Hematopoietic Stem And Progenitor Cells Homing Research Articles

Development of VLA4 and CXCR4 Antagonists for the Mobilization of Hematopoietic Stem and Progenitor Cells.

The treatment of patients diagnosed with hematologic malignancies typically includes hematopoietic stem cell transplantation (HSCT) as part of a therapeutic standard of care. The primary graft source of hematopoietic stem and progenitor cells (HSPCs) for HSCT is mobilized from the bone marrow into the peripheral blood of allogeneic donors or patients. More recently, these mobilized HSPCs have also been the source for gene editing strategies to treat diseases such as sickle-cell anemia. For a HSCT to be successful, it requires the infusion of a sufficient number of HSPCs that are capable of adequate homing to the bone marrow niche and the subsequent regeneration of stable trilineage hematopoiesis in a timely manner. Granulocyte-colony-stimulating factor (G-CSF) is currently the most frequently used agent for HSPC mobilization. However, it requires five or more daily infusions to produce an adequate number of HSPCs and the use of G-CSF alone often results in suboptimal stem cell yields in a significant number of patients. Furthermore, there are several undesirable side effects associated with G-CSF, and it is contraindicated for use in sickle-cell anemia patients, where it has been linked to serious vaso-occlusive and thrombotic events. The chemokine receptor CXCR4 and the cell surface integrin α4β1 (very late antigen 4 (VLA4)) are both involved in the homing and retention of HSPCs within the bone marrow microenvironment. Preclinical and/or clinical studies have shown that targeted disruption of the interaction of the CXCR4 or VLA4 receptors with their endogenous ligands within the bone marrow niche results in the rapid and reversible mobilization of HSPCs into the peripheral circulation and is synergistic when combined with G-CSF. In this review, we discuss the roles CXCR4 and VLA4 play in bone marrow homing and retention and will summarize more recent development of small-molecule CXCR4 and VLA4 inhibitors that, when combined, can synergistically improve the magnitude, quality and convenience of HSPC mobilization for stem cell transplantation and ex vivo gene therapy after the administration of just a single dose. This optimized regimen has the potential to afford a superior alternative to G-CSF for HSPC mobilization.

Silencing endomucin in bone marrow sinusoids improves hematopoietic stem and progenitor cell homing during transplantation.

Efficient homing of infused hematopoietic stem and progenitor cells (HSPCs) into the bone marrow (BM) is the prerequisite for successful hematopoietic stem cell transplantation. However, only a small part of infused HSPCs find their way to the BM niche. A better understanding of the mechanisms that facilitate HSPC homing will help to develop strategies to improve the initial HSPC engraftment and subsequent hematopoietic regeneration. Here, we show that irradiation upregulates the endomucin expression of endothelial cells in vivo and in vitro. Furthermore, depletion of endomucin in irradiated endothelial cells with short-interfering RNA (siRNA) increases the HSPC-endothelial cell adhesion in vitro. To abrogate the endomucin of BM sinusoidal endothelial cells (BM-SECs) in vivo, we develop a siRNA-loaded bovine serum albumin nanoparticle for targeted delivery. Nanoparticle-mediated siRNA delivery successfully silences endomucin expression in BM-SECs and improves HSPC homing during transplantation. These results reveal that endomucin plays a critical role in HSPC homing during transplantation and that gene-based manipulation of BM-SEC endomucin in vivo can be exploited to improve the efficacy of HSPC transplantation.

Inducible CXCL12/CXCR4–dependent extramedullary hematopoietic niches in the adrenal gland

Adult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic niche, which regulates HSPC quiescence, self-renewal, and commitment in a demand-adapted manner. Although the complex BM niche is responsible for adult hematopoiesis, evidence exists for simpler, albeit functional and more accessible, extramedullary hematopoietic niches. Inspired by the anecdotal description of retroperitoneal hematopoietic masses occurring at higher frequency upon hormonal dysregulation within the adrenal gland, we hypothesized that the adult adrenal gland could be induced into a hematopoietic-supportive environment in a systematic manner, thus revealing mechanisms underlying de novo niche formation in the adult. Here, we show that upon splenectomy and hormonal stimulation, the adult adrenal gland of mice can be induced to recruit and host functional HSPCs, capable of serial transplantation, and that this phenomenon is associated with de novo formation of platelet-derived growth factor receptor α/leptin receptor (PDGFRα+/LEPR+/-)-expressing stromal nodules. We further show in CXCL12-green fluorescent protein reporter mice that adrenal glands contain a stromal population reminiscent of the CXCL12-abundant reticular cells, which compose the BM HSPC niche. Mechanistically, HSPC homing to hormonally induced adrenal glands was found dependent on the CXCR4-CXCL12 axis. Mirroring our findings in mice, we found reticular CXCL12+ cells coexpressing master niche regulator FOXC1 in primary samples from human adrenal myelolipomas, a benign tumor composed of adipose and hematopoietic tissue. Our findings reignite long-standing questions regarding hormonal regulation of hematopoiesis and provide a novel model to facilitate the study of adult-specific inducible hematopoietic niches, which may pave the way to therapeutic applications.

