scholarly journals Release from Quiescence of Primitive Human Hematopoietic Stem/Progenitor Cells by Blocking Their Cell-Surface TGF-β Type II Receptor in a Short-Term In Vitro Assay

Stem Cells ◽  
2000 ◽  
Vol 18 (2) ◽  
pp. 102-111 ◽  
Author(s):  
Nicolas Fortunel ◽  
Jacques Hatzfeld ◽  
Sergueï Kisselev ◽  
Marie-Noëlle Monier ◽  
Karin Ducos ◽  
...  
Keyword(s):  
Cell Surface ◽  
Progenitor Cells ◽  
In Vitro Assay ◽  
Type Ii ◽  
Short Term ◽  
Vitro Assay ◽  
Hematopoietic Stem

scholarly journals Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells

2020 ◽  
Vol 90 ◽  
pp. 52-64.e11 ◽  
Author(s):  
Hanyang Lin ◽  
Jackie E. Damen ◽  
Marta A. Walasek ◽  
Stephen J. Szilvassy ◽  
Ali G. Turhan ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Serum Free

scholarly journals Dietary Preference of Newly Weaned Pigs and Nutrient Interactions According to Copper Levels and Sources with Different Solubility Characteristics

Animals ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1133 ◽  
Author(s):  
Sandra Villagómez-Estrada ◽  
José Francisco Pérez ◽  
Sandra van Kuijk ◽  
Diego Melo-Durán ◽  
Razzagh Karimirad ◽  
...  
Keyword(s):  
Phytic Acid ◽  
Feeding Preference ◽  
In Vitro Assay ◽  
Nutrient Interactions ◽  
Short Term ◽  
Vitro Assay ◽  
Dietary Preference ◽  
Weaning Pigs ◽  
Weaned Pig

Two feeding preference experiments and an in vitro assay were performed to assess the weaned pig preference for Cu doses and sources based on their sensorial perception and on the likely post-ingestive effects of Cu. At day 7 post-weaning, a total of 828 pigs were distributed into two different experiments. In Exp.1 (dose preference) a diet with a nutritional Cu level (15 mg/kg) of Cu sulfate (SF) was pair offered with higher Cu levels (150 mg/kg) of either SF or hydroxychloride (HCl). In Exp.2 (source preference), a diet supplemented with Cu-SF at 150 mg/kg was compared to a Cu-HCl (150 mg/kg) diet. At the short-term (day 7–9) and for the entire experimental week (day 7–14), pigs preferred diets with a high Cu level than with Cu at a nutritional dose (p < 0.05). Likewise, pigs preferred diets supplemented with a Cu-HCl source compared to diets with Cu-SF (p < 0.05). In vitro assay results showed a greater solubility and interaction of Cu-SF with phytic acid compared to Cu-HCl. In conclusion, pigs chose diets with higher levels of Cu probably to re-establish homeostasis after weaning. Pigs preferred diets with Cu-HCl compared to Cu-SF probably due to their solubilities and chemical differences.

Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P&lt;0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

scholarly journals A novel micronucleus in vitro assay utilizing human hematopoietic stem cells

2015 ◽  
Vol 29 (7) ◽  
pp. 1897-1905 ◽  
Author(s):  
N. Kotova ◽  
N. Hebert ◽  
E.-L. Härnwall ◽  
D. Vare ◽  
C. Mazurier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Hematopoietic Stem

Novel Double Labeled Mixed Lymphocyte in Vitro Assay to Predict the Dominant Graft in 2 Unit UCB Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4699-4699
Author(s):  
Shicheng Yang ◽  
Xiao Huang ◽  
Hongyan Lu ◽  
Amandeep Salhotra ◽  
Alexander Wendling ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
In Vitro Assay ◽  
Proliferation Index ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Ifn Γ

