Megakaryocyte Growth and Maturation from Purified Peripheral Blood Progenitors in Unilineage Serum-Free Liquid Culture

1996 ◽  
pp. 445-452
Author(s):  
H. J. Hassan ◽  
R. Guerriero ◽  
U. Testa ◽  
M. Gabbianelli ◽  
G. Mattia ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Liquid Culture ◽  
Serum Free

Enrichment and detection of fetal erythroid cells from maternal peripheral blood using liquid culture

2001 ◽  
Vol 21 (1) ◽  
pp. 22-26 ◽  
Author(s):  
Jin-Yeong Han ◽  
Young-Hee Lee ◽  
Sang-Dong Sin ◽  
Joo-In Park ◽  
In-Hoo Kim ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Liquid Culture ◽  
Erythroid Cells ◽  
Maternal Peripheral Blood

Animal Serum-Free Culture of Purified Human CD34+ Cells: Amplification of Progenitors from G-CSF and GM-CSF-Mobilized Peripheral Blood

1997 ◽  
Vol 6 (4) ◽  
pp. 335-349 ◽  
Author(s):  
LISA R. SCHAIN ◽  
SONIA JAIN ◽  
MICHELLE WYSOCKI ◽  
MELINDA HALL ◽  
BARBARA DADEY ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Serum Free ◽  
Gm Csf ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Juvenile Myelo-Monocytic Leukemia (JMML): the Effect of Granulocyte Monocyte-Colony Stimulating Factor (GM-CSF) on Wilms Tumor Gene (WT1) by Quantitative Real Time-Polymerase Chain Reaction (q-RT-PCR) and Flow Cytometry

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5063-5063
Author(s):  
Morris Kletzel ◽  
Sana Khan ◽  
Wei Huang ◽  
Marie Olszewski
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Liquid Culture ◽  
Surrogate Marker ◽  
Wt1 Gene ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Total Rna ◽  
Gm Csf

Abstract Abstract 5063 JMML is a rare disease of early childhood, frequently associated with high white cell count with increased monocytes in peripheral blood and splenomegaly. WT1 gene expression is high in most acute and chronic leukemia, including JMML. We have considered WT1 gene expression as a surrogate marker for proliferation. However, there are few reports which suggest that WT1 gene expression contributes to the pathogenesis and prognosis of JMML. In this study we correlate the WT1 gene expression in samples from patients with JMML to establish a direct association of its expression with other markers of proliferation (CD133/CD34 and CD71/34). Samples obtained from 5 newly diagnosed patients with JMML were compared to samples of peripheral blood (PB) obtained from healthy donors (n=3) each sample was placed in liquid culture with or without the addition of GM-CSF at D-0. Cultures were assessed for WT1 gene expression (q-RT-PCR) and flow cytometry at D+7 and D+14, data only presented for D+14. q-RT-PCR: Total RNA was extracted using QIAmp RNA Blood mini kit following manufacture's procedure. A 2-step reverse transcriptase reaction was conducted in a 20 ul reaction volume containing 1ug of total RNA from each sample. Reverse transcription was incubated at 42 degrees C for 1 hour, followed by denaturation at 96 degrees C for 10 min. Ten micro liter of cDNA was amplified during first round of PCR, the second round was carried out by taking 1 ul of the 1st round of amplified product using SyberGreen. After the completion of PCR, the LightCycler Software through extrapolation from the external curve calculated the concentration of target molecule. Flow cytometry: Staining with phenotype markers known to express in early hematopoietic stem cells and proliferation were paired with monoclonal antibodies: CD133/CD34 and CD71/CD34. All determinations were performed in triplicate. Statistical analysis was performed by column statistics and the comparison between groups was done by parametric student t test (Graph Pad by PRISM). The WT1 gene expression was no different at day 14 between the non-GM-CSF and the GM-CSF (see table). WT1 gene expression and early proliferative markers where high in all samples of JMML patients, both in the non-GM-CSF and in the GM-CSF stimulated conditions when compared to the normal controls but, not as high as the K562 cell line. In JMML samples at D+14 there is a 2 fold rise in WT1 gene expression, 12 fold rise in CD 133/34 and a 6 fold rise in CD 71/34. Our findings suggest no difference in WT1 gene expression in JMML cells, and that GM-CSF does not induce higher proliferation of these cells in liquid culture. Thus, our study supports the hypothesis that WT1 gene expression is a surrogate marker of proliferation and may not have any role in the pathogenesis of JMML. GM-CSF does not play a role in the WT1 gene expression. Thus, allowing us to use it as a marker of Minimal Residual Disease (MRD) and its persistence correlate with poor prognosis. Disclosures: No relevant conflicts of interest to declare.

