Exosomes released from Shiga toxin 2a–treated human macrophages modulate inflammatory responses and induce cell death in toxin receptor expressing human cells

Shiga toxins (Stxs) produced by Stx‐producing Escherichia coli are the primarily virulence factors of hemolytic uremic syndrome and central nervous system (CNS) impairment. Although the precise mechanisms of toxin dissemination remain unclear, Stxs bind to extracellular vesicles (EVs). Exosomes, a subset of EVs, may play a key role in Stx‐mediated renal injury. To test this hypothesis, we isolated exosomes from monocyte‐derived macrophages in the presence of Stx2a or Stx2 toxoids. Macrophage‐like differentiated THP‐1 cells treated with Stxs secreted Stx‐associated exosomes (Stx‐Exo) of 90–130 nm in diameter, which induced cytotoxicity in recipient cells in a toxin receptor globotriaosylceramide (Gb₃)‐dependent manner. Stx2‐Exo engulfed by Gb₃‐positive cells were translocated to the endoplasmic reticulum in the human proximal tubule epithelial cell line HK‐2. Stx2‐Exo contained pro‐inflammatory cytokine mRNAs and proteins and induced more severe inflammation than purified Stx2a accompanied by greater death of target cells such as human renal or retinal pigment epithelial cells. Blockade of exosome biogenesis using the pharmacological inhibitor GW4869 reduced Stx2‐Exo‐mediated human renal cell death. Stx2‐Exo isolated from human primary monocyte–derived macrophages activated caspase 3/7 and resulted in significant cell death in primary human renal cortical epithelial cells. Based on these results, we speculate that Stx‐containing exosomes derived from macrophages may exacerbate cytotoxicity and inflammation and trigger cell death in toxin‐sensitive cells. Therapeutic interventions targeting Stx‐containing exosomes may prevent or ameliorate Stx‐mediated acute vascular dysfunction.     

Epidermal growth factor regulates the development of stem and progenitor Leydig cells in rats

Epidermal growth factor (EGF) has many physiological roles. However, its effects on stem and progenitor Leydig cell development remain unclear. Rat stem and progenitor Leydig cells were cultured with different concentrations of EGF alone or in combination with EGF antagonist, erlotinib or cetuximab. EGF (1 and 10 ng/mL) stimulated the proliferation of stem Leydig cells on the surface of seminiferous tubules and isolated CD90⁺ stem Leydig cells and progenitor Leydig cells but it blocked their differentiation. EGF also exerted anti‐apoptotic effects of progenitor Leydig cells. Erlotinib and cetuximab are able to reverse EGF‐mediated action. Gene microarray and qPCR of EGF‐treated progenitor Leydig cells revealed that the down‐regulation of steroidogenesis‐related proteins (Star and Hsd3b1) and antioxidative genes. It was found that EGF acted as a proliferative agent via increasing phosphorylation of AKT1. In conclusion, EGF stimulates the proliferation of rat stem and progenitor Leydig cells but blocks their differentiation.     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

Inhibition of MEK/ERK signalling pathway promotes erythroid differentiation and reduces HSCs engraftment in ex vivo expanded haematopoietic stem cells

The MEK/ERK pathway is found to be important in regulating different biological processes such as proliferation, differentiation and survival in a wide variety of cells. However, its role in self‐renewal of haematopoietic stem cells is controversial and remains to be clarified. The aim of this study was to understand the role of MEK/ERK pathway in ex vivo expansion of mononuclear cells (MNCs) and purified CD34⁺ cells, both derived from human umbilical cord blood (hUCB). Based on our results, culturing the cells in the presence of an inhibitor of MEK/ERK pathway—PD0325901 (PD)—significantly reduces the expansion of CD34⁺ and CD34⁺ CD38^? cells, while there is no change in the expression of stemness‐related genes (HOXB4, BMI1). Moreover, in vivo analysis demonstrates that PD reduces engraftment capacity of ex vivo expanded CD34⁺ cells. Notably, when ERK pathway is blocked in UCB‐MNCs, spontaneous erythroid differentiation is promoted, found in concomitant with increasing number of burst‐forming unit‐erythroid colony (BFU‐E) as well as enhancement of erythroid glycophorin‐A marker. These results are in total conformity with up‐regulation of some erythroid enhancer genes (TAL1, GATA2, LMO2) and down‐regulation of some erythroid repressor genes (JUN, PU1) as well. Taken together, our results support the idea that MEK/ERK pathway has a critical role in achieving the correct balance between self‐renewal and differentiation of UCB cells. Also, we suggest that inhibition of ERK signalling could likely be a new key for erythroid induction of UCB‐haematopoietic progenitor cells.     

