Inhibition of HIV-1 replication in chronically infected cell lines and peripheral blood mononuclear cells by retrovirus-mediated antitat gene transfer

To investigate epidemiological and pathogenetic features of HTLV-I infection, a cohort of carriers has been followed at the USP Teaching Hospital since 1991. This study describes the establishment of cell lines from peripheral blood mononuclear cells (PBMC) of infected subjects. Ex vivo PBMC were cultured with those from a seronegative donor and morphologic evidence of cell transformation was obtained after 90 days with detection of multinucleated cells exhibiting cerebriform nuclei. Integration of HTLV-I proviral DNA and expression of viral antigens was demonstrated in culture by PCR and immunofluorescence. Cell lines were maintained for 240 days, gradually weaned from exogenous IL-2. Immunophenotyping of cell lines on flow cytometry yielded evidence of cell activation. Establishment of HTLV-I-infected cell lines from ex vivo PBMC is feasible and may be useful for studies on lymphocyte phenotypic changes and on mechanisms of HTLV-induced cell proliferation. Moreover they may be used with diagnostic purposes in immunofluorescence tests.

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

Cells ◽

10.3390/cells10030523 ◽

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.

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