Novel Small Molecule VLA-4 Inhibitors Optimized for Extended Hematopoietic and Progenitor Cell Mobilization

Background: Hematopoietic stem and progenitor cell (HSPC) transplant is an essential treatment for a variety of blood disorders and malignancies, and more recently, for gene editing therapies such as sickle cell anemia. A key step in this procedure is the effective mobilization of a large quantity of primitive donor HSPCs to ensure a durable and long lasting engraftment capable of developing into all blood cell lineages. The most commonly used regimen for donor mobilization is a 5-day course of Granulocyte Colony Stimulating Factor (G-CSF). However, G-CSF does not effectively mobilize a sufficient number or quality of stem cells in all donors, can have unwanted side effects, and is contraindicated in sickle cell disease. Therefore, it is desirable to identify an alternative treatment that is safe, rapid and cost effective. The cell surface integrin receptor VLA-4 (a4b1) and the chemokine receptor CXCR4 are essential for HSPC homing and retention in the bone marrow. Inhibition of these receptors rapidly mobilizes HSPCs within an hour, as opposed to the multi-day process using G-CSF. Small molecule inhibitors of VLA-4 have been reported previously, but most have poor pharmacokinetic properties and/or weak inhibition of VLA4. These inhibitors rapidly mobilize HSPCs into the peripheral blood of mice after a single s.c. injection, but their effect is short lived, yielding an inadequate number of HSPCs necessary for stem cell transplantation or for gene therapy. Therefore, VLA4 inhibitors with improved pharmacokinetic properties imparting extended and greater HSPC mobilization after a single dose is desirable. Methods: We designed and synthesized multiple novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. Inhibitors determined to be most potent with improved aqueous solubility were then tested in vivo in DBA mice for extended HSPC mobilization beyond 4 hours after a single SC injection, alone and in combination with the CXCR4 inhibitors. Results: We developed two main core molecules that each incorporate key VLA4 receptor binding motifs critical for potent inhibition of VLA4 binding to VCAM-1. Our first generation of inhibitors were quite potent and rapidly mobilized HSPCs, but their effect was short lived and yielded insufficient numbers of HSPCs for gene editing and engraftment studies. Most had poor solubility and very short plasma half-lives after s.c. injection in mice. We designed later generation analogues that strategically exploited a region of each optimized core, allowing for covalent addition of polyethylene glycol (PEG) chains of increasing length without decreasing potency. We determined that a minimum PEG length of 24 PEG units was required for extended HSPC mobilization in mice. WU-106, one of our VLA-4 inhibitors containing 24 PEG units, exhibited excellent aqueous solubility, longer plasma half-life after s.c. dosing, and mobilized 2-fold more murine HSPCs for a longer time than previously published best-in-class VLA-4 inhibitors or our first generation VLA4 inhibitor leads. Importantly, a synergistic effect on HSPC mobilization was achieved when WU-106 was co-administered with CXCR4 inhibitors (Plerixafor or BL-8040). Further optimization by the addition of longer PEG chains, even up to 40KD, retained sub nM potency and extended mobilization beyond 24 hours after a single injection. Our 20kD PEG analogues attached to our two different core molecules (WU-125 and WU-133) allowed for dosing of the drug in the evening, with maximum mobilization still present the next morning, thereby enabling HSPC collection to initiate soon (2-4 hours) after CXCR4 inhibitor dosing in the morning. The quantity of HSPCs obtained using this staggered dosing regimen was greater than simultaneous administration of the VLA-4 and CXCR4 inhibitors ( Fig. 1). Furthermore, our novel, long-acting PEG analogues demonstrated superior HSPC mobilization compared to an alpha 4-targeted antibody (anti-CD49d) in mice ( Fig.2). Conclusion: We have developed novel, potent PEGylated small molecule VLA-4 inhibitors that have excellent aqueous solubility and pharmacokinetics. The kinetics and magnitude of HSPC mobilization by our PEGylated inhibitors are superior to other previously described VLA-4 inhibitors and an antibody to alpha-4(anti-CD49d).