Monoclonal antibodies against Stx1B subunit of Escherichia coli O157:H7 distinguish the bacterium from other bacteria

Aims: The Shiga‐like toxins (Stx) are critical virulence factors of enterohaemorrhagic Escherichia coli (EHEC). Stx1B subunit plays important roles in EHEC infection. This work aims to generate and characterize monoclonal antibodies (mAbs) against the Stx1B and to investigate their utility in discrimination ELISA. Methods and Results: Two newly identified mAbs (designated 2H8 and 1B10, respectively) against the Stx1B protein were prepared via hybridoma techniques. The immunoreactivity of both mAbs to the Stx1B protein was confirmed in ELISA and Western blot. Moreover, they differentiate EHEC from Salmonella enteritis, non‐Stx1‐producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae and Staphylococcus aureus. Conclusions: The anti‐STx1B mAbs are valuable diagnostic reagents for distinguishing EHEC from other bacteria. Significance and Impact of the Study: This is the first report regarding the usage of anti‐STx1B mAbs in discrimination ELISA. The established ELISA may have potential in clinical surveillance of EHEC infection.     

Foe turned friend: multiple functional roles attributable to hyper-activating stem cell factor receptor mutant in regeneration of the haematopoietic cell compartment

Objectives: Stem cell factor receptor, c‐kit, is considered to be the master signalling molecule of haematopoietic stem cells. It develops the orchestral pattern of haematopoietic cell lineages, seen by its varying degree of omnipresence in progenitors, lineage committed and mature cells. We have investigated the effect of over‐expressing c‐kit on early recovery of the haematopoietic compartment, in irradiated hosts. Materials and methods: Normal bone marrow cells (BMCs) were transfected with Kit_wt (wild‐type c‐kit) or its variant Kit_mu (asp814tyr) by electroporation. Lethally irradiated mice were transplanted with normal or transfected congeneic BMCs. The effect of ectopic expression of c‐kit on haematopoietic cell recovery was determined by analysing donor‐derived cells. Furthermore, effects of both types of c‐kit over‐expression on progenitor and lineage‐committed cells were examined by flow cytometric analysis of Sca‐1 and lineage‐committed (Lin⁺) cells respectively. Results: Hyper‐activating Kit_mu significantly improved recovery of the haematopoietic system in irradiated hosts. In vivo results showed that the donor‐derived c‐kit⁺ cell population was increased to more than 3‐fold in the case of Kit_mu‐transfected cells compared to normal and Kit_wt over‐expressing BMCs. In general, survival of progenitor and committed cell was improved in the Kit_mu over‐expressing system compared to the other two cohorts. Conclusion: These results suggest that recruitment of the hyper‐activating variant of c‐kit (Kit_mu) lead to early recovery of the bone marrow of lethally irradiated mice.     

Candida albicans stimulates in vivo differentiation of haematopoietic stem and progenitor cells towards macrophages by a TLR2‐dependent signalling

