanted with BMNNCs exposed to NF499 or recipient mice pretreated with this inhibitor demonstrated defective homing and engraftment as compared to control animals transplanted with cells not exposed to P2X1 inhibitor. Similar effects were noticed for control recipient mice that were not exposed to NF499. This study demonstrates for the first time the novel role of the P2X1 receptor in HSPCs trafficking in the mouse. Furthermore, it supports an important role of purinergic signaling engaging its downstream target Nlrp3 inflammasome in the mobilization, homing and engraftment of HSPCs.

Myeloablative Conditioning for Transplantation Induces State of Sterile Inflammation in the Bone Marrow: Implications for Optimizing Homing and Engraftment of Hematopoietic Stem Cells.

Significance: The success rate of hematopoietic stem cell transplantation depends mainly on the number of transplanted hematopoietic stem/progenitor cells (HSPCs) followed by the speed of their engraftment in the myeloablated transplant recipient. Therefore, clinical outcomes will significantly benefit from accelerating the homing and engraftment of these cells. This is, in particular, important when the number of cells available for the transplantation of HSPCs is limited. Recent Advances: We postulated that myeloablative conditioning for hematopoietic transplantation by radio- or chemotherapy induces a state of sterile inflammation in transplant recipient peripheral blood (PB) and bone marrow (BM). This state is mediated by activation of the BM stromal and innate immunity cells that survive myeloablative conditioning and respond to danger-associated molecular patterns released from the cells damaged by myeloablative conditioning. As a result of this, several factors are released that promote proper navigation of HSPCs infused into PB of transplant recipient and prime recipient BM to receive transplanted cells. Critical Issues: We will present data that cellular innate immunity arm and soluble arm comprised complement cascade proteins, promoting the induction of the BM sterile inflammation state that facilitates the navigation, homing, and engraftment of HSPCs. Future Directions: Deciphering these mechanisms would allow us to better understand the mechanisms that govern hematopoietic recovery after transplantation and, in parallel, provide important information on how to optimize this process in the clinic by employing small molecular modifiers of innate immunity and purinergic signaling. Antioxid. Redox Signal. 37, 1254-1265.

Modulation of Purinergic Signaling Prior to Transplantation Both in Hematopoietic Stem Progenitor Cells (HSPCs) or Recipient Bone Marrow (BM) Microenvironment - As Novel Strategies to Accelerate and Facilitate HSPCs Homing and Engraftment

Background. The success rate of hematopoietic stem cell transplantation strongly depends on the number of transplanted hematopoietic stem/progenitor cells (HSPCs) and their speed of engraftment after infusion to the myeloablated transplant recipient. Therefore, clinical outcomes will benefit from accelerating the speed of homing and engraftment rate of transplanted HSPCs. This is important when the number of available HSPCs is low, as seen after poor harvest from BM, poor mobilization efficiency of the donor, and a low number of HSPCs present in the available umbilical cord blood (UCB) unit for an adult recipient. Our recent research demonstrated that purinergic signaling involving extracellular adenosine triphosphate (eATP) and its extracellular metabolite adenosine (eAdo) play a significant opposite role in homing/engraftment of HSPCs - reviewed in Curr Opin Hematol 2021, 28:251-261. To explain this eATP released from the cells of conditioned for transplantation by myeloablation recipient's BM facilitates homing of HSPCs, and subsequently becomes metabolized by cell surface ectonucleotidases CD39 and CD73 to eAdo, that inhibits this process. Therefore, eATP and eAdo upregulated in PB and BM modulate homing/engraftment in i) infused to the recipient donor-derived HSPCs and ii) in recipient BM microenvironment - in an opposite way. We also reported that the beneficial effect of eATP on homing/engraftment of HSPCs depends on the promotion of membrane lipid raft formation on the surface of HSPCs that incorporate homing receptors for their optimal interaction with BM released homing chemoattractants. This process is promoted by eATP activated Nlrp3 inflammasome. On the other hand, Nlrp3 inflammasome and membrane lipid raft formation are inhibited by eAdo in heme oxygenase-1 (HO-1)-dependent manner (Leukemia 2020; 34:1512-1523). Hypothesis. We hypothesized that proper modulation of eATP - eAdo signaling both at the level of transplanted HSPCs and recipient BM microenvironment will speed up the seeding efficiency of transplanted cells to BM niches. Material and Methods. We exposed HSPCs before transplantation ex vivo to i) exogenous eATP or ii) small molecular CD39 and CD73 inhibitors. We also inhibited CD39 and CD73 in transplant recipients BM at the time of myeloablative conditioning. In addition, we also activated ex vivo Nlrp3 inflammasome in HSPCs to be transplanted by specific activator nigericin. In control experiments, eATP stimulated Nlrp3 inflammasome activity was inhibited by the HO-1 activator that is CoPP. Homing of HSPCs was evaluated by measuring a number of donor-derived fluorochrome-labeled cells and clonogenic progenitors in BM of myeloablated hosts at 24 hours after transplantation. Early engraftment was assessed by counting the number of CFU-S and clonogeneic progenitors 12 days after transplantation and by evaluating kinetics of recovery of PB hematopoietic cell counts. Finally, while activation of Nlrp3 inflammasome was assessed by immunofluorescence assay, membrane lipid raft formation was evaluated by confocal microscopy. Results. We noticed that homing and engraftment of HSPCs was significantly accelerated after i) short exposure before transplantation to eATP, ii) inhibition of eAdo formation by CD39, and CD73 inhibitors, and iii) activation of Nlrp3 inflammasome by nigericin. Similarly, inhibition of eAdo formation in recipient BM microenvironment of transplanted mice by CD39 and CD73 inhibitors also improved homing and engraftment efficiency. This correlated with activation in the eATP-dependent manner of Nlrp3 inflammasome in HSPCs followed by membrane lipid raft formation. In the BM microenvironment, upregulation of eATP and inhibition of eAdo also enhanced expression of homing chemoattractants. Conclusions. Since all purinergic signaling modifiers employed in our studies are non-toxic against HSPCs, our data obtained in the animal model indicates that modulation of purinergic signaling before transplantation in HSPCs as well as in BM of the myeloablated recipient would significantly accelerate hematopoietic recovery after hematopoietic transplantation. DisclosuresNo relevant conflicts of interest to declare.

