ALBERT

Description: Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P 〈 .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P 〈 .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

Description: The stromal cell–derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing and engraftment of hematopoietic stem/progenitor cells (HSCs) during bone marrow transplantation. To investigate the transcriptional regulation provided by this axis, we performed the first differential transcriptome profiling of human cord blood CD34+ cells in response to short-term exposure to SDF-1 and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family of proteins, was expressed in CD34+CD38−/lo and CD34+CD38+ cells. CD9 levels were enhanced by SDF-1, which simultaneously down-regulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expression was modulated via CXCR4, G-protein, protein kinase C, phospholipase C, extracellular signal-regulated kinase, and Janus kinase 2 signals. Pretreatment of CD34+ cells with the anti-CD9 monoclonal antibody ALB6 significantly inhibited SDF-1–mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells was enhanced. Pretreatment of CD34+ cells with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID (nonobese diabetic/severe combined immune-deficient) mice. Sorted CD34+CD9− cells displayed lower bone marrow homing capacity compared with that of total CD34+ cells. CD9 expression on homed CD34+ cells was significantly up-regulated in vivo. Our results indicate that CD9 might possess specific functions in HSC homing.

Description: RGS family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to stromal cell-derived factor-1 (SDF-1) 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 = 10) expressed 7 out of 10 R4 RGS proteins at mRNA level (RGS1-3, 5, 13, 16 and 18), whereas expressions of RGS4, 8 and 21 were undetectable. Exposure of CB CD34+ cells to SDF-1 significantly increased RGS1, 2, 13 and 16 expressions and decreased RGS3 and 18 expressions (P ≤ 0.0402, n = 5). Expressions of RGS1, 13 and 16 were significantly higher in bone marrow (BM, n = 10) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 5) CD34+ cells (P ≤ 0.0160), while RGS3 and 18 expressions were lower in BM CD34+ cells (P ≤ 0.0471), suggesting a SDF-1- and niche-dependent regulation of RGS expressions. To investigate the potential involvement of RGS proteins in SDF-1-mediated homing-related functions, we introduced RGS overexpression constructs into CB CD34+ cells by lentiviral transduction. With 〉80% transduction efficiency, we showed that overexpression of RGS1, 13 and 16 but not RGS2 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.0391, n = 4-5). Similarly, RGS1, 13 and 16 overexpression suppressed SDF-1-induced Akt phosphorylation (n = 2), but none of them affected SDF-1-mediated actin polymerization (n = 3). In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing was impaired in RGS1- (16.3% reduction), RGS13- (12.7% reduction) or RGS16-overexpressing CD34+ cells (33.7% reduction). Taken together, we provided the first evidence that expressions of R4 RGS proteins are regulated by the SDF-1/CXCR4 axis in human CD34+ HSC. We also presented evidence that specific R4 RGS proteins (RGS1, 13 and 16) negatively regulate in vitro SDF-1-mediated responses and in vivo homing of CD34+ cells, suggesting that RGS proteins may serve as a feedback mechanism to regulate SDF-1/CXCR4 signaling. Strategies to inhibit RGS signaling could thus be a potential method for enhancing efficiency of HSC homing and long-term engraftment, which is particularly important in the setting of CB transplantation. Disclosures No relevant conflicts of interest to declare.

