Differentiation Of Cord Blood CD34 Research Articles

Screening and Identifying Reference Genes for Erythrocyte Production from Cord Blood CD34+ Cells Exposed to Hypoxia.

Cord blood (CB) CD34+ cells have the potential to be used to achieve artificial hematopoiesis because of their ability to expand and differentiate in multiple directions. However, the mechanism and molecular changes underlying such differentiation are still unclear. The differentiation of CB CD34+ cells is generally driven by subtle changes in gene expression. A crucial method for examining gene expression is quantitative real-time polymerase chain reaction, but the accuracy of the results is dependent on the use of reliable reference genes. Here, the transcription levels of 10 novel candidate reference genes (EIF4G2, DYNC1H1, LUC7L3, CD46, POLR1D, WSB1, GAPVD1, HGS, LGALS8, and RBM5) and 8 traditional reference genes (GAPDH, YWHAZ, ACTB, B2MG, TBP, HMBS, PPIA, HPRT1) in CB CD34+ cells under different oxygen concentrations were screened and evaluated by using the geNorm and NormFinder algorithms. Comprehensive analysis conducted by RefFinder online tool showed that TBP (a traditional reference gene) and EIF4G2 (a novel reference gene) had the most stable expression, whereas GAPDH and HMBS were the least suitable reference genes under these conditions. These results may serve as a basis for selecting reference genes with stable expression for more accurate normalization under different oxygen concentration stimulation during CB CD34+ cells differentiation.

Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells.

Understanding physiologic T-cell development from hematopoietic stem (HSCs) and progenitor cells (HPCs) is essential for development of improved hematopoietic cell transplantation (HCT) and emerging T-cell therapies. Factors in the thymic niche, including Notch 1 receptor ligand, guide HSCs and HPCs through T-cell development in vitro. We report that physiologically relevant oxygen concentration (5% O2 , physioxia), an important environmental thymic factor, promotes differentiation of cord blood CD34+ cells into progenitor T (proT) cells in serum-free and feeder-free culture system. This effect is enhanced by a potent reducing and antioxidant agent, ascorbic acid. Human CD34+ cell-derived proT cells in suspension cultures maturate into CD3+ T cells in an artificial thymic organoid (ATO) culture system more efficiently when maintained under physioxia, compared to ambient air. Low oxygen tension acts as a positive regulator of HSC commitment and HPC differentiation toward proT cells in the feeder-free culture system and for further maturation into T cells in the ATO. Culturing HSCs/HPCs in physioxia is an enhanced method of effective progenitor T and mature T-cell production ex vivo and may be of future use for HCT and T-cell immunotherapies.

Human mesenchymal stem cells support B-cell and T-cell differentiation of cord blood CD34+ cells under optimized conditions

In vitro assays for T/B/NK lymphopoietic differentiation could be helpful to assess the differentiation potential of hematopoietic stem cells (HSCs) from patients with immune deficiencies before and after genetic correction. Current protocols involve the use of murine Op9/Op9-DL1 cell lines. However, these protocols are suboptimal for differentiation of human HSCs. NOTCH1 signaling occurs through its ligands Delta-Like 1 (DL1) and is important for T cell differentiation. Here, we developed immortalized cell lines using human bone marrow-derived mesenchymal stem cells (MSCs) and 3rd generation self-inactivating (SIN) lentiviral vectors carrying a codon-optimized gene for human telomerase reverse transcriptase (hTERTco) and eGFP. Subclones were transduced with codon-optimized human hDL1co-Tomato. The hTERTco cell line was used for B-cell differentiation; the hTERTco/hDL1co cell line was used for T-cell differentiation. CD34+ UCB cells were cultured for 6 weeks in presence of differentiation medium containing Flt3 ligand, IL-7 and c-fms inhibitor III, to inhibit M-CSF signaling. Once weekly, 10% of the hematopoietic cells was collected and analyzed, remaining cells were replated on fresh hTERTco or hTERTco/hDL1co feeder layers. After 6 weeks of co-culturing on hTERTco, >20% of cells expressed the B-cell marker CD20. Co-culturing on hTERTco/hDL1co resulted in robust T-cell differentiation (CD3+/CD4+ and CD3+/CD8+ cells) and when stimulated for 4 days with PHA, these cells displayed typical clonal expansion. In conclusion, functional B and T-cell differentiation on human MSC cell lines is feasible under controlled culture conditions.