Novel Evidence That Alternative Activation Pathway of Complement Cascade (ComC) Regulates Optimal Homing and Engraftment of Hematopoietic Stem/Progenitor Cells (HSPCs) in Reactive Oxygen Species (ROS) - Nlrp3 Inflammasome-Dependent Manner

Background. We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required not only for mobilization of hematopoietic stem progenitor cells (HSPCs) but also for their proper homing and engraftment after transplantation to bone marrow (BM) (Leukemia 2012; 26:106-16). The ComC consists of zymogen proteins that become activated in a cascade-mediated manner by the i) classical, ii) mannan-binding lectin (MBL), or iii) alternative pathway. However, it is not clear which of these pathways plays a crucial role in inducing state of sterile inflammation in recipient BM conditioned for transplantation. Interestingly, the alternative pathway of ComC activation, in contrast to the other two pathways, is not triggered by antibodies or specific structures expressed on the surface of invading microorganisms, but is continuously activated and “ticking” by the spontaneous hydrolysis of the third component of the ComC (C3), which is the most abundant complement protein present in blood plasma. This process of C3 hydrolysis is hyperactivated in response to tissue/organ damage and changes the structure of C3 in order to promote binding of factor B (FB) that initiates the amplification process by which more C3b molecules and C3b-Bb convertases are created responsible for activation of the ComC. We also recently demonstrated that homing and engraftment of HSPCs to BM is mediated by activation of innate immunity pattern recognition receptor Nlrp3 inflammasome (Leukemia 2020; 34:1512-1523). On the other hand reactive oxygen species (ROS) are known activators of Nlrp3 inflammasome. Hypothesis. We hypothesized that sensitive and “continuously ticking” in peripheral blood (PB) alternative pathway of ComC activation facilitates homing and engraftment of HSPCs in response to myeloablation triggered sterile inflammation of hematopoietic microenvironment in donor BM. We also hypothesized that this depends on the activation of the Nlrp3 inflammasome in ROS-dependent manner. Materials and Methods. Activation of ComC and Nlrp3 inflammasome in BM of conditioned for hematopoietic transplantation by lethal irradiation mice was evaluated by C5a ELISA assay and immunofluorescence glow assay measuring activation of Nlrp3 inflammasome product that is activated caspase-1, respectively. We also run shotgun proteomic analysis with BM conditioned media and BMMNCs extracts. The release of ROS was measured by a colorimetric assay. To assess the involvement of alternative pathway of ComC activation, we performed homing and engraftment experiments in wild-type (WT) and FB-deficient mice, that have defect in alternative pathway of ComC activation. FB-KO and WT animals were transplanted with WT BMMNC. We also analyzed changes in the BM microenvironment in response to lethal irradiation in WT and FB-KO mice at mRNA and protein level. Results. We demonstrate for a first time that conditioning for transplantation by myeloablative irradiation induces in BM state of sterile inflammation reflected by activation of ComC, the release of reactive oxygen species (ROS), and activation of Nlrp3 inflammasome in ROS-dependent manner. Moreover, as compared to WT animals, all these pathways were significantly inhibited in FB-KO mice. As a result of this FB-KO animals displayed defective homing and engraftment after transplantation of WT BMMNC. This correlated at molecular level by the decreased expression of cell adhesion molecules and group of structural proteins involved in so called “docking structures” necessary for cell migration and homing as well as for Nlrp3 inflammasome complex and caspase 1, 2, 4 and 6. Moreover, FB-KO mice demonstrated lower level of SDF-1 and KL in BM after myeloablative conditioning for transplantation. Conclusions. We provide for a first time an evidence that myeloablative conditioning for transplantation by lethal irradiation activates in ComC-ROS-Nlrp3 inflammasome - dependent manner a state of sterile inflammation in the BM microenvironment, required for optimal homing and engraftment. This data also explains this phenomenon at molecular level and provides an evidence for a crucial involvement of alternative pathway of ComC activation. DisclosuresNo relevant conflicts of interest to declare.