Thymic innate lymphoid cells promote thymocyte differentiation of transplanted hematopoietic progenitors in immunodeficient mice.

Abstract Patients with combined immunodeficiencies can be cured by intravenous transfer of allogeneic hematopoietic stem and progenitor cells (HSPC). However, complications such as an impaired and delayed T cell differentiation are unfortunately common, notably due to the defective thymic architecture in these patients and the requirement of HSPC homing to the thymus. To circumvent these problems, we have developed an innovative approach based on the direct targeting of HSPC into the thymus by intrathymic (IT) injection. Using ZAP70-deficient mice as a paradigm, we previously showed that IT injection of HSPC can support a long-term thymus-autonomous differentiation. Here, we show that the IT injection of WT HSPC results in a rapid engraftment and differentiation. Donor-derived thymocytes expand >100-fold, accounting for >13% of thymus cellularity by 3 weeks post IT transfer and importantly, engraftment occurs in the absence of cytoreductive conditioning. While mature CD4 thymocyte differentiation occurs in 3 weeks, the differentiation of mature Treg thymocytes requires 4 weeks. The kinetics of thymic Treg development parallels the generation of a thymic medulla, with the appearance of mature medullary thymic epithelial cells at 3–4 weeks post transplantation. Notably, the generation of the thymic medulla is associated with the differentiation of donor-derived RORγT+ innate lymphoid cells (ILC). While ILC are not required for physiological thymopoiesis, we find that they are critical for T cell differentiation in ZAP70-deficient mice presenting with an absence of the thymic medulla. These results may open new therapeutic avenues, promoting the differentiation of ILC via IT-mediated HSPC transfer, enhancing T cell differentiation. Supported by grants from ANR (CHIC) and AFM-Telethon

Clinical Progress and Preclinical Insights Into Umbilical Cord Blood Transplantation Improvement.

The application of umbilical cord blood (UCB) as an important source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution in the clinical context has steadily grown worldwide in the past 30 years. UCB has advantages that include rapid availability of donors, less strict HLA-matching demands, and low rates of graft-versus-host disease (GVHD) versus bone marrow (BM) and mobilized peripheral blood (PB). However, the limited number of HSPCs within a single UCB unit often leads to delayed hematopoietic engraftment, increased risk of transplant-related infection and mortality, and proneness to graft failure, thus hindering wide clinical application. Many strategies have been developed to improve UCB engraftment, most of which are based on 2 approaches: increasing the HSPC number ex vivo before transplantation and enhancing HSPC homing to the recipient BM niche after transplantation. Recently, several methods have shown promising progress in UCB engraftment improvement. Here, we review the current situations of UCB manipulation in preclinical and clinical settings and discuss challenges and future directions.

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

The Laminin Receptors Basal Cell Adhesion Molecule/Lutheran and Integrin α7β1 on Human Hematopoietic Stem Cells.

In the adult organism, hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow (BM) in specialized hematopoietic stem cell niches of which the extracellular matrix (ECM) is an integral component. Laminins (LM) are a family of heterotrimeric ECM molecules of which mainly family members containing an α4 or α5 chain are expressed in cells from BM niches and involved in HSPC homing and proliferation. Various integrin and non-integrin laminin receptors have been identified and characterized. Among these, the integrins α6β1 and α3β1 were reported to be strongly expressed on human and mouse HSPC. In the present study, we focus on two further specific laminin receptors, namely integrin α7β1 and basal cell adhesion molecule/Lutheran (BCAM/Lu). Using RT-PCR analyses, immunofluorescence staining, immunoblotting and flow cytometry, we show that both are strongly expressed by human lineage-negative CD34 + HSPC. Treatment with function-blocking antibodies against BCAM/Lu neither inhibits the strong adhesive interaction of CD34 + HSPC with LM-511/LM-521 nor the LM-511/LM-521 mediated changes in CD34 + HSPC proliferation, but however, influences the cytokine-induced differentiation of HSPC in colony formation assays. In addition, stromal-derived factor (SDF) 1α-mediated transmigration of CD34 + HSPC through an endothelial cell layer was effectively diminished by BCAM/Lu antibodies, suggesting a direct involvement of BCAM/Lu in the migration process. This study indicates that both laminin receptors newly identified on human CD34 + HSPC should be taken into consideration in future studies.