In vitro differentiated plasmacytoid dendritic cells as a tool to induce anti-leukemia activity of natural killer cells

Acute lymphoblastic leukemia (ALL) is believed to be resistant to NK cell-mediated killing. To overcome this resistance, we developed an innovative approach based on NK cell stimulation with Toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDC). The translation of this approach into the clinic requires the production of high numbers of human pDC. Herein, we show that in vitro differentiation of cord blood CD34+ progenitors in the presence of aryl hydrocarbon receptor antagonists gives rise to clinically relevant numbers of pDC, as about 108 pDC can be produced from a typical cord blood unit. Blocking the aryl hydrocarbon receptor (AHR) pathway significantly increased the yield of pDC. When compared to pDC isolated from peripheral blood, in vitro differentiated pDC (ivD-pDC) exhibited an increased capacity to induce NK cell-mediated killing of ALL. Although ivD-pDC produced lower amounts of IFN-α than peripheral blood pDC upon TLR activation, they produced more IFN-λ2, known to play a critical role in the induction of anti-tumoral NK cell functions. Both TLR-9 and TLR-7 ligands triggered pDC-induced NK cell activation, offering the possibility to use any clinical-grade TLR-7 or TLR-9 ligands in future clinical trials. Finally, adoptive transfer of ivD-pDC cultured in the presence of an AHR antagonist cured humanized mice with minimal ALL disease. Collectively, our results pave the way to clinical-grade production of sufficient numbers of human pDC for innate immunotherapy against ALL and other refractory malignancies.

IL-4 and IL-13 regulate TLR expression and eosinophil-basophil differentiation of cord blood CD34+ progenitor cells

Background Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent allergic disease development [1]. We have previously shown that cord blood (CB) progenitor cells of high atopic risk infants have reduced toll-like receptor (TLR) expression and produce fewer lipopolysaccharide (LPS)-stimulated eosinophil-basophil (Eo/B) colonies, compared to lowatopic risk infants. In the present study, we investigated whether a surrogate ex vivo TH2 milieu (i.e., either IL-4 or IL-13), could represent an underlying mechanism to explain our previous findings.

IL-4 and IL-13 differentially regulate TLR-induced eosinophil-basophil differentiation of cord blood CD34+ progenitor cells.

Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent allergic disease development. We have previously shown that fewer lipopolysaccharide (LPS)-stimulated eosinophil-basophil (Eo/B) colonies grow from cord blood (CB) of high-atopic risk infants, compared to low-atopic risk infants. In the present study, we investigated whether a surrogate ex vivo TH2 milieu (i.e., either IL-4 or IL-13) could represent an underlying mechanism to explain our previous findings. CB CD34+ cells from healthy donors were cultured with IL-4 or IL-13 (in combination with LPS) and assessed for Eo/B differentiation using methylcellulose cultures and flow cytometry for related intracellular signalling pathways. Pharmacological inhibitors were added to the methylcellulose cultures to determine the effect of blocking intracellular signalling in CB CD34+ cells in relation to Eo/B colony forming unit (CFU) formation. Stimulation of CD34+ cells with IL-4, but not IL-13, reduced Eo/B CFU formation in the presence of LPS; this was found to be dependent on IL-4Rα and not IL-13Rα1. Additionally, IL-4 reduced the expression of ERK 1/2 after LPS stimulation, which was recovered by inhibition of IL-4Rα. While IL-13 did not have an inhibitory effect on ERK 1/2 expression, inhibition of ERK 1/2 significantly reduced Eo/B CFU formation. Thus, the responsiveness of CB CD34+ progenitor cells to LPS is differentially regulated by the TH2 cytokines, IL-4 and IL-13. This may have implications for in utero interactions between placental-derived pro-allergic cytokines and neonatal progenitor cells influencing Eo/B-mediated inflammatory responses in early life.

Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells

Hematopoiesis is a complex process regulated by sets of transcription factors in a stage-specific and context-dependent manner. THAP11 is a transcription factor involved in cell growth, ES cell pluripotency, and embryogenesis. Here we showed that THAP11 was down-regulated during erythroid differentiation but up-regulated during megakaryocytic differentiation of cord blood CD34+ cells. Overexpression of THAP11 in K562 cells inhibited the erythroid differentiation induced by hemin with decreased numbers of benzidine-positive cells and decreased mRNA levels of α-globin (HBA) and glycophorin A (GPA), and knockdown of THAP11 enhanced the erythroid differentiation. Conversely, THAP11 overexpression accelerated the megakaryocytic differentiation induced by phorbol myristate acetate (PMA) with increased percentage of CD41+ cells, increased numbers of 4N cells, and elevated CD61 mRNA levels, and THAP11 knockdown attenuated the megakaryocytic differentiation. The expression levels of transcription factors such as c-Myc, c-Myb, GATA-2, and Fli1 were changed by THAP11 overexpression. In this way, our results suggested that THAP11 reversibly regulated erythroid and megakaryocytic differentiation.