The P2X4 purinergic receptor has emerged as a potent regulator of hematopoietic stem/progenitor cell mobilization and homing-a novel view of P2X4 and P2X7 receptor interaction in orchestrating stem cell trafficking.

Recent evidence indicates that extracellular adenosine triphosphate (eATP), as a major mediator of purinergic signaling, plays an important role in regulating the mobilization and homing of hematopoietic stem progenitor cells (HSPCs). In our previous work we demonstrated that eATP activates the P2X7 ion channel receptor in HSPCs and that its deficiency impairs stem cell trafficking. To learn more about the role of the P2X purinergic receptor family in hematopoiesis, we phenotyped murine and human HSPCs with respect to the seven P2X receptors and observed that, these cells also highly express P2X4 receptors, which shows ~50% sequence similarity to P2X7 subtypes, but that P2X4 cells are more sensitive to eATP and signal much more rapidly. Using the selective P2X4 receptor antagonist PSB12054 as well as P2X4-KO mice, we found that the P2X4 receptor, similar to P2X7 receptor, promotes trafficking of HSPCs in that its deficiency leads to impaired chemotaxis of HSPCs in response to a stromal-derived factor 1 (SDF-1) gradient, less effective pharmacological mobilization, and defective homing and engraftment of HSPCs after transplantation into myeloablated hosts. This correlated with a decrease in SDF-1 expression in the BM microenvironment. Overall, our results confirm the proposed cooperative dependence of both receptors in response to eATP signaling. In G-CSF-induced mobilization, a lack of one receptor is not compensated by the presence of the other one, which supports their mutual dependence in regulating HSPC trafficking.

The neurotransmitter receptor Gabbr1 regulates proliferation and function of hematopoietic stem and progenitor cells

AbstractHematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.

A Novel Underappreciated Role for the Extracellular Adenosine Triphosphate (ATP)-P2X4 Purinergic Receptor Axis in the Homing and Engraftment of HSPCs

Background. Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but when released from activated cells into the extracellular space as extracellular ATP (eATP) it becomes a crucial mediator of the purinergic signaling network. Purinergic receptors for extracellular nucleotides (EXNs), expressed on the surface of all cells in the body, are represented by the P1, P2X, and P2Y receptor families, which are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for eATP signaling and consists of seven members (P2X1-7). We found that human and murine hematopoietic stem progenitor cells (HSPCs) highly express two members of this family, the P2X4 and P2X7 receptors. We recently reported that both are involved in optimal mobilization of HSPCs by activating Nlrp3 inflammasome (Leukemia 2020 Jun;34(6):1512-1523 and Stem Cell Rev Rep. 2019 Jun;15(3):391-403). We also reported that the P2X7 receptor expressed on the surface of HSPCs facilitates the homing and engraftment of HSPCs by increasing their responsiveness to SDF-1 gradients. Interestingly, it has been proposed that both receptors heterodimerize to exert optimal activity. Hypothesis. Since, the P2X4 and P2X7 receptors show several similar biological effects in non-hematopoietic cells, we became interested in the role of the P2X4 receptor in homing and engraftment of HSPCs.Materials and Methods. To test this hypothesis, we isolated SKL cells from P2X4-KO mice and tested them for migration in response to BM chemoattractants, including the major homing factor SDF-1. Next, we tested the short- and long-term homing of mouse BM cells after exposure to the P2X4-specific inhibitor PBS12054 in normal mice by evaluating the number of donor-derived PKH67-labeled BMMNCs and CFU-GM clonogenic progenitors isolated from recipient mouse BM 24 hours after transplantation as well as the number of day-12 colony-forming units in spleen (CFU-S) and day-12 CFU-GM clonogenic progenitors. These data were confirmed in transplant studies employing P2X4-KO bone marrow cells. In parallel, we also evaluated the recovery kinetics of leukocytes and blood platelets in the PB of transplanted animals. Finally, we also perturbed P2X4 expression in transplanted mice with PBS12054 and studied the effect on homing and engraftment of normal BM cells, as described above. Results. We found that P2X4-KO mouse HSPCs have a defect in migration in response to BM chemoattractants involved in BM homing, including the major homing factor SDF-1 as well as the supportive factors S1P and eATP. Perturbation of P2X4 expression on the surface of HSPCs led to significant defective homing and engraftment of HSPCs. Moreover, inhibition of P2X4 in the recipient mouse BM microenvironment had a similar effect. Conclusions. We identified for the first time the role of eATP-P2X4 signaling in the homing and engraftment of HSPCs. To explain this result, we conclude that the eATP-P2X4 axis is, like the eATP-P2X7 axis, a potent activator of Nlrp3 inflammasomes and that defective eATP-P2X4 signaling impairs the role of purinergic signaling and the Nlrp3 inflammasome in homing and engraftment. Moreover, our results show a similar homing and engraftment phenotype for P2X4-KO mice as that seen in P2X7-KO animals, which provides functional support for the proposed dimerization of P2X7 with P2X4 receptors and the necessary presence of both receptors for optimal function. This question is currently being addressed in our laboratory by employing the fluorescence resonance energy transfer (FRET) technique. Finally, we provide additional evidence that, in addition to SDF-1 and S1P, eATP and purinergic signaling involving P2X4 and P2X7 receptors is an important and underappreciated regulator of HSPC trafficking and a potential target for molecular optimization of both processes. Disclosures No relevant conflicts of interest to declare.