Toll‐like receptors (TLRs) are expressed by haematopoietic stem and progenitor cells (HSPCs), and may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that (i) inactivated yeasts of Candida albicans induce in vitro differentiation of HSPCs towards the myeloid lineage, and (ii) soluble TLR agonists induce in vivo their differentiation towards macrophages. In this work, using an in vivo model of HSPCs transplantation, we report for the first time that HSPCs sense C. albicans in vivo and subsequently are directed to produce macrophages by a TLR2‐dependent signalling. Purified lineage‐negative cells (Lin^?) from bone marrow of C57BL/6 mice (CD45.2 alloantigen) were transplanted into B6Ly5.1 mice (CD45.1 alloantigen), which were then injected with viable or inactivated C. albicans yeasts. Transplanted cells were detected in the spleen and in the bone marrow of recipient mice, and they differentiate preferentially to macrophages, both in response to infection or in response to inactivated yeasts. The generation of macrophages was dependent on TLR2 but independent of TLR4, as transplanted Lin^? cells from TLR2^?/? mice did not give rise to macrophages, whereas Lin^? cells from TLR4^?/? mice generated macrophages similarly to control cells. Interestingly, the absence of TLR2, or in a minor extent TLR4, gives Lin^? cells an advantage in transplantation assays, as increases the percentage of transplanted recovered cells. Our results indicatethat TLR‐mediated recognition of C. albicans by HSPCs may help replace and/or increase cells that constitute the first line of defence against the fungus, and suggest that TLR‐mediated signalling may lead to reprogramming early progenitors to rapidly replenishing the innate immune system and generate the most necessary mature cells to deal with the pathogen.     

Induction of foetal haemoglobin synthesis in erythroid progenitor stem cells: mediated by water‐soluble components of Terminalia catappa

Current novel therapeutic agents for the treatment of sickle cell anaemia (SCA) focus on increasing foetal haemoglobin (HbF) levels in SCA patients. Unfortunately, the only approved HbF‐inducing agent, hydroxyurea, has long‐term unpredictable side effects. Studies have shown the potential of plant compounds to modulate HbF synthesis in primary erythroid progenitor stem cells. We isolated a novel HbF‐inducing Terminalia catappa distilled water active fraction (TCDWF) from Terminalia catappa leaves that induced the commitment of erythroid progenitor stem cells to the erythroid lineage and relatively higher HbF synthesis of 9.2‐ and 6.8‐fold increases in both erythropoietin (EPO)‐independent and EPO‐dependent progenitor stem cells respectively. TCDWF was differentially cytotoxic to EPO‐dependent and EPO‐independent erythroid progenitor stem cell cultures as revealed by lactate dehydrogenase release from the cells. TCDWF demonstrated a protective effect on EPO‐dependent and not EPO‐independent progenitor cells. TCDWF induced a modest increase in caspase 3 activity in EPO‐independent erythroid progenitor stem cell cultures compared with a significantly higher (P?0.05) caspase 3 activity in EPO‐dependent ones. The results demonstrate that TCDWF may hold promising HbF‐inducing compounds, which work synergistically, and suggest a dual modulatory effect on erythropoiesis inherent in this active fraction. Copyright © 2014 John Wiley & Sons, Ltd.     

Enterohemorrhagic Escherichia coli trivalent recombinant vaccine containing EspA, intimin and Stx2 induces strong humoral immune response and confers protection in mice

Enterohemorrhagic Escherichia coli (EHEC) is an important food-borne pathogen, which causes a wide spectrum of diseases ranging from hemorrhagic colitis to life-threatening hemolytic uremic syndrome (HUS). Currently, insufficient measures to prevent and treat EHEC infection make a vaccine against EHEC in great demand. EspA (E. coli secreted protein A), intimin, and Stx2 (Shiga toxin 2) are three predominant virulence factors of EHEC, and each of them has proved to be capable of inducing partial protective immunity. In this study, we constructed a trivalent recombinant protein designated EIS that is composed of EspA (E), C-terminal 300 amino acids of intimin (I) and B subunit of Stx2 (S), and tested it as vaccine using a mouse model. Our results showed that immunization of EIS induced strong humoral response to EspA, intimin and Stx2 and protected mice against the challenges with live EHEC or EHEC sonicated lysate. Moreover, it enhanced clearance of intestinally colonized bacteria. This work suggests that for EHEC vaccines using a combination of EspA, intimin and Stx2 antigens appears to be more effective than using any of these immunogens alone.     