IL-4 and IL-13 Differentially Regulate TLR-Induced Eosinophil-Basophil Differentiation Of Cord Blood CD34+ Progenitor Cells

Intrauterine environmental exposures have been shown to influence neonatal immunity and subsequent atopy. The effect of “pro-allergic” TH2 cytokines on neonatal cord blood (CB) hematopoietic progenitor cells, the phenotype and differentiation of which are associated with atopic risk, is currently unknown. Since we have previously shown that high-atopic risk infants have decreased eosinophil-basophil (Eo/B) differentiation after stimulation with LPS, we investigated whether a TH2 milieu (IL-4 or IL-13) could influence LPS-induced Eo/B colony forming units (CFU), and the mechanism(s) through which this might occur.

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.

Detection of a CD4+CD8−CD3− cell subpopulation during the differentiation of cord blood CD34+ cells into T cells in vitro

Umbilical cord blood contains relatively abundant primitive CD34(+) hematopoietic progenitor cells which can differentiate into T lymphocytes ex vivo In this study, thymic stromal cells (TSCs) were isolated from aborted fetuses and a monolayer culture system was established. Highly-purified CD34(+) cells from umbilical cord blood were cultured on the TSCs after limiting-dilution. The cells were then harvested and evaluated for CD4, CD8, and CD3 expression at different time points. CD4(+)CD8(-)CD3(-) lymphoid progenitor cells that could differentiate into mature T lymphocytes were observed after 15 days when a cocktail of cytokines, including Flt-3 ligand, stem cell factor, interleukin (IL)-12, and IL-2, was added. These results thus show that CD4(+)CD8(-)CD3(-) cells can be derived from CD34(+) cells in vitro when cultured on TSCs. We showed that CD4(+)CD8(-)CD3(-) cells can be derived from highly purified CD34(+) cells on TSCs during T-cell lymphopoiesis in vitro.

PANEL REACTIVE ANTIBODY IN THALASSEMIC SERUM INHIBITS PROLIFERATION AND DIFFERENTIATION OF CORD BLOOD CD34+ CELLS IN VITRO

Thalassemic children are at a high risk of graft rejection in cord blood transplantation. To investigate this possible mechanism, the authors evaluated the effect of panel reactive antibody on the growth of CD34+ cells in vitro. On semisolid medium, CD34+ cells derived from cord blood were incubated with thalassemic serum, and colony-forming units were counted after 10 days of culture. After incubation with serum-specific panel reactive antibody, profound decreases were found in the numbers of CFU-GM, CFU-GEMM and BFU-E compared with controls. The results indicated that serum-specific panel reactive antibody might have an apparent inhibition effect on proliferation and differentiation of cord blood CD34+ cells.

Characterization of Monocyte Subpopulations (CD14+CD16− and CD14++CD16+) generated from Cord Blood Haematopoietic Progenitor CD34+

