scholarly journals Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion

2019 ◽  
Vol 94 (9) ◽  
pp. 963-974 ◽  
Author(s):  
Erik J. Scully ◽  
Estela Shabani ◽  
Gabriel W. Rangel ◽  
Christof Grüring ◽  
Usheer Kanjee ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Cell Line ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Model System ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell ◽  
Progenitor Cell Line

scholarly journals Generation of an Immortalized Erythroid Progenitor Cell Line Directly from Peripheral Blood Mononuclear Cells without Using Mobilized CD34+ Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1032-1032
Author(s):  
Abhirup Bagchi ◽  
Aneesha Nath ◽  
Vignesh Rajendiran ◽  
Madhavi Maddali ◽  
Ekta Jajodia ◽  
...  
Keyword(s):  
Cell Line ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Mononuclear Cells ◽  
Normal Donor ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Differentiation Potential ◽  
Erythroid Progenitor Cell ◽  
Progenitor Cell Line

Abstract A reliable stable human erythroid progenitor cell line that can differentiate to the later stages of erythropoiesis is an important cellular model for studying molecular mechanisms of human erythropoiesis in physiological and pathological situations. An erythroid progenitor cell line (HUDEP) was derived from cord blood haematopoietic stem cells (HSCs) by doxycycline inducible expression of HPV E6/E7 gene (Kurita et al., 2013). This cell line could be differentiated further to terminally differentiated red cells, and it has been extensively used for studying transcriptional regulation of human erythropoiesis. Using the same strategy, immortalized erythroid progenitors could also be generated from adult HSCs (Trakarnsanga et al, 2017). However, generation of immortalized erythroid cells from patients using this protocol is challenging as obtaining sufficient number of adult HSCs requires mobilization of HSCs using GCSF. Peripheral blood mononuclear cells (PBMNCs) contain a small number of erythroid progenitors, which can be expanded and differentiated in culture. Till date, there are no reports on the generation of immortalized erythroid progenitors directly from PBMNCs. In this study, we established a protocol for the generation of immortalized erythroid progenitors from PBMNCs of a normal donor. The PBMNCs isolated from 10ml of blood from a normal donor were cultured for 24 hours in the primary erythroid expansion medium as described earlier (Trakarnsanga et al, 2017). These cells were then transduced with lentiviruses to express HPV E6/E7 gene and a fluorescent protein hKO1. After 3 days, the cells were cultured in a serum free medium containing the cytokines (stem cell factor and erythropoietin) and dexamethasone in the presence of doxycycline for 15 days. The cells that expressed hKO1 were sorted by FACS, and they were cultured in the same medium till the immortalization was complete. We continuously monitored the cells for the kinetics in the expression of erythroid cell surface markers, CD36, CD71 and CD235a, till >95% of the cells expressed all these markers. On day 50, all the cells expressed high levels of the erythroid markers and the cell morphology analysis using Giemsa staining showed that 65% of the cells were pronormoblasts, 22% were basophilic normoblasts and the rest of the cells were at the later stages of differentiation. To evaluate the differentiation potential of these cells, the cells were cultured using the media and conditions described by Hawksworth et al, 2018. After the removal of doxycycline from the culture medium, the cells showed haemoglobinization and the morphology analysis showed that 10% of the cells were in the polychromatic stage and 88% of the cells were in the orthochromatic stage, suggesting robust erythroid differentiation of the immortalized erythroid progenitors with the suitable cell culture conditions. These data showed that immortalized erythroid progenitors with differentiation potential could be generated directly from peripheral blood without using mobilized haematopoietic stem cells. This protocol is suitable for the generation of immortalized erythroid cells from the patients with rare red cell genetic disorders for studying disease mechanisms. Disclosures No relevant conflicts of interest to declare.

scholarly journals Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Steven Mayers ◽  
Pablo Diego Moço ◽  
Talha Maqbool ◽  
Pamuditha N. Silva ◽  
Dawn M. Kilkenny ◽  
...  
Keyword(s):  
Cell Line ◽  
Progenitor Cell ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell ◽  
Progenitor Cell Line

Application of immortalized human erythroid progenitor cell line in serologic tests to detect red blood cell alloantibodies