CD45+/CD133+ positive cells expanded from umbilical cord blood expressing PDX‐1 and markers of pluripotency

UCB (human umbilical cord blood) contains cells able to differentiate into non‐haematopoietic cell lineages. It also contains cells similar to primitive ESCs (embryonic stem cells) that can differentiate into pancreatic‐like cells. However, few data have been reported regarding the possibility of expanding these cells or the differential gene expression occurring in vitro. In this study, we expanded formerly frozen UCB cells by treatment with SCF (stem cell factor) and GM‐CSF (granulocyte–macrophage colony stimulating factor) in the presence of VPA (valproic acid). Gene expression profiles for beta cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT‐PCR and immunocytochemistry. The results show a dramatic expansion (>150‐fold) of haematopoietic progenitors (CD45⁺/CD133⁺) which also expressed embryo markers of pluripotency (nanog, kfl‐4, sox‐2, oct‐3/4 andc‐myc), nestin, and pancreatic markers such as pax‐4, ngn‐3, pdx‐1 and syt‐1 (that is regulated by pdx‐1 and provides the cells with a Ca⁺⁺ regulation mechanism essential for insulin exocytosis). Our results show that UCB cells can be expanded to produce large numbers of cells of haematopoietic lineage that naturally (without the need of retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Further investigations are necessary to clarify, first, whether in this context, the embryogenes expressed are functional or not, and secondly, since these cells are safer than cells transfected with retroviral vectors or transposons, whether they would represent a potential tool for clinical application.     

Enterohaemorrhagic Escherichia coli O157:H7 Shiga‐like toxin 1 is required for full pathogenicity and activation of the p38 mitogen‐activated protein kinase pathway in Caenorhabditis elegans

Enterohaemorrhagic Escherichia coli (EHEC) causes life‐threatening infections in humans as a consequence of the production of Shiga‐like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga‐like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen‐activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1‐dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

Characterization of the hemopoietic defect in early stages of the myelodysplastic syndromes

Myelodysplasia (MDS) is a heterogeneous disorder characterised by bone marrow failure and progression to acute myeloid leukaemia where the molecular and cellular haematopoietic defects are poorly understood. To gain insight into this the pathogenesis of this condition, we analyzed gene expression profiles of bone marrow CD34⁺ stem/progenitor cells from patients with MDS at a early stage in the disease and compared them with profiles from CD34⁺ cells from age-matched controls and patients with non-MDS anaemia. Given the heterogeneity of early MDS, a surprisingly consistent finding was decreased expression of B-cell lineage affiliated genes in MDS patients compared to normal controls and samples with non-MDS anaemia. These findings were then confirmed in the original samples and further samples from a new MDS patient group by Taqman Real Time PCR. Flow cytometry on unfractionated marrow from independent samples also demonstrated reduced B-cell progenitors in MDS patients compared to normal controls. These novel findings suggest a common perturbation in early MDS haematopoiesis. They also provide the rationale for a larger study to evaluate the diagnostic utility of reduced B-cell progenitor number as a diagnostic biomarker of early low risk MDS which can pose a diagnostic challenge.     

Protein profile of basal prostate epithelial progenitor cells—stage‐specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo

The long‐term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin^?Sca‐1⁺ CD49f⁺ Trop2^high‐phenotype) and human (Lin^? CD49f⁺ TROP2^high) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti‐human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single‐cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f⁺/TROP2^high phenotype of basal prostate progenitor cells and characterized by in vivo sandwich‐transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9⁺/CD24⁺/CD29⁺/CD44⁺/CD47⁺/CD49f⁺/CD104⁺/CD147⁺/CD326⁺/Trop2^high of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan‐1 and stage‐specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f⁺ TROP2⁺ basal prostate progenitor cells. Transplantation experiments suggest that CD49f⁺ TROP2^high SSEA‐4^high human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f⁺ TROP2^high or CD49f⁺ TROP2^high SSEA‐4^low cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA‐4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage.     