Introduction. In the past decade significant advances in ex vivo expansion of haematopoietic stem cells were made. Protocols of differentiating CD34+ cells into cells from hematopoietic linages, especially dendritic cells, were described. However, little is known about in vitro differentiation of CD34+ cells to monocytes. In peripheral blood there are two main populations of monocytes CD14++CD16− and CD14+CD16+. They represent about 10 percent of total blood monocytes. Recently we have showed that expansion and differentiation of cord blood CD34+ cells to monocytes/macrophages leads to generation of CD14+CD16− and CD14++CD16+ monocyte subpopulations (Stec et al, J Leukoc Biol. 2007).Aim of study. To compare two monocyte subpopulation: CD14+CD16− and CD14++CD16+ obtained from our expansion and differentiation protocol of CD34+ cord blood cells.Materials and methods. After 3–10 days expansion in X-VIVO10 medium with FBS, SCF, IL-3, FLT-3L, TPO and then 7–10 days differentiation in IMDM medium with FBS, M-CSF, FLT-3L, IL-3, SCF, CD14+CD16− and CD14++CD16+ subpopulations were isolated by FACS sorting (FACSVantage). Release of IL-6, IL8, IL-12, IP-10, MIP1 α, MIP-1 β, RANTES, VEGF and FGF after LPS/INFγ or cancer cells stimulation were analysed using CBA (Cytometric Bead Array) method. Cytotoxic activity against cancer cells were assessed by MTT test and migration capacity was assesed with uncoated porous filters (8 μm) in a 24-well Boyden chamber. The adhesion capacity of subpopulations on human umbilical vein endothelial cells (HUVEC) were assessed with 5(6)-CFDA, SE (Molecular Probes) application and fluorescent microscope. NOD-SCID mice were used to evaluate the influence of subpopulations on angiogenesis and tumour growth.Results. CD14++CD16+ monocytes released more IL-8, IL-12, IP-10, MIP1-α, MIP1-β, and RANTES than CD14+CD16− monocytes after LPS/INFγ or cancer cells stimulation. After 24 hours culture in medium without cytokines and than stimulation with LPS/INFγ or cancer cells, CD14++CD16+ subpopulation released more RANTES, TNF, MIP1-α, IL10 than CD14+CD16−cells. CD14+CD16− subpopulation exhibited greater chemotaxis to SDF-1 and MIP1-α than CD14++CD16+ (12,2%±5,5% versus 2,2%±1%). At the same time CD14++CD16+ monocytes showed significantly higher cytotoxic activity against tumour cells in vitro than CD14+CD16− (34,04±17,45% versus 15,56±13,66%). CD14++16+ population showed increased adhesion to HUVEC than CD14+16− population (79±20 cells versus 30±13 cells). In in vivo tests in NODSCID mice both subpopulations together inhibited angiogenesis, but CD14+CD16− subpopulation inhibited stronger tumour growth than CD14++CD16+ subpopulation.Conclusions. Using our expansion protocol it was possible to obtain two subpopulations of monocytes: CD14+CD16− and CD14++CD16+ which showed the differences in the release of cytokines/chemokines following stimulation with LPS/INFγ or tumour cells, chemotactic and antiangiogenic activity and cytotoxicity and are clearly distinct from known main subpopulations of blood monocytes CD14+CD16+ and CD14++. Additional tests, which are still in progress in our lab will provide evidence if both populations are functional monocytes.

Effects of Rosa roxburghii Extract on Proliferation and Differentiation in Human Hepatoma SMMC-7721 Cells and CD34+ Haematopoietic Cells

Rosa roxburghii Tratt is an herbal medicine with anticancer potential. This study investigated the effects of an ethanol extracts and a triterpene (CiLi triterpene) of Rosa roxburghii on proliferation and differentiation of human hepatoma SMMC-7721 cells and in umbilical cord blood CD34+ hematopoietic progenitor cells. The CiLi triterpene and ethanol extracts inhibited the proliferation of hepatoma cells in a concentration- and time-dependent manner and decreased the release of alpha-fetoprotein from hepatoma cells. Apoptosis was increased only at the highest dose of the ethanol extract in hepatoma cells. The Cili triterpene and ethanol extracts of Rosa roxburghii did not affect the differentiation of cord blood CD34+ cells to granulocytes and monocytes, as evidenced by flow cytometry analysis of CD11b and CD15. Thus the Cili triterpene and ethanol extracts of Rosa roxburghii are effective in the inhibition of human hepatoma SMMC-7721 cell growth, without affecting the differentiation of CD34+ cells.

Retinoic acid modulates IL-5 receptor expression and selectively inhibits eosinophil-basophil differentiation of hemopoietic progenitor cells