Abstract Abstract 4699 Introduction: Umbilical cord blood cells (UCB) from allogeneic donors have been established as an alternative source for HSC transplantation in patients who lack suitably HLA matched bone marrow or peripheral blood stem cells from adult donors. Transplantation using 2 unit UCB has been shown to compensate the low engraftment and slow hematopoietic recovery resulting from 1 unit UCB transplantation in full stature adult patients. At present, there are no unit specific factors that reliably predicts for the “winning unit” in 2 unit UCB transplantation, e.g. cell viability, number of infused total nucleated cells, CD34+ or CD3+ cells, sex mismatch, ABO blood group, and degree of HLA mismatch. In vivo mouse models suggest that CD34 negative subsets play an important role. Among CD34 negative subsets, CD8 T subset accounts for approximately 34.0+/−23.3% of T lymphocytes from UCB. In bone marrow transplantation CD8 T cells have been found to facilitate donor hematopoietic cell engraftment. Moreover, it has been reported that 1 dominant unit coincides with a specific CD8 T cell response against the non-engrafted unit which was not observed from CD4 or NK cells. Methods: In this study, we used volunteer donated UCB research units (kindly provided by P. Rubinstein, MD, New York Blood Center). Mononuclear cells (MNC) were purified by Ficoll gradient centrifugation, and CD3 T cells were isolated with CD3 MicroBeads (Miltenyi Biotec; autoMACS). The purified CD3 (confirmed by FACS >95% purity) cells were labeled with CFSE and DDAO-SE. After labeling, the cells from two different donors were mixed in 96-well U-bottom plates for continued culture in 37 °C 5% CO2. The expansion from each labeled donor cells was evaluated using flow cytometry; the dead cells were gated out using propidium iodide, and the data was analyzed using FlowJo software. For proper T cells activation, we also compared different activation conditions using i.) anti-CD3/CD28 Beads, ii.) anti-CD3 antibody plus anti-CD28 antibody, and iii.) cytokine IL-2. The schematic illustration of methods is shown in Figure 1. Results and discussion: We noted that T cells from UCB are primarily at naïve stage as determined by CD45RA (93.8 +/− 7.11%) and CCR7 (84.9 +/− 12.0%) expression. We also determined the optimal activation condition using a modified mixed lymphocyte reaction from 2 UCB units. Four days after incubation, the proliferation from 2 units labeled with CFSE and DDAO-SE could be reproducibly distinguished using FL1 channel for CFSE and FL4 channel for DDAO-SE (Figure 1). The optimal concentration for labeling using CFSE (1 mM) and DDAO (1 μM or 3 mM) was determined by titration. To avoid cell toxicity resulting from CFSE and DDAO-SE labeling, as well as self-crossing from each donor using two dyes, we examined additional mixed lymphocyte analyses in which each donor was labeled with CFSE or DDAO-SE respectively and vice versa. As shown in Figure 1, we found consistently that the predicated dominant unit accounted for the majority of culture (73.2% stained with DDAO; 63.5% stained with CFSE) after 4 days co-culture. The dominance was not correlated with cell proliferation indicated by the proliferation index (1.12 for dominant and 1.48 for another unit). After confirmation of this in vitro assay, further studies were conducted to evaluate the IFN-γ release of 2 UCB units in this optimized mixed lymphocyte assay in the condition using cytokine IL-2. Interestingly, we could only detect IFN-γ by intracellular staining in one unit when co-culture was set-up using CD3 T cells from each unit; the expression of IFN-γ was not detected when we used CD3 T cells from 1 unit. The correlation between dominance and the expression of IFN-γ is currently under investigation. Conclusion: UCB Transplantation is an important alternative for patients lacking bone marrow or peripheral blood stem cell donors. With the establishment of this novel modified mixed lymphocyte in vitro assay for prediction of the “winning” immune dominant unit, routine analyses can be performed to guide unit selection. Further interventions can be exploited to preferentially treat the expected dominant unit with glycosylation, cytokines, prostaglandins, or C3a compliments to further enhance hematopoietic stem cells trafficking and engraftment to the marrow. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The effect of oxygen tension on the in vitro assay of human osteoblastic connective tissue progenitor cells

