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. 2021:2244:83-101.
doi: 10.1007/978-1-0716-1111-1_5.

Human Hematopoietic Long-Term Culture (hLTC) for Human Cytomegalovirus Latency and Reactivation

Megan Peppenelli  1 , Jason Buehler  2 , Felicia Goodrum  3   4
Affiliations

Human Hematopoietic Long-Term Culture (hLTC) for Human Cytomegalovirus Latency and Reactivation

Megan Peppenelli et al. Methods Mol Biol. 2021.

Abstract

Of the many research challenges posed by the study of human cytomegalovirus (HCMV) latency, one of the most notable is the requirement for the use of primary hematopoietic cell culture. Culturing hematopoietic progenitor subpopulations requires that consideration be given to maintaining their physiological relevance. We describe a long-standing primary CD34+ hematopoietic progenitor cell (HPC) system as an in vitro model to study HCMV latent infection. Key aspects of the model include infection of primary human CD34+ HPCs prior to ex vivo expansion, a long-term culture with a stromal cell support designed to maintain the ability of stem cells to support hematopoietic reconstitution, and an assay to quantify infectious centers produced prior to and following a reactivation stimulus. Importantly, this system has been used to identify a number of viral determinants of latency or reactivation and findings have been recapitulated in vivo using a humanized mouse model for HCMV latency. Therefore, this system offers a powerful approach to defining virus-host interactions and mechanisms important for HCMV latency and reactivation.

Keywords: CD34+ hematopoietic cells; Extreme limiting dilution analysis; HPCs; Human Cytomegalovirus; Latency; Progenitor cells; Reactivation.

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Figures

Figure 1.
Figure 1.. The potential latency phenotypes in CD34+ hematopoietic progenitor cell model.
Pre-reactivation is the virus produced prior to the reactivation as determined by measuring infectious centers in the lysate controls (light grey bars). The frequency of reactivation (dark grey bars) is determined by infectious centers obtained by the co-culture of viable, latently infected CD34+ cells with fibroblast monolayers.
Figure 2.
Figure 2.. Purification of CD34+ cells from cord blood.
The mononuclear cells from any blood or bone marrow source are separated by Ficoll gradients and CD34+ cells are subsequently purified from the mononuclear fraction using the CD34 MicroBead kit.
Figure 3.
Figure 3.. Schematic of Infectious Centers Assay.
The infected CD34+ cells or the equivalent cell lysate from the long term culture are diluted two-fold in deep well dishes and transferred to monolayers of MRC5 cell in 96 well dishes for the infectious centers assay.
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