A Novel View of the Role of Prostaglandin E2 (PGE2) in Facilitating Engraftment of HSPCs By Activating the NOX2-ROS-Nlrp3 Inflammasome Axis to Incorporate the CXCR4 Receptor into Membrane Lipid Rafts

Background . It is known that prostaglandin E2 (PGE2) increases the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs). However, aside from its role in upregulation of CXCR4 receptor expression on the surface of these cells, the exact mechanism has not been proposed. We have demonstrated in the past that an important step enabling the migration of HSPCs is the incorporation of CXCR4 into membrane lipid rafts on the leading surface (leading edge, in two dimensions) of migrating cells, which facilitates its interaction with cell migration signaling pathways (Wysoczynski M et al. Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient. Blood. 2005;105(1):40-48). Recently, we reported that Nlrp3 inflammasome-deficient HSPCs show a defect in lipid raft formation that results in defective migration of these cells in response to an SDF-1 gradient and their defective homing and engraftment after transplantation (Adamiak, M et al. Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts. Stem Cell Rev and Rep (2020). https://doi.org/10.1007/s12015-020-10005-w). An important activator of Nlrp3 inflammasomes is reactive oxygen species (ROS). Importantly, the enzyme that generates ROS, known as NADPH oxidase 2 (NOX2), is also associated with cell membrane lipid rafts. Hypothesis. Given the known roles of PGE2, membrane lipid rafts, and the Nlrp3 inflammasome in migration, homing, and engraftment of HSPCs, we hypothesized that PGE2 signaling promotes Nlrp3 inflammasome activation in a Nox2-ROS-dependent manner that results in incorporation of CXCR4 into membrane lipid rafts, which better explains the role of PGE2 in these phenomena.Materials and Methods. To test this hypothesis, murine SKL and human CD34+ cells enriched for HSPCs were stimulated with PGE2 to evaluate activation of genes of the Nlrp3 inflammasome complex at the mRNA and protein levels. Next, HSPCs from Nox2-KO mice were tested for membrane lipid raft formation in functional chemotaxis assays in response to SDF-1 gradients under conditions promoting membrane lipid raft formation. Formation of membrane lipid rafts in Nox2-KO cells was also evaluated by confocal analysis in the presence or absence of PGE2. Finally, the effect of the PGE2-Nox2-Nlrp3 inflammasome axis on the formation of membrane lipid rafts was evaluated in the presence of the ROS scavenger N-acethyl-cysteine (NAC). Results. We provide for the first time evidence that PGE2 activates Nlrp3 inflammasomes in HSPCs in a Nox2-ROS-dependent manner. This Nlrp3 inflammasome activation increases at the leading surface of migrating HSPCs with incorporation of the CXCR4 receptor into membrane lipid rafts. Formation of membrane lipid rafts was absent in Nox2-KO and Nlrp3-KO mouse HSPCs and in normal wild type cells after their exposure to NAC. Moreover, we also observed that Nox2-KO and Nlrp3-KO mice had a lower basal level of CXCR4 expression. Conclusions. Our results for the first time explain the role of PGE2 in promoting homing and migration of HSPCs, which occurs in response to PGE2 by activation of the Nox2-ROS-Nlrp3 inflammasome axis and thereby promotes incorporation of the CXCR4 receptor into membrane lipid rafts. Moreover, basal expression of the CXCR4 receptor was at a low level on the surface of HSPCs from Nlrp3-KO mice. Thus, our results provide evidence for the importance of the Nox2-ROS-Nlrp3 inflammasome axis in PGE2-mediated homing and engraftment of HSPCs and the role of PGE2-mediated lipid raft formation for optimal responsiveness of CXCR4 to SDF-1 in the BM microenvironment. Disclosures No relevant conflicts of interest to declare.

Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor\xa0into Membrane Lipid Rafts

Fast and efficient homing and engraftment of hematopoietic stem progenitor cells (HSPCs) is crucial for positive clinical outcomes from transplantation. We found that this process depends on activation of the Nlrp3 inflammasome, both in the HSPCs to be transplanted and in the cells in the recipient bone marrow (BM) microenvironment. For the first time we provide evidence that functional deficiency in the Nlrp3 inflammasome in transplanted cells or in the host microenvironment leads to defective homing and engraftment. At the molecular level, functional deficiency of the Nlrp3 inflammasome in HSPCs leads to their defective migration in response to the major BM homing chemoattractant stromal-derived factor 1 (SDF-1) and to other supportive chemoattractants, including sphingosine-1-phosphate (S1P) and extracellular adenosine triphosphate (eATP). We report that activation of the Nlrp3 inflammasome increases autocrine release of eATP, which promotes incorporation of the CXCR4 receptor into membrane lipid rafts at the leading surface of migrating cells. On the other hand, a lack of Nlrp3 inflammasome expression in BM conditioned for transplantation leads to a decrease in expression of SDF-1 and danger-associated molecular pattern molecules (DAMPs), which are responsible for activation of the complement cascade (ComC), which in turn facilitates the homing and engraftment of HSPCs.

Pannexin-1 channel \u201cfuels\u201d by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells

An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1+ and CD11b+ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

A Novel Evidence That Mannan Binding Lectin (MBL) Pathway of Complement Cascade Activation is Involved in Homing and Engraftment of Hematopoietic Stem Progenitor Cells (HSPCs)

Delayed homing and engraftment of hematopoietic stem progenitor cells (HSPCs) or even failure to engraft at all is significant clinical problem after hematopoietic transplant. Therefore, in order to develop more efficient homing and engraftment facilitating strategies it is important to learn more about this process. Our team has postulated that myeloablative conditioning for transplantation induces in bone marrow (BM) microenvironment a state of sterile inflammation in which elements of innate immunity activated by radio- or chemotherapy conditioning for transplant play an important role. In frame with this claim we reported that a significant role in this process plays activation of complement cascade (ComC). Accordingly, mice that that lack a fifth component (C5) of ComC turned out to engraft poorly with normal syngeneic BM cells as compared to normal control animals. In extension of our previous studies we provide for first time evidence that mannan binding lectin (MBL) pathway is involved in activation of ComC in myeloablated transplant recipient BM and thus plays an important role in homing and engraftment of HSPCs. To support this MBL-KO mice show significant defect in hematopoietic reconstitution after hematopoietic transplantation. This correlates with a decrease in expression of stromal derived factor-1 (SDF-1) and impaired activation of Nlrp3 inflammasome in irradiated BM of these mice.

Galactosyl carbohydrate residues on hematopoietic stem/progenitor cells are essential for homing and engraftment to the bone marrow

The role of carbohydrate chains in leukocyte migration to inflamed sites during inflammation and trafficking to the lymph nodes under physiological conditions has been extensively characterized. Here, we report that carbohydrate chains also mediate the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow (BM). In particular, we found that transplanted BM cells deficient in β-1,4-galactosyltransferase-1 (β4GalT-1) could not support survival in mice exposed to a lethal dose of irradiation. BM cells obtained from mice deficient in β4GalT-1 showed normal colony-forming activity and hematopoietic stem cell numbers. However, colony-forming cells were markedly rare in the BM of recipient mice 24 h after transplantation of β4GalT-1-deficient BM cells, suggesting that β4GalT-1 deficiency severely impairs homing. Similarly, BM cells with a point mutation in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, encoding a key enzyme in sialic acid biosynthesis, showed mildly impaired homing and engraftment abilities. These results imply that the galactosyl, but not sialyl residues in glycoproteins, are essential for the homing and engraftment of HSPCs to the BM. These findings suggest the possibility of modifying carbohydrate structures on the surface of HSPCs to improve their homing and engraftment to the BM in clinical application.