Fetal hematopoietic stem cell homing is controlled by VEGF regulating the integrity and oxidative status of the stromal-vascular bone marrow niches.

SummaryHematopoietic stem and progenitor cell (HSPC) engraftment after transplantation during anticancer treatment depends on support from the recipient bone marrow (BM) microenvironment. Here, by studying physiological homing of fetal HSPCs, we show the critical requirement of balanced local crosstalk within the skeletal niche for successful HSPC settlement in BM. Transgene-induced overproduction of vascular endothelial growth factor (VEGF) by osteoprogenitor cells elicits stromal and endothelial hyperactivation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSPC homing and survival are drastically impaired. Transcriptome profiling, flow cytometry, and high-resolution imaging indicate alterations in perivascular and endothelial cell characteristics, vascular function and cellular metabolism, associated with increased oxidative stress within the VEGF-enriched BM environment. Thus, developmental HSPC homing to bone is controlled by local stromal-vascular integrity and the oxidative-metabolic status of the recipient milieu. Interestingly, irradiation of adult mice also induces stromal VEGF expression and similar osteo-angiogenic niche changes, underscoring that our findings may contribute targets for improving stem cell therapies.

IFNg Increases HSC Homing and Induces Relocalization within the Bone Marrow Niche

Abstract Infections affect hematopoietic stem cell (HSC) biology, with significant implications in ageing, cancer development, and bone marrow failure syndromes. We previously showed that some infections induce HSCs to proliferate through a cell autonomous response to interferon gamma (IFNg). Given that interactions of HSCs with their bone marrow niche are a critical factor in maintaining quiescence, we considered how IFNg affects the interaction between HSCs and the niche. We hypothesized that infection would displace HSCs from the niche and reduce homing in an IFNg-dependent manner, thereby contributing to activation. We used a Mycobacterium avium model of infection to characterize the effects of infection on HSC homing. In this model HSCs are activated to divide and differentiate via an IFNg-dependent mechanism with depletion of HSCs after several months of infection. We compared the homing of hematopoietic stem and progenitor cells (HSPCs) from M. avium infected versus non-infected animals by IV administration of CSFE-labeled cKit+ cells into lethally irradiated recipient mice. Homing was determined by the frequency with which labeled cells could be found in the bone marrow 17 hours later, relative to a DDAO-stained internal control. Surprisingly, HSPCs from M. avium -infected animals displayed improved homing compared to control HSPCs. A similar phenotype was seen for cKit+ cells isolated from mice treated with recombinant IFNg, whereas cKit+ cells from Ifngr1-/- mice showed decreased homing compared to wildtype. Using transplantation assays, we found decreased engraftment of Ifngr1-deficient whole bone marrow when cells were administered intravenously; but engraftment was normal when cells were infused intrafemorally. Altogether, these findings show that basal IFNg signaling contributes to normal HSPC homing and that IFNg induced during M. avium infection increases homing to the bone marrow. The above studies reflect homing of HSPCs to the bone marrow, not cellular interactions within the niche. Since proximity to Cxcl12-expressing cells in the perivascular niche can affect HSC activation status, we used 3-D intravital imaging to study the effects of IFNg on HSC-niche interactions. We injected CMTMR-stained HSCs into Cxcl12-GFP transgenic mice and measured the distance of the HSCs from quiescence-associated Cxcl12-abundant reticular (CAR) cells. CMTMR-stained HSCs were seen moving upon IFNg treatment, and 24hrs post-IFNg HSCs were located significantly further away from CAR cells compared to untreated or TGF-b treated controls. These results suggest that IFNg can induce movement away from CAR cells, thereby allowing HSC proliferation during inflammatory conditions. To define the mechanism by which IFNg causes HSC relocalization, we performed gene expression profiling of HSCs from control or IFNg-treated mice. Gene set enrichment analysis of differentially regulated genes showed modulation of the expected immune and cytokine response pathways, but also genes related to adhesion and extracellular matrix remodeling. Of note, the glycoprotein Bst2 (Tetherin) was upregulated in HSCs following IFNg treatment. Bst2 has been shown to bind to E-selectin, and localization to E-selectin+ endothelial cells is associated with increased HSC proliferation. Thus we hypothesized that IFNg-induced upregulation of surface Bst2 may facilitate proliferation by promoting HSC binding to E-selectin. We confirmed induction of Bst2 mRNA and protein by qPCR and flow cytometry, and we found that Bst2 was upregulated following M. avium infection via an IFNg-dependent mechanism. In vitro binding assays showed increased HSPC binding to recombinant E-selectin following IFNg treatment. Thus, IFNg-dependent cell surface expression of Bst2 may facilitate the displacement of HSCs from the quiescence-enforcing CAR niche to an active E-selectin-positive niche. Because E-selectin expression along blood vessel walls is also important for adhesion and transendothelial migration of HSCs, increased Bst2 on HSCs may also contribute to more efficient homing. In summary, IFNg improves HSPC homing but displaces HSCs from quiescence-enforcing CAR cells in the niche, potentially by inducing Bst2-mediated movement to the E-selectin rich proliferative niche. Future studies will ascertain whether blocking Bst2 is a feasible method to protect HSCs from the deleterious effects of chronic inflammation. Disclosures No relevant conflicts of interest to declare.