Description: The stromal cell-derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis is crucial to migration and homing of hematopoietic stem/progenitor cells (HSC). In a previous study, we reported the expression profile of human cord blood CD34+ cells in response to a short-term exposure of SDF-1 (Leung et al, Blood, 2011). We further identified that expression levels of several R4 Regulator of G-protein signaling (RGS) family proteins were upregulated upon SDF-1 treatment. RGS family proteins are known to negatively control G-protein-coupled receptor signal transduction through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes), responses to SDF-1 are subjected to regulation by RGS proteins. However, their expression patterns and functional roles in HSC are poorly characterized. We first assessed the basal expressions of 10 R4 RGS proteins by quantitative RT-PCR. Our results demonstrated that cord blood CD34+ cells expressed relatively high mRNA level of RGS1, RGS2 and RGS3, and moderate level of RGS5, RGS13, RGS16 and RGS18 (n = 3). Expression of RGS8 was barely detectable, while RGS4 and RGS21 expression was not detectable. A short-term exposure (1-24 hours) of CD34+ cells to SDF-1 (100 ng/mL, n = 4) significantly increased expressions of RGS1 (1.5 fold, P = .002), RGS13 (1.8 fold, P = .044), and to a lesser extent RGS2 and RGS3 (both 1.3 fold; P 〈 .032). Expressions of RGS5, RGS16 and RGS18 were not affected by SDF-1 stimulation. Preincubation of CD34+ cells with the CXCR4 antagonist AMD3100 resulted in 82.2% and 56.2% inhibition of SDF-1-induced RGS1 and RGS13 expressions respectively. To investigate the functional roles of RGS1 and RGS13 in SDF-1-mediated responses, we introduced GFP-tagged cDNA clones into cord blood CD34+ cells by lentiviral transduction. Using the spleen focus-forming virus (SFFV) promoter, we achieved 55.2% transduction efficiency in control GFP vector-transduced cells, 39.4% in RGS1-transduced cells and 51.7% in RGS13-transduced cells. Quantitative RT-PCR further confirmed RGS1 (76 fold) and RGS13 (3,641 fold) overexpression in transduced CD34 cells. We showed that SDF-1-directed migration was inhibited by 18.5% in RGS1-transduced cells, but not in RGS13-transduced cells. In RGS1-transduced cells, significantly less GFP+ cells was found in the migrating fraction (51% in non-migrating vs 28% in migrating fraction). We further demonstrated that actin polymerization, an early response to SDF-1 stimulus, was reduced by 9.5% in RGS13-transduced cells but not in RGS1-transduced cells. In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing of CD34+ cells was impaired in RGS1-overexpressing (54.4% reduction) or RGS13-overexpressing (28.1% reduction) cells, when compared to control GFP-transduced cells. Splenic homing of CD34+ cells was not affected by either RGS1 or RGS13. Taken together, we provided the first expression profile of R4 RGS proteins in cord blood CD34+ cells, and their expressional changes in response to SDF-1 stimulation. We also provided evidence that RGS1 and RGS13 regulate specific SDF-1-mediated responses and, importantly, both negatively affect homing of CD34+ cells. Strategies to inhibit RGS1 or RGS13 signaling could be a potential method for enhancing HSC homing and their long-term engraftment for immunologic reconstitution. Disclosures No relevant conflicts of interest to declare.

Description: Background:In postnatal life, hematopoietic stem/progenitor cells (HSC) mostly reside in specialized bone marrow niches, however a low number of HSC is constantly released into the peripheral circulation. Emerging evidence suggests that these circulating HSC may play an active role in the host defense mechanism against bacterial infection by fostering the local production of tissue-resident innate immune cells. In adult patients with sepsis, CD34+ cells were significantly elevated in their circulation, which correlated with monocyte production and survival. Due to the immaturity of the immune system, preterm infants are vulnerable to life-threatening systemic infection. Although neonatal blood contains relatively high levels of CD34+ cells, there has been no knowledge on whether the same regulation of circulating CD34+ cells occurs in preterm infants in response to systemic infection, as in adults. Methods: We collected peripheral blood from preterm, very low birth weight (72 hours of age) infection requiring full sepsis evaluation and antibiotic treatment. Circulating CD34+ cells were assessed using 3-color flow cytometry (CD34, CD45 and 7-AAD), and enumerated using the ISHAGE gating strategy. Results: Of 39 episodes of suspected clinical sepsis investigated, 12 were blood culture-confirmed sepsis and 27 were non-infected episodes. There were no significant differences between infected and non-infected infants in gestational age, birth weight, postnatal age at the time of sepsis screening, Apgar scores at 1 and 5 minutes, male/female ratio or white cell count. However, there was a significant decrease in the frequency of CD34+ cells (percentage of total CD45+ cells) in infected infants [median (interquartile range), 0.08 (0.06-0.16) %], when compared to non-infected episodes [0.19 (0.12-0.34) %, P = 0.001]. Concomitant increase in the frequency of neutrophils [69 (59-75) % vs. 45 (34-53) %, P 〈 0.001] and decrease in lymphocytes [18 (13-22) vs. 32 (27-41) %, P 〈 0.001] were observed during these infection episodes. In addition, an inverse correlation was found between the frequencies of CD34+ cells and neutrophils (r = -0.471, P = 0.004), whereas a positive correlation was observed between the frequencies of CD34+ cells and lymphocytes (r = 0.485, P = 0.003). Interestingly, a rebound of CD34+ cells was evidenced at 24 hours after the commencement of antibiotic treatment in the sepsis group [0.12 (0.07-0.20) %, P = 0.012] but not in the non-infected group [0.21 (0.16-0.27) %, P= 0.847], suggesting that the level of circulating CD34+ cells may be under active control and correlate with clinical progress. Conclusions: This is the first evaluation of circulating CD34+ cells in preterm infants during an episode of systemic infection. Our data revealed a drop in the frequency of CD34+ cells in these infants at the onset of sepsis, which may be linked to the preferential commitment to neutrophilic lineage at the expense of lymphocyte production. Moreover, there is an opposite direction in the regulation of CD34+ cells in preterm infants when compared with adults affected by sepsis or other inflammatory conditions. This phenomenon may be attributed by defective mobilization, lower plasma levels of stem cell-mobilizing cytokines or insufficient stem cell pool in the bone marrow, which warrants further investigation. Disclosures No relevant conflicts of interest to declare.