Novel Evidence That the Ectonucleotidases CD39 and CD73, Which Are Expressed on Hematopoietic Stem/Progenitor Cells (HSPCs), Regulate Mobilization and Homing - Studies in CD39-/- and CD73-/- Mice and with Small-Molecule CD39 and CD73 Inhibitors

Background. Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but it is also released from activated cells into the extracellular space as a crucial component of the purinergic signaling network. Purinergic signaling is an ancient form of extracellular signaling that is mediated by extracellular nucleotides (EXNs), including most importantly the purine ATP and its nucleoside metabolite, adenosine. Purinergic receptors for EXNs are expressed on the surface of all cells in the body; are represented by several families of P1, P2X, and P2Y receptors; and are among the most abundant receptors in living organisms. ATP, the most important EXN, is secreted from cells through pannexin and connexin channels and is processed in the extracellular space to ADP, AMP and finally adenosine by the cell-surface-expressed ectonucleotidases CD39 and CD73. We recently reported that the ATP-adenosine axis regulates the mobilization of hematopoietic stem progenitor cells (HSPCs) (Leukemia 2018, in press,doi: 10.1038/s41375-018-0122-0). Hypothesis. We hypothesized that the ectonucleotidases CD39 and CD73 are important modulators of the ATP-adenosine axis and are new and underappreciated modulators, not only of mobilization but also of bone marrow (BM) homing of HSPCs. Materials and Methods. To shed more light on the role of purinergic signaling in the trafficking of HSPCs, we i) phenotyped murine and human HSPCs for expression of CD39 and CD73 ectonucleotidases by FACS, ii) performed mobilization and homing studies in CD39-/- and CD73-/- mice, and iii) blocked CD39 and CD73 activity by employing the small-molecule inhibitors ARL 67156 and AMP-CP, respectively. CD39- and CD73-deficient mice were mobilized with G-CSF or AMD3100, and wild type (WT) animals were exposed to CD39 and CD73 inhibitors in addition to mobilizing agents. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. To address the involvement of CD39 and CD73 expressed on the surface of HSPCs or in the hematopoietic microenvironment, we created irradiation chimeras that were subsequently mobilized with G-CSF or AMD3100. Bone marrow (BM) transplantation studies were performed by transplanting WT mice with WT BM cells or BM cells from ectonucleotidase-deficient mice. Homing efficiency was evaluated by determining i) the number of fluorochrome-labeled cells in BM 24 hours after transplantation ii) the number of CFU-S and CFU-GM progenitors that had engrafted in BM on day 12, and iii) the recovery of peripheral blood counts. Results. We found that CD39 and CD73 are expressed by murine and human HSPCs and process ATP to adenosine, which as we observed, is a novel and potent inhibitor of HSPC trafficking. In support of this finding, CD73-/- and CD39-/- mice, with reduced extracellular adenosine levels, mobilize HSPCs much better than their normal control littermates. A similar effect was observed in WT mice exposed to CD39 and CD73 small-molecule inhibitors during mobilization. Studies in irradiation chimeras revealed that this effect depended on expression of CD39 and CD73 on the surface of HSPCs and not in the BM microenvironment. Moreover, adenosine also inhibited the homing of HSPCs to BM, as cells from CD39-/- or CD73-/- mice engrafted better than WT cells. Furthermore, homing was also inhibited in the presence of exogenously added adenosine. That ectonucleotidase-deficient mice engrafted BM cells from WT littermates as efficiently as did WT recipients indicates the involvement of HSPC-expressed CD39 and CD73 in regulating the homing process. Conclusions. We demonstrate for the first time that HSPC-expressed CD39 and CD39 ectonucleotidases modulate the trafficking of HSPCs. By processing ATP to adenosine, these enzymes negatively affect both the mobilization and the homing processes. Based on this finding, future strategies employing small-molecule inhibitors of both ectonucleotidases could find practical application in improving mobilization in poor mobilizers and accelerating the engraftment of HSPCs after transplantation. DisclosuresNo relevant conflicts of interest to declare.

Novel Evidence That Hematopoietic-Specific PLC-β2 Is Required for Normal Homing and Engraftment of Hematopoietic Stem Cells