2008 ◽  
Vol 26 (10) ◽  
pp. 1390-1397 ◽  
Author(s):  
Sandra M. Villarruel ◽  
Cynthia A. Boehm ◽  
Mark Pennington ◽  
Jason A. Bryan ◽  
Kimerly A. Powell ◽  
...  
Keyword(s):  
Connective Tissue ◽  
Progenitor Cells ◽  
Oxygen Tension ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Effect Of Oxygen ◽  
Connective Tissue Progenitor Cells

scholarly journals Heptad Repeat-Derived Peptides Block Protease-Mediated Direct Entry from the Cell Surface of Severe Acute Respiratory Syndrome Coronavirus but Not Entry via the Endosomal Pathway

2007 ◽  
Vol 82 (1) ◽  
pp. 588-592 ◽  
Author(s):  
Makoto Ujike ◽  
Hiroki Nishikawa ◽  
Akira Otaka ◽  
Naoki Yamamoto ◽  
Norio Yamamoto ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Cell Surface ◽  
Inhibitory Effect ◽  
In Vitro Assay ◽  
Heptad Repeat ◽  
Vitro Assay ◽  
Strong Inhibitory Effect ◽  
Endosomal Pathway ◽  
Heptad Repeats

ABSTRACT The peptides derived from the heptad repeat (HRP) of severe acute respiratory syndrome coronavirus (SCoV) spike protein (sHRPs) are known to inhibit SCoV infection, yet their efficacies are fairly low. Recently our research showed that some proteases facilitated SCoV's direct entry from the cell surface, resulting in a more efficient infection than the previously known infection via endosomal entry. To compare the inhibitory effect of the sHRP in each pathway, we selected two sHRPs, which showed a strong inhibitory effect on the interaction of two heptad repeats in a rapid and virus-free in vitro assay system. We found that they efficiently inhibited SCoV infection of the protease-mediated cell surface pathway but had little effect on the endosomal pathway. This finding suggests that sHRPs may effectively prevent infection in the lungs, where SCoV infection could be enhanced by proteases produced in this organ. This is the first observation that HRP exhibits different effects on virus that takes the endosomal pathway and virus that enters directly from the cell surface.

BEHAVIOURAL ACTION OF ANDROGEN IN THE DOVE: EFFECTS OF LONG-TERM CASTRATION ON RESPONSE SPECIFICITY AND BRAIN AROMATIZATION

1981 ◽  
Vol 90 (2) ◽  
pp. 167-178 ◽  
Author(s):  
J. B. HUTCHISON ◽  
TH. STEIMER ◽  
R. DUNCAN
Keyword(s):  
Preoptic Area ◽  
In Vitro Assay ◽  
Aromatase Activity ◽  
Male Courtship ◽  
Short Term ◽  
Vitro Assay ◽  
Vocal Behaviour ◽  
Response Specificity

Differences in the effectiveness of oestradiol-17β and testosterone on male courtship and vocal behaviour were examined in long-term castrated doves. Nest-orientated behaviour was restored by intramuscular injection of oestradiol-17β. Testosterone was effective in restoring aggressive courtship and vocal behaviour, but not for the nest-orientated behaviour. The effects of these hormones were separable, therefore, under conditions of prolonged androgen deficit, suggesting differences in their specificity of action. In-vitro assay of brain enzyme activity indicated that aromatization of testosterone to oestradiol-17β occurred in the preoptic area of long-term castrated doves. Preoptic aromatase activity of long- and short-term castrated doves did not differ. The ineffectiveness of testosterone in restoring nest-orientated behaviour in long-term castrated doves did not appear, therefore, to be due to a difference between the groups in the basal rate of testosterone aromatization in the preoptic area.

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