Defining the Transcriptional Code That Specifies Sinusoidal Endothelial Cells in the HSPC Niche

Abstract Hematopoietic stem and progenitor cell (HSPC) niches are specialized microenvironments that support the maintenance and self-renewal of HSPCs. In the fetal liver, bone marrow and spleen, vascular endothelial cells are thought to function as core components of the HSPC niche. In the bone marrow, sinusoidal endothelial cells are known to secrete soluble factors and express cell surface adhesion molecules that promote the recruitment, maintenance and function of HSPCs. Using tomo-seq (RNA tomography) - a technique that involves extracting and barcoding the RNA from sequentially collected cryosections and then performing RNA-seq - we identified ~300 genes enriched in the zebrafish caudal hematopoietic tissue (CHT; the hematopoietic equivalent of the mammalian fetal liver). We also used FACS with tissue-specific fluorescent reporter transgenes to isolate different cell populations from whole embryos and then performed RNA-seq. This included endothelial cells, macrophages, neutrophils and erythrocytes. By overlaying these datasets with the tomo-seq results, we determined the cell types in which many of the CHT-enriched genes were expressed. Unexpectedly, we found a set of ~20 genes that appeared to be enriched specifically in endothelial cells within the CHT. Whole embryo in situ hybridization revealed that these genes had similar spatial and temporal expression patterns, being specifically enriched in sinusoidal endothelial cells in the CHT but absent in other endothelial cells throughout the body. We cloned the upstream promoter regions for several of these genes, fused them to a GFP reporter and then injected them into embryos. Of note, we found that a 5.3 kb sequence upstream of selectin-e (sele ; a gene known to be expressed by bone marrow endothelial cells where it promotes HSPC homing) drove GFP expression specifically in sinusoidal endothelial cells in the CHT. By crossing this sele:GFP transgene to a pan-endothelial marker (kdrl:mCherry), we were able to use FACS to isolate CHT endothelial cells, as well as endothelial cells from tissues outside the CHT. We performed ATAC-seq - a transposon-based method used to detect regions of open chromatin - on these two cell populations and identified 6,710 peaks (regions of open chromatin) that were unique to sele:GFP+; kdrl:mCherry+ CHT niche endothelial cells. Several of these peaks were associated with CHT endothelial genes we had initially identified by tomo-seq, including mrc1a, gpr182, stab1, stab2 and exoc3l2a . Informatic analysis of the sequences under the 6,710 CHT endothelial peaks identified Ets, Sox, Nuclear Hormone and Gata sites as the most enriched transcription factor binding motifs compared to peaks from the non-CHT endothelial cells. These results suggested a transcription factor code involving members of these families might specify sinusoidal endothelial cells in the HSPC niche. Upon closer examination of the sele locus itself, we observed a narrow ATAC-seq peak within the 5.3 kb upstream sequence that was uniquely open in the sele:GFP+; kdrl:mCherry+ CHT niche endothelial cells. Promoter truncation experiments identified a short 158 bp sequence that perfectly aligned with this unique ATAC-seq peak and which was sufficient to drive GFP expression in CHT endothelial cells. This 158 bp sequence contained three Ets, three Sox and one RORA (nuclear hormone receptor) binding sites. Synthetic variants were made in which the different transcription factor binding sites within the 158 bp sequence were disrupted. Injection of these variants driving GFP demonstrated that the Sox and RORA sites were required for expression in CHT endothelial cells. Disruption of the Ets sites, in contrast, led to dramatically enhanced GFP expression, suggesting these sites could be repressive. Together these studies demonstrate that the transcriptional regulation of niche endothelial cells include transcription factors that bind to Ets, Sox, and RORA motifs. The results of this work has important implications for culturing HSPCs in vitro with endothelial cells or for modulating the HSPC niche in the context of human disease or transplantation. Disclosures Zon: Fate, Inc.: Consultancy, Equity Ownership; Marauder, Inc.: Consultancy, Equity Ownership; Scholar Rock, Inc: Consultancy, Equity Ownership; Stemgent: Consultancy.