8: SERUM FREE EX VIVO EXPANSION RESTORES HUMAN DIABETIC PERIPHERAL BLOOD ENDOTHELIAL PROGENITOR CELL FUNCTION

2010 ◽  
Vol 125 (Supplement) ◽  
pp. 13
Author(s):  
R Tanaka ◽  
H Masuda ◽  
Y Kurihara ◽  
R Ito ◽  
M Kobori ◽  
...  
Keyword(s):  
Endothelial Progenitor Cell ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Cell Function ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Endothelial Progenitor ◽  
Serum Free

scholarly journals Production of erythropoietic bursts by progenitor cells from adult human peripheral blood in an improved serum-free medium: role of insulinlike growth factor 1

Blood ◽  
1991 ◽  
Vol 78 (11) ◽  
pp. 2823-2833 ◽  
Author(s):  
PN Correa ◽  
AA Axelrad
Keyword(s):  
Growth Factor ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Erythroid Progenitors ◽  
Retinyl Acetate ◽  
Serum Free ◽  
Adult Human ◽  
Insulinlike Growth Factor ◽  
Igf I

Several culture media for the growth of human circulating erythroid burst-forming units (BFU-E) that have been claimed to be “serum-free” (“SF”) have actually included albumin preparations known to be contaminated with an undefined burst-promoting activity (BPA); a BPA has also been found in the preparations of other “SF” medium components. This has precluded reliable investigation of the growth factor (GF) requirements of these progenitors. Using a defatted, BPA- free bovine serum albumin (BSA) and the recombinant human growth factors (GFs) erythropoietin (rHu Epo), insulinlike growth factor 1 (rHu IGF-1), and interleukin-3 (rHu IL-3), we have developed an improved serum-free (SF) medium for the production of erythroid bursts from normal adult human peripheral blood mononuclear cells (PBMNC), which requires both hemin and retinyl acetate for its optimal performance. In the presence of BSA without IL-3 or Epo, no burst or colony formation was observed. With IL-3 and Epo alone, only a small number of day 14 erythroid colonies was obtained (12 +/- 1/10(5) PBMNC). Addition of hemin (0.1 mmol/L) allowed the direct scoring of day 14 hemoglobinized colonies and increased their number sevenfold (86 +/- 5). Inclusion of retinyl acetate at physiologic concentrations further augmented the number of colonies threefold to fourfold. Under these apparently optimal conditions, we found that IGF-I could entirely replace Epo. However, IGF-I required a 100-fold higher molar concentration than that of Epo to reach maximal stimulation. The combined effect of Epo and IGF-I was found to be less than the sum of their individual effects, suggesting an overlap in the sensitivities of erythroid progenitors to these GFs. The colony-forming efficiencies of erythroid progenitors in the improved SF medium was very high: 700 single, day 14 erythroid colonies/10(5) PB MNC (at 0.25 mmol/L hemin) distributed as 126 clusters (bursts), with a mean of 5.6 component colonies per burst. These findings show that IGF-I has an Epo-like activity that targets circulating early erythroid progenitors or their progeny, providing strong evidence for the existence of an Epo- independent pathway for normal human adult erythropoiesis, possibly operative when Epo levels are low.

scholarly journals Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 566-574 ◽  
Author(s):  
M Flasshove ◽  
D Banerjee ◽  
S Mineishi ◽  
MX Li ◽  
JR Bertino ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Dihydrofolate Reductase ◽  
Peripheral Blood ◽  
Liquid Culture ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Precursor Cells ◽  
Retroviral Gene Transfer ◽  
Selection Of

Abstract Retroviral gene transfer into human myeloid precursor cells allows introduction of marker genes as well as genes conferring resistance to chemotherapeutic drugs. We transduced a human mutant dihydrofolate reductase (DHFR) cDNA into CD34 antigen-positive peripheral blood cells from patients with breast or ovarian cancer obtained after treatment with chemotherapy and granulocyte colony-stimulating factor (G-CSF). This mutant DHFR has been shown to confer resistance to methotrexate (MTX) in murine bone marrow. We established a transduction protocol that permitted ex vivo expansion and selection of transduced early progenitor cells. The number of progenitor cells from transduced CD34- positive cells increased 50-fold after cytokine prestimulation with interleukin-1 (IL-1), c-kit ligand (KL; stem cell factor), and IL-3 and 2 weeks in liquid culture. Transduced colony-forming unit-granulocyte- macrophage (CFU-GM), assayed directly after the transduction procedure, were protected completely against 2 x 10(-8) mol/L MTX, a concentration that significantly reduced the CFU-GM detected in the control population. Gene transfer of the mutant DHFR led to a twofold selective advantage for a pre-CFU population after exposure to MTX in liquid culture (P < .001). Polybrene, in contrast with protamine, significantly inhibited the expansion of progenitors. The presence of proviral DNA was monitored by polymerase chain reaction (PCR) and was detected in greater than 80% of CFU-GM and ex vivo expanded pre-CFU. We have demonstrated that human hematopoietic precursor cells can be expanded extensively after retroviral gene transfer. The same population of early progenitors can be selected ex vivo with low-dose MTX. As long-term expression of transduced genes in human hematopoietic cells remains a problem in vivo, these results may have implications for future clinical trials, especially for the introduction of nonselectable genes.

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