Background. We recently identified phospholipase C-β2 (PLC-β2) as the first lipolytic enzyme known to be involved in the mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB) (Leukemia 2016, 4:919-28). The phospholipase C (PLC) family of enzymes consists of 13 members in six subfamilies, including the PLC-δ (1, 3, 4), -β (1-4), -γ (1, 2), -ε, -ζ, and -η isoforms. PLC enzymes are associated with cell surface receptors that convert phosphatidyloinositol-4,5-biphosphate into two important second messengers, diacylglycerol (DAG) and inositol-1,4.5-triphosphate (IP3). Among these isoforms, PLC-β2 is somewhat unique in being a hematopoietic-specific enzyme. Moreover, PLC-β2, when released extracellularly from granulocytes and HSPCs upon stimulation, cleaves glycolipid glycosylphosphatidylinositol anchor (GPI-A) in cell membranes and thus disrupts the structure of membrane lipid rafts, which are important in the retention of HSPCs in BM niches. It is well known that the major BM-retention receptors for HSPCs, CXCR4 and VLA-4, are membrane lipid raft-associated receptors. What is important for the current study, while performing mobilization studies, we previously found that BM cells from these animals show somewhat reduced chemotaxis in response to several chemoattractants involved in BM homing.Aim of the Study. Based on this latter observation, we became interested in the potential role of PLC-β2 in regulating the migration of HSPCs, as this enzyme could be potentially involved in BM homing and engraftment of HSPCs after transplantation. We also explored the potential relationship between PLC-β2 and heme oxygenase 1 (HO-1), which is, as we recently demonstrated, a negative regulator of HSPC trafficking (Cell Transplant. 2016 epub).Materials and Methods. We performed i) homing, ii) short-term engraftment, and iii) long-term competitive engraftment studies in WT mice transplanted with BM cells derived from PLC-β2-/- mice and control (WT) animals. Chemotaxis and adhesion of PLC-β2-/- HSPCs was evaluated in Transwell dishes in response to SDF-1, S1P, C1P, and ATP. The expression of HO-1 was evaluated by real-time PCR and western blotting. Finally, both HSPCs and Gr-1+ cells were evaluated for release of Ca2+ upon stimulation by homing factors.Results. We report here for first time that, besides its role in the release of HSPCs from BM niches, PLC-β2 also regulates the migration of HSPCs in response to chemotactic gradients of BM homing factors, including stromal derived factor-1 (SDF-1), sphingosine-1 phosphate (S1P), ceramide-1 phosphate C1P, and adenosine triphosphate (ATP). Specifically, HSPCs from PLC-β2-KO mice show impaired homing and engraftment in vivo after transplantation into lethally irradiated mice. This decrease in migration of HSPCs can be explained by impaired calcium release and calcium signaling in cells from PLC-β2-KO mice and a high baseline level of HO-1, an enzyme that negatively regulates cell migration, in PLC-β2-KO mouse cells. Downregulation of HO-1 activity in HSPCs improved homing of PLC-β2-/- BM cells.Conclusions. PLC-β2 plays an important role in homing and engraftment of HSPCs. The decrease in migration of HSPCs in PLC-β2-KO mice can be explained by impaired calcium release and a high baseline level of HO-1, an enzyme that negatively regulates cell migration and our data provides evidence that downregulation of HO-1 activity in HSPCs by small molecular inhibitors could become a new strategy to improve homing and engraftment after transplantation. DisclosuresNo relevant conflicts of interest to declare.

Downregulation of Heme Oxygenase 1 (HO-1) Activity in Hematopoietic Cells Enhances Their Engraftment After Transplantation.

Heme oxygenase 1 (HO-1) is an inducible stress-response enzyme that not only catalyzes the degradation of heme (e.g., released from erythrocytes) but also has an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury. HO-1 has a well-documented anti-inflammatory potential, and HO-1 has been reported to have a negative effect on adhesion and migration of neutrophils in acute inflammation in a model of peritonitis. This finding is supported by our recent observation that hematopoietic stem progenitor cells (HSPCs) from HO-1 KO mice are easy mobilizers, since they respond better to peripheral blood chemotactic gradients than wild-type littermates. Based on these findings, we hypothesized that transient inhibition of HO-1 by nontoxic small-molecule inhibitors would enhance migration of HSPCs in response to bone marrow chemoattractants and thereby facilitate their homing. To directly address this issue, we generated several human hematopoietic cell lines in which HO-1 was upregulated or downregulated. We also exposed murine and human BM-derived cells to small-molecule activators and inhibitors of HO-1. Our results indicate that HO-1 is an inhibitor of hematopoietic cell migration in response to crucial BM homing chemoattractants such as stromal-derived factor 1 (SDF-1) and sphingosine-1-phosphate (S1P). Most importantly, our in vitro and in vivo animal experiments demonstrate for the first time that transiently inhibiting HO-1 activity in HSPCs by small-molecule inhibitors improves HSPC engraftment. We propose that this simple and inexpensive strategy could be employed in the clinical setting to improve engraftment of HSPCs, particularly in those situations in which the number of HSPCs available for transplant is limited (e.g., when transplanting umbilical cord blood).

38. CCR5 Gene Edited Hematopoietic Stem Cells Engraft in Diverse Anatomical Locales and Undergo SHIV-Dependent Positive Selection in Nonhuman Primates