Engraftment and SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells Are Regulated By R4 Rgs Subfamily Proteins

Abstract Regulator of G-protein Signaling (RGS) family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of mature hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to chemokines are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 12) expressed RGS1 , RGS2 , RGS3 , RGS5 , RGS13 , RGS16 and RGS18 at mRNA level, whereas RGS4 , RGS8 and RGS21 were undetectable. Exposure of CB CD34+ cells to the critical homing-related chemokine stromal cell-derived factor-1 (SDF-1) significantly increased RGS1 , RGS2 , RGS13 and RGS16 , and decreased RGS3 and RGS18 expressions (P ≤ 0.040, n = 5). Expressions of RGS1 , RGS13 and RGS16 were significantly higher in bone marrow (BM, n = 14) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 14) CD34+ cells (P ≤ 0.002), while RGS3 and RGS18 expressions were lower in BM CD34+ cells (P ≤ 0.036), suggesting a SDF-1 and niche-dependent regulation of RGS expressions. Lentivirus-mediated overexpression of RGS1 , RGS13 or RGS16 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.039, n = 4-5) butdid not alter SDF-1-induced actin polymerization (n = 3). In addition, phosphorylation of signaling molecules downstream of the SDF-1/CXCR4 axis, including Akt, ERK and STAT3 was significantly suppressed in RGS1 - and RGS16 -overexpressing CD34+ cells. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1 , RGS13 or RGS16 significantly reduced BM engraftment of CD34+ cells by 98% (P = 0.023, n = 7), 89% (P = 0.014, n = 8) and 87% (P = 0.015, n = 8) respectively. Transcriptome profiling (n = 4) of CD34+ cells identified 312 genes with differential expressions upon overexpression of RGS1 , RGS13 or RGS16 , with 32 genes commonly regulated. Gene function enrichment analysis revealed significant downregulation of genes involved in the migration (PROS1 , CCL1 , THBS1 , F2RL2 ), complement activation (C3AR1 , C5AR1 , C5AR2 ), and proteolysis (MMP14 , TIMP3 ) pathways, which were validated by qPCR (n = 8). Taken together, we provided the first evidence that R4 RGS members are expressed and regulated by the SDF-1 in human CD34+ HSC. We also presented data showing that RGS1 , RGS13 or RGS16 negatively regulated in vitro SDF-1-mediated migration and signaling as well as in vivo engraftment of CD34+ cells, suggesting that these RGS members might serve as a feedback mechanism to regulate the SDF-1/CXCR4 axis. Strategies to inhibit specific R4 RGS proteins could potentially enhance efficiency of HSC homing and engraftment, which are particularly important in the setting of CB transplantation. Disclosures No relevant conflicts of interest to declare.

Stem cell homing: From physiology to therapeutics.

Stem cell homing is a multistep endogenous physiologic process that is also used by exogenously administered hematopoietic stem and progenitor cells (HSPCs). This multistep process involves cell migration and is essential for hematopoietic stem cell transplantation. The process can be manipulated to enhance ultimate engraftment potential, and understanding stem cell homing is also important to the understanding of stem cell mobilization. Homing is also of potential importance in the recruitment of marrow mesenchymal stem and stromal cells (MSCs) to sites of injury and regeneration. This process is less understood but assumes importance when these cells are used for repair purposes. In this review, the process of HSPC and MSC homing is examined, as are methods to enhance this process.

CXCR4 expression in the bone marrow microenvironment is required for hematopoietic stem and progenitor cell maintenance and early hematopoietic regeneration after myeloablation.