Background: IL-5 plays a central role in eosinophil and basophil differentiation, exerting its effects through the IL-5 receptor (IL-5Rα). Currently, little is known concerning regulation of IL-5Rα expression in the context of commitment of hemopoietic progenitor cells to the eosinophil and basophil lineages. Objective: Because all-trans retinoic acid (ATRA) is known to modulate some aspects of hemopoietic differentiation, we examined the effects of ATRA on eosinophil-basophil differentiation and IL-5Rα expression. Methods: Progenitor cells were obtained from bone marrow aspirates and cord blood samples. Enriched populations of CD34+ cells were isolated by means of positive immunomagnetic selection with MACS beads. Results: In semisolid methylcellulose cultures of normal human bone marrow, ATRA (10−6 mol/L) selectively suppressed eosinophil-basophil colony-forming units but had no effect on granulocyte-macrophage colony-forming units. Similarly, ATRA (10−6 mol/L) inhibited eosinophil-basophil differentiation of cord blood CD34+ cells in liquid culture, whereas neutrophil differentiation proceeded without impediment. Most importantly, these effects of ATRA (10−8 to 10−6 mol/L) on CD34+ cells were associated with a dose-dependent inhibition of IL-5Rα but no change in GM-CSF receptor expression, as detected with flow cytometry. Conclusions: These findings indicate that retinoids can differentially regulate expression of IL-5Rα, but not GM-CSF receptor, and that these effects have functional consequences in vitro on eosinophil and basophil differentiation. (J Allergy Clin Immunol 2002;109:307-13.)

Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow

AbstractHuman mesenchymal stem/progenitor cells (MSCs) have been identified in adult bone marrow, but little is known about their presence during fetal life. MSCs were isolated and characterized in first-trimester fetal blood, liver, and bone marrow. When 106 fetal blood nucleated cells (median gestational age, 10+2 weeks [10 weeks, 2 days]) were cultured in 10% fetal bovine serum, the mean number (± SEM) of adherent fibroblastlike colonies was 8.2 ± 0.6/106 nucleated cells (69.6 ± 10/μL fetal blood). Frequency declined with advancing gestation. Fetal blood MSCs could be expanded for at least 20 passages with a mean cumulative population doubling of 50.3 ± 4.5. In their undifferentiated state, fetal blood MSCs were CD29+, CD44+, SH2+, SH3+, and SH4+; produced prolyl-4-hydroxylase, α-smooth muscle actin, fibronectin, laminin, and vimentin; and were CD45−, CD34−, CD14−, CD68−, vWF−, and HLA-DR−. Fetal blood MSCs cultured in adipogenic, osteogenic, or chondrogenic media differentiated, respectively, into adipocytes, osteocytes, and chondrocytes. Fetal blood MSCs supported the proliferation and differentiation of cord blood CD34+cells in long-term culture. MSCs were also detected in first-trimester fetal liver (11.3 ± 2.0/106 nucleated cells) and bone marrow (12.6 ± 3.6/106 nucleated cells). Their morphology, growth kinetics, and immunophenotype were comparable to those of fetal blood-derived MSCs and similarly differentiated along adipogenic, osteogenic, and chondrogenic lineages, even after sorting and expansion of a single mesenchymal cell. MSCs similar to those derived from adult bone marrow, fetal liver, and fetal bone marrow circulate in first-trimester human blood and may provide novel targets for in utero cellular and gene therapy.

Regulation of IL-5 Receptor on Eosinophil Progenitors in Allergic Inflammation: Role of Retinoic Acid

We and others have shown that IL-5 plays a central role in eosinophil and basophil differentiation, exerting its effects through the IL-5 receptor (IL-5R). Little is currently known concerning regulation of IL-5Rα gene transcription in the context of commitment of hemopoietic progenitor cells to the eosinophil and basophil lineages; recent studies have indicated that IL-5 itself can regulate IL-5Rα expression on mature eosinophils. We now provide evidence to indicate that IL-5 can upregulate IL-5Rα on bone marrow CD34+ progenitors in vitro, as we have demonstrated in vivo in atopic asthmatics. Given that all-trans retinoic acid (ATRA) is known to modulate some granulopoiesis, causing neutrophilic differentiation, we examined the effects of ATRA on eosinophil/basophil differentiation and IL-5Rα expression. In cultures of normal human bone marrow, ATRA selectively suppressed eosinophil/basophil differentiation. Similarly, ATRA inhibited eosinophil/basophil differentiation of cord blood CD34+ cells, while neutrophil differentiation proceeded without impediment. Most importantly, these effects of ATRA on CD34+ cells were associated with selective, dose-dependent inhibition of membrane-bound IL-5Rα, upregulation of soluble IL-5Rα transcription, but no change in GM-CSF receptor expression. These findings indicate that retinoids can differentially regulate membrane and soluble isoforms of IL-5Rα, and that these effects have functional consequences in vitro on eosinophil and basophil differentiation. ATRA may be of therapeutic benefit in allergic inflammatory disorders in which eosinophil differentiation and membrane-bound IL-5R are upregulated.