Background: Gene editing in hematopoietic stem/progenitor cells (HSPCs) has rapidly emerged as a promising therapy for a number of diseases, including human immunodeficiency virus (HIV) infection. We have previously demonstrated the feasibility of this approach in nonhuman primates. Here, we leverage our expertise with gene editing in large animal models to interrogate the clonal persistence, trafficking, and antiviral efficacy of CCR5-edited cells in the pigtailed macaque, M. nemestrina. Our objectives were to map the tissue distribution of HSPC-derived, gene edited progeny, understand how individual gene edited HSPCs persist following autologous transplantation, and develop strategies to select for these cells in vivo.Methods: Zinc Finger Nucleases (ZFNs) are used to target the CCR5 locus in macaque HSPCs. Engraftment and persistence of these autologous stem cells, and stem cell-derived lymphoid and myeloid cells, are measured ex vivo and in vivo. Animals are challenged with simian/human immunodeficiency virus (SHIV). Gene edited HSPCs are transplanted either prior to SHIV infection, or in SHIV-infected animals that are treated with combination antiretroviral therapy (cART) in order to approximate a well-suppressed HIV+ patient. Edited cells are measured longitudinally in peripheral blood, bone marrow, gastrointestinal (GI) tract, and lymph nodes, and at necropsy in a panel of 25 tissues, using methods including deep sequencing.Results: We observe up to 14-fold enrichment of CCR5-gene edited memory CD4+ T-cells in SHIV-infected animals, consistent with virus-dependent selection against CCR5 wt memory CD4+ T-cells. Gene edited cells are found in a broad array of anatomical sites, including GI tract and lymph nodes. Spatial and temporal tracking of CCR5 mutations suggests that gene edited cells persist in an analogous fashion to control lentivirus gene-marked cells. Homology directed repair (HDR) pathways can be exploited in macaque CD34+ HSPCs, facilitating knock-in of selectable markers at the disrupted CCR5 locus.Conclusions: Our results in SHIV-infected animals reinforce that this gene editing strategy results in stable engraftment of CCR5-mutated and SHIV-resistant HSPCs and their progeny. Additionally. gene edited CD4+ T-cells undergo positive selection during active infection, further supporting the validity of this approach in the clinic. Moreover, our preliminary ex vivo HDR data suggest that these gene edited cells could be engineered to undergo virus-independent selection.

Defining Engraftment Patterns and Interactions Between Hematopoietic Stem Cells and Stromal Cells after in Utero Transplantation

In utero hematopoietic stem cell transplantation (IUHSCT) has only been fully successful in recipients with severe combined immunodeficiency disorders. Because IUHSCT must be performed without myeloablation or immunosuppression, immunologic barriers and absence of stress-induced signaling have been considered significant contributors to the limited success of this procedure. Other potential barriers to successful engraftment of allogeneic adult donor hematopoietic stem/progenitor cells (HSC) in a fetal recipient result from the unique characteristics of fetal HSC biology and the fetal microenvironment. Transplanted adult cells could be out-competed by fetal HSC, and the fetal microenvironment might not support engraftment and/or expansion of HSC derived from ontogenically disparate sources, as differences in membrane composition and response to cytokines exist between fetal and adult cells. We have previously shown that increased levels of HSC engraftment can be achieved in a fetal setting, if bone marrow (BM)-derived mesenchymal stromal cells (MSC) or (BM)-derived endothelial progenitor cells (EPC) are administered prior to, or at the time of, HSC transplantation. Using a fetal sheep model of IUHSCT, we interrogated mechanisms by which transplanting sheep EPC or MSC prior to, or concurrently with, sheep HSC leads to statistically significant higher levels of HSC engraftment when compared to HSC alone. Using confocal microscopy and fluorescently-labeled cells, we first determined the sites of engraftment/localization of donor EPC, MSC, and HSC at 2-2.5 months after IUHSCT when transplanted at the does of 7.1 x 106 /Kg, 2.5 x 106 /kg, and 2.1 x 106 /kg, respectively, at 60-65 days of gestation (human equivalent of 16 gestational weeks). We demonstrated that EPC and MSC engraft at different sites after IUHSCT than the transplanted HSC. MSC and EPC engrafted in the diaphysis, where they integrated into the vascular and perivascular niches and produced SCF and CXCL12. Confocal analysis demonstrated that transplanted EPC produced higher levels of CXCL12 than endogenous cells, explaining, at least in part, the higher levels of hematopoietic engraftment seen in the BM and PB of animals transplanted with EPC 3 days prior to HSC. HSC engrafted mainly in the metaphysis, a metabolically active tissue in the fetus, which is comprised of large sinusoidal veins expressing high levels of hyaluronan (HA) synthase. In addition, donor HSC in the metaphysis were actively cycling (>90%) as determined by Ki67 staining. Fewer donor HSC were found in the diaphysis, and only a small percentage of these cells were dividing, but they localized in clusters in close proximity to donor MSC or EPC. Moreover, a direct correlation was found between the levels of donor-derived perivascular cells engrafted within the vasculature, and the numbers of donor-derived HSC present in the perivascular areas (defined as being within a distance of 5 cell nuclei from vessel), suggesting that, in the diaphysis, adult MSC and EPC provided support/attracted adult HSC. In summary, we have shown that, in a non-myeloablative fetal setting without stress-induced signaling, allogeneic adult donor HSC engraft and proliferate efficiently along with endogenous hematopoietic cells within the metaphysis, and that the high levels of CXCL12 produced by adult EPC were potentially responsible for their ability to significantly enhance HSC engraftment after IUHSCT. DisclosuresNo relevant conflicts of interest to declare.