Transfusion ◽  
2018 ◽  
Vol 58 (11) ◽  
pp. 2675-2682 ◽  
Author(s):  
Go Kikuchi ◽  
Ryo Kurita ◽  
Kenichi Ogasawara ◽  
Kazumi Isa ◽  
Hatsue Tsuneyama ◽  
...  
Keyword(s):  
Blood Cell ◽  
Cell Line ◽  
Red Blood Cell ◽  
Progenitor Cell ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell ◽  
Serologic Tests ◽  
Progenitor Cell Line

scholarly journals Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 523
Author(s):  
Abhirup Bagchi ◽  
Aneesha Nath ◽  
Vasanth Thamodaran ◽  
Smitha Ijee ◽  
Dhavapriya Palani ◽  
...  
Keyword(s):  
Cell Lines ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Mononuclear Cells ◽  
Red Cell ◽  
Peripheral Blood Mononuclear ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell ◽  
Blood Mononuclear Cells

Reliable human erythroid progenitor cell (EPC) lines that can differentiate to the later stages of erythropoiesis are important cellular models for studying molecular mechanisms of human erythropoiesis in normal and pathological conditions. Two immortalized erythroid progenitor cells (iEPCs), HUDEP-2 and BEL-A, generated from CD34+ hematopoietic progenitors by the doxycycline (dox) inducible expression of human papillomavirus E6 and E7 (HEE) genes, are currently being used extensively to study transcriptional regulation of human erythropoiesis and identify novel therapeutic targets for red cell diseases. However, the generation of iEPCs from patients with red cell diseases is challenging as obtaining a sufficient number of CD34+ cells require bone marrow aspiration or their mobilization to peripheral blood using drugs. This study established a protocol for culturing early-stage EPCs from peripheral blood (PB) and their immortalization by expressing HEE genes. We generated two iEPCs, PBiEPC-1 and PBiEPC-2, from the peripheral blood mononuclear cells (PBMNCs) of two healthy donors. These cell lines showed stable doubling times with the properties of erythroid progenitors. PBiEPC-1 showed robust terminal differentiation with high enucleation efficiency, and it could be successfully gene manipulated by gene knockdown and knockout strategies with high efficiencies without affecting its differentiation. This protocol is suitable for generating a bank of iEPCs from patients with rare red cell genetic disorders for studying disease mechanisms and drug discovery.

Enhancement of erythroid progenitor cell growth in medium conditioned by a human cancer cell line, KONT

1985 ◽  
Vol 3 (1) ◽  
pp. 22-32 ◽  
Author(s):  
Yoshiyuki Niho ◽  
Nobuhiro Kimura ◽  
Yujiro Yamano ◽  
Yusei Yamamoto ◽  
Yuichi Hirota ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Cancer Cell ◽  
Progenitor Cell ◽  
Human Cancer ◽  
Cancer Cell Line ◽  
Human Cancer Cell Line ◽  
Human Cancer Cell ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell

Downregulation of c-kit (Stem Cell Factor Receptor) in Transformed Hematopoietic Precursor Cells by Stroma Cells

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 554-563 ◽  
Author(s):  
Christoph Heberlein ◽  
Jutta Friel ◽  
Christine Laker ◽  
Dorothee von Laer ◽  
Ulla Bergholz ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Line ◽  
Stem Cell Factor ◽  
Progenitor Cell ◽  
Receptor Expression ◽  
General Mechanism ◽  
Ligand Interaction ◽  
Kit Ligand ◽  
Kit Receptor ◽  
Progenitor Cell Line

Abstract We show a dramatic downregulation of the stem cell factor (SCF) receptor in different hematopoietic cell lines by murine stroma. Growth of the human erythroid/macrophage progenitor cell line TF-1 is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). However, TF-1 cells clone and proliferate equally well on stroma. Independent stroma-dependent TF-1 clones (TF-1S) were generated on MS-5 stroma. Growth of TF-1S and TF-1 cells on stroma still requires interaction between c-kit (SCF receptor) and its ligand SCF, because antibodies against c-kit inhibit growth to less than 2%. Surprisingly, c-kit receptor expression (RNA and protein) was downregulated by 2 to 3 orders of magnitude in TF-1S and TF-1 cells grown on stroma. This stroma-dependent regulation of the kit receptor in TF-1 was also observed on exposure to kit ligand-negative stroma, thus indicating the need for heterologous receptor ligand interaction. Removal of stroma induced upregulation by 2 to 4 orders of magnitude. Downregulation and upregulation of c-kit expression could also be shown for the megakaryocytic progenitor cell line M-07e and was comparable to that of TF-1, indicating that stroma-dependent regulation of c-kit is a general mechanism. Downregulation may be an economic way to compensate for the increased sensitivity of the c-kit/ligand interaction on stroma. The stroma-dependent c-kit regulation most likely occurs at the transcriptional level, because mechanisms, such as splicing, attenuation, differential promoter usage, or mRNA stability, could be excluded.

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