The bone marrow (BM) microenvironment/niche plays a key role in regulating hematopoietic stem and progenitor cell (HSPC) activities; however, mechanisms regulating niche cell function are not well understood. In this study, we show that niche intrinsic expression of the CXCR4 chemokine receptor critically regulates HSPC maintenance during steady state, and promotes early hematopoietic regeneration after myeloablative irradiation. At steady state, chimeric mice with wild-type (WT) HSPC and marrow stroma that lack CXCR4 show decreased HSPC quiescence, and their repopulation capacity was markedly reduced. Mesenchymal stromal cells (MSC) were significantly reduced in the BM of CXCR4 deficient mice, which was accompanied by decreased levels of the HSPC supporting factors stromal cell-derived factor-1 (SDF-1) and stem cell factor (SCF). CXCR4 also plays a crucial role in survival and restoration of BM stromal cells after myeloablative irradiation, where the loss of BM stromal cells was more severe in CXCR4-deficient mice compared to WT mice. In addition, transplantation of WT donor HSPC into CXCR4-deficient recipient mice demonstrated reduced HSPC homing and early hematopoietic reconstitution. We found that CXCR4 signaling attenuates irradiation-induced BM stromal cell loss by upregulating the expression of the antiapoptotic protein Survivin via the PI3K pathway. Our study suggests that SDF-1-CXCR4 signaling in the stromal microenvironment cells plays a crucial role in maintenance of HSPCs during homeostasis, and promotes niche regeneration and early hematopoietic reconstitution after transplantation. Modulation of CXCR4 signaling in the HSPC microenvironment could be a means to enhance hematopoietic recovery after clinical hematopoietic cell transplantation.

Thrombopoietin enhances hematopoietic stem and progenitor cell homing by impeding matrix metalloproteinase 9 expression.

We reported a novel function of recombinant human thrombopoietin (TPO) in increasing hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM). Single doses of TPO treatment to the recipients immediately after BM transplantation showed significantly improved homing of HSPCs to the BM, which subsequently resulted in enhanced short‐ and long‐term engraftment of HSPCs in mice. We found that TPO could downregulate the expression and secretion of matrix metalloproteinase 9 in BM cells. As a result, SDF‐1α level was increased in the BM niche. Blocking the interaction of SDF‐1α and CXCR4 on HSPCs by using AMD3100 could significantly reverse the TPO‐enhanced HSPC homing effect. More importantly, a single dose of TPO remarkably promoted human HSPC homing and subsequent engraftment to the BM of nonobese diabetic/severe combined immunodeficiency mice. We then performed a clinical trial to evaluate the effect of TPO treatment in patients receiving haploidentical BM and mobilized peripheral blood transplantation. Surprisingly, single doses of TPO treatment to patients followed by hematopoietic stem cell transplantation significantly improved platelet engraftment in the cohort of patients with severe aplastic anemia (SAA). The mean volume of platelet and red blood cell transfusion was remarkably reduced in the cohort of patients with SAA or hematological malignancies receiving TPO treatment. Thus, our data provide a simple, feasible, and efficient approach to improve clinical outcomes in patients with allogenic hematopoietic stem cell transplantation. The clinical trial was registered in the Chinese Clinical Trial Registry website (http://www.chictr.org.cn) as ChiCTR‐OIN‐1701083.

Hematopoietic stem and progenitor cells use podosomes to transcellularly cross the bone marrow endothelium.

Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells (HSPC) upon transplantation, but surprisingly little is known on how the bone marrow (BM) endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted HSPC to the BM of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced BM permeability, but did not prevent HSPC cells migration into the BM, suggesting that HSPC use the transcellular migration route to enter BM. Indeed, using an in vitromigration assay, we show that human HSPC cells predominantly cross BM endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of HSPC. These findings are important in the development of potential therapeutic targets to improve HSPC homing strategies.

Intravital Imaging of Blood Flow and HSPC Homing in Bone Marrow Microvessels.

Two-photon intravital microscopy (2P-IVM) is an advanced imaging technique that allows the visualization of dynamic cellular behavior deeply inside tissues and organs of living animals. Due to the deep tissue penetration, imaging of highly light-scattering tissue as the bone becomes feasible at subcellular resolution.To better understand the influence of blood flow on hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM) microvasculature of the calvarial bone, we analyzed blood flow dynamics and the influence of flow on the early homing behavior of HSPCs during their passage through BM microvessels. Here, we describe a 2P-IVM approach for direct measurements of red blood cell (RBC) velocities in the BM microvasculature using repetitive centerline scans at the level of individual arterial vessels and sinusoidal capillaries to obtain a detailed flow profile map. Furthermore, we explain the isolation and enrichment of HSPCs from long bones and the transplantation of these cells to study the early homing behavior of HSPCs in BM sinusoids at cellular resolution. This is achieved by high-resolution spatiotemporal imaging through a chronic cranial window using transgenic reporter mice.