Hemopoietic progenitor cells with limited potential for differentiation: erythropoietic function of mouse marrow "lymphocytes"

Filtration of mouse marrow cell suspensions over columns of glass wool increased the frequency of small and medium-sized lymphocytes (SML) and of erythropoietic progenitor units (EPU) by about the same factor. Identical results were obtained when erythropoiesis was assayed by isotope uptake (⁵⁹FeCl₃ and ¹²⁵IUdR) or by the spleen-colony techniques. Transfusion of prospective donor mice with erythrocytes virtually eliminated morphologically recognizable erythroid cells from marrow without affecting the frequency of EPU. Injection of prospective donors with cortisol decreased the frequency of SML in marrow but not that of EPU or erythropoietin-sensitive cells. However, glass wool filtration of lymphocyte-poor marrow taken from mice pretreated with cortisol resulted in a similar increase in frequency of residual SML and of EPU. Therefore, it appears that a subpopulation of marrow SML are EPU. Whereas glass wool filtration increased the frequency of erythropoietic progenitor and colony-forming units, the filtration failed to change the frequency of leukopoietic progenitor or colony-forming units (assayed in mice hypertransfused with erythrocytes to suppress erythropoiesis). It follows that separate progenitor cells for erythropoiesis and leukopoiesis are present in bone marrow of adult mice, in addition to pluripotent stem cells.     

Chromosomal stability during ex vivo expansion of UCB CD34(+) cells

Objectives: Ex vivo expansion is a feasible strategy, which may overcome limitation of the very low frequency of haematopoietic stem/progenitor cells, in umbilical cord blood (UCB). However, both quality of cells and safety of expanded population are critical issues to be addressed for their clinical application. Hence, in this study, we evaluated genetic stability of UCB‐derived CD34⁺ cells during ex vivo culture, based on karyotype analysis, as well as its effect on cell proliferation characteristics. Materials and methods: CD34⁺ cells were isolated from human UCB samples by immunomagnetic separation and were expanded ex vivo over a 28‐day period. Expansion of total nucleate cells, CD34⁺ cells and CD34⁺ CD38^? cells was investigated. Karyotype analysis of the expanded cells from six randomly selected UCB samples was performed to evaluate their genetic stability. Results: Chromosomal abnormality of expanded cells mainly appeared by day 14, but was seldom sustained until day 28. None of the chromosomal abnormal samples displayed neoplastic proliferation, and expanded cells with altered chromosomes did not show obvious transformation phenomena according to soft agar assay. Conclusions: Ex vivo expansion could lead to occurrence of chromosomal abnormality, although here it did not produce excessive proliferative advantage of the expended cells. Importantly, chromosomal alteration seemed not to be inheritable and unlikely to result in malignant transformation. However, further in‐depth evaluation of potential clinical risks of chromosomal abnormality is warranted.     

Erythropoiesis Suppression Is Associated with Anthrax Lethal Toxin-Mediated Pathogenic Progression

Anthrax is a disease caused by the bacterium Bacillus anthracis, which results in high mortality in animals and humans. Although some of the mechanisms are already known such as asphyxia, extensive knowledge of molecular pathogenesis of this disease is deficient and remains to be further investigated. Lethal toxin (LT) is a major virulence factor of B. anthracis and a specific inhibitor/protease of mitogen-activated protein kinase kinases (MAPKKs). Anthrax LT causes lethality and induces certain anthrax-like symptoms, such as anemia and hypoxia, in experimental mice. Mitogen-activated protein kinases (MAPKs) are the downstream pathways of MAPKKs, and are important for erythropoiesis. This prompted us to hypothesize that anemia and hypoxia may in part be exacerbated by erythropoietic dysfunction. As revealed by colony-forming cell assays in this study, LT challenges significantly reduced mouse erythroid progenitor cells. In addition, in a proteolytic activity-dependent manner, LT suppressed cell survival and differentiation of cord blood CD34⁺-derived erythroblasts in vitro. Suppression of cell numbers and the percentage of erythroblasts in the bone marrow were detected in LT-challenged C57BL/6J mice. In contrast, erythropoiesis was provoked through treatments of erythropoietin, significantly ameliorating the anemia and reducing the mortality of LT-treated mice. These data suggested that suppressed erythropoiesis is part of the pathophysiology of LT-mediated intoxication. Because specific treatments to overcome LT-mediated pathogenesis are still lacking, these efforts may help the development of effective treatments against anthrax.     

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.