Description: Ex vivo expansion of hematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. In this study, we investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis on prolonged maintenance and expansion of cord blood CD34+ cells. Enriched CD34+ cells (1 x 105/mL, n=5) or mononuclear cells (1 x 106/mL, n=8) were cultured in X-VIVO-10 medium for 14, 21, 28 and 35 days without supplementary cytokine or medium changing. Our results showed that the presence of NTL (200 ng/mL) or FL-3 ligand (FL, 40 ng/mL) significantly preserved populations of early stem/progenitor cells (total CFU, BFU/CFU-E, CFU-GM, CFU-GEMM) in these cultures, compared with respective controls at various time points. In the ex vivo expansion study (n=16), the presence of stem cell factor (S, 50 ng/mL), thrombopoietin (T, 50 ng/mL), FL (F, 80 ng/mL) effectively expanded total nucleated cells (TNC) at day 8 (116 ± 20.2 fold) and day 12 (424 ± 68.8 fold), as well as all subsets of progenitor cells as demonstrated by flow cytometry and CFU assays. The presence of NTL (200 ng/mL) significantly increased TNC (148 ± 24.5 fold at day 8; 572 ± 91.9 fold at day 12; P 〈 0.01) and expansion of early progenitor cells (CD34+, CD34+CD38−, CFU-GEMM) and committed CFU of the myeloid (CFU-GM), erythroid (BFU/CFU-E) and the megakaryocytic lineage (CFU-MK) (P 〈 0.01 compared with respective TSF cultures). There was also slight but consistent increase of CD61+CD41+ cells in the presence of NTL (8.58 ± 2.14 x 105 vs. 7.30 ± 1.82 x 105 cells/mL, P 〈 0.001). Significantly, the increased expansion was not only contributed by the higher TNC, but also by the increase in the proportion of CD34+ cells, CD34+CD38− cells and the density of differential CFU. Six weeks after enriched CD34+ cells at day 0 or expanded cells at day 12 were infused into sub-lethally irradiated NOD/SCID mice, human CD45+ cells were detectable in the BM, spleen and PB of the mice. In the BM, there were engraftments of human hematopoietic cells of the early (CD34+), myeloid (CD33+, CD14+), B-lymphoid (CD19+) and megakaryocytic (CD61+) lineages. In animals that received day 12 expanded cells in the TSF + NTL group, there was a significant increase of human CD45+ cells in the BM (19.3% vs. 11.5%, P = 0.03, n = 15) when compared with those only exposed to TSF, and a trend of increased engraftment in their spleen (P = 0.07, n = 14). Comparison of the complete amino acid sequences of NTL and FRIL (a dicot mannose-binding lectin shown to preserve hematopoietic stem cells, PNAS, 96, 646–650, 1999) showed 10.2% identity and both peptides contain putative functional/structural sites such as those for N-myristoylation, casein kinase II phosphorylation, protein kinase C phosphorylation and N-glycosylation. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34+ cells for transplantation.

Description: Gram-negative bacterial infection is a serious condition in neonates which could lead to septicemic shock, disseminated intravascular coagulation and death. LPS, the cell wall component of Gram-negative bacteria is a potent stimulator of the host immune response system, mediated by binding to toll-like receptor (TLR)-4 and CD14 receptors. Neutrophils are the first line of innate defense which possess antimicrobial and acute inflammatory activities. Neonatal neutrophils, though incompletely characterized, are suggested to be functionally immature and compromised in their bactericidal capacity compared with adults. We performed a comparative genome-wide expression array analysis on purified (〉95%) human cord blood (term delivery) neutrophils upon challenge by LPS for 4 hours at 100 ng/mL. With the criteria of 2-fold differences and statistical significance (P