The tetraspanin CD82 regulates bone marrow homing and engraftment of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cell (HSPC) transplantation represents a treatment option for patients with malignant and nonmalignant hematological diseases. Initial steps in transplantation involve the bone marrow homing and engraftment of peripheral blood–injected HSPCs. In recent work, we identified the tetraspanin CD82 as a potential regulator of HSPC homing to the bone marrow, although its mechanism remains unclear. In the present study, using a CD82 knockout (CD82KO) mouse model, we determined that CD82 modulates HSPC bone marrow maintenance, homing, and engraftment. Bone marrow characterization identified a significant decrease in the number of long-term hematopoietic stem cells in the CD82KO mice, which we linked to cell cycle activation and reduced stem cell quiescence. Additionally, we demonstrate that CD82 deficiency disrupts bone marrow homing and engraftment, with in vitro analysis identifying further defects in migration and cell spreading. Moreover, we find that the CD82KO HSPC homing defect is due at least in part to the hyperactivation of Rac1, as Rac1 inhibition rescues homing capacity. Together, these data provide evidence that CD82 is an important regulator of HSPC bone marrow maintenance, homing, and engraftment and suggest exploiting the CD82 scaffold as a therapeutic target for improved efficacy of stem cell transplants.

Effects of Mesenchymal Stem Cell Derivatives on Hematopoiesis and Hematopoietic Stem Cells.

Hematopoiesis is a balance among quiescence, self-renewal, proliferation, and differentiation, which is believed to be firmly adjusted through interactions between hematopoietic stem and progenitor cells (HSPCs) with the microenvironment. This microenvironment is derived from a common progenitor of mesenchymal origin and its signals should be capable of regulating the cellular memory of transcriptional situation and lead to an exchange of stem cell genes expression. Mesenchymal stem cells (MSCs) have self-renewal and differentiation capacity into tissues of mesodermal origin, and these cells can support hematopoiesis through release various molecules that play a crucial role in migration, homing, self-renewal, proliferation, and differentiation of HSPCs. Studies on the effects of MSCs on HSPC differentiation can develop modern solutions in the treatment of patients with hematologic disorders for more effective Bone Marrow (BM) transplantation in the near future. However, considerable challenges remain on realization of how paracrine mechanisms of MSCs act on the target tissues, and how to design a therapeutic regimen with various paracrine factors in order to achieve optimal results for tissue conservation and regeneration. The aim of this review is to characterize and consider the related aspects of the ability of MSCs secretome in protection of hematopoiesis.

Granulocyte–Colony Stimulating Factor plus Plerixafor in Patients with β-thalassemia Major Results in the Effective Mobilization of Primitive CD34+ Cells with Specific Gene Expression Profile

Successful gene therapy for β-thalassemia requires optimal numbers of autologous gene-transduced hematopoietic stem and progenitor cells (HSPCs) with high repopulating capacity. Previous studies suggested superior mobilization in these patients by the combination of granulocyte–colony stimulating factor (G-CSF) plus plerixafor over single agents. We mobilized four adult patients using G-CSF+plerixafor to assess the intra-individual variation of the circulating CD34+ cells number and subtypes preand post-plerixafor administration. The procedure was well-tolerated and the target cell dose of ≥8 × 106 CD34+ cells/kg was achieved in three of them with one apheresis procedure. The addition of plerixafor unanimously increased the number of circulating CD34+ cells, and the frequency of the most primitive CD34+ subtypes: CD34+/38− and CD34+/133+/38− as well as the in vitro clonogenic potency. Microarray analyses of CD34+ cells purified from the leukapheresis of one patient mobilized twice, with G-CSF and with G-CSF+plerixafor, highlighted in G-CSF+plerixafor-mobilized CD34+ cells, higher levels of expression genes involved in HSPC motility, homing, and cell cycles. In conclusion, G-CSF+plerixafor in β-thalassemia patients mobilizes optimal numbers of HSPCs with characteristics that suggest high capacity of engraftment after transplantation.