Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Summary

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

Abstract

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Introduction

The hematopoietic stem and progenitor population supplies the organism with a large range of mature cells, including cells from the myeloid lineage (neutrophils, eosinophils, basophils, and monocytes). The process that drives the production of myeloid cells from hematopoietic stem cells is known as myelopoiesis, and adequate production of mature myeloid cells in response to changing demands is a prerequisite for proper coping of the organism with stress conditions, such as infections and blood loss. Insufficient production of mature myeloid cells may lead to inability to eliminate pathogens, reduced blood coagulation, and other life-threatening conditions

Protocol

NOTE: Steps describing expansion, differentiation, and transduction of 32D/G-CSF-R cells are presented below.

1. Preparation

1. Media preparation
   1. Prepare 250 mL of culture medium: RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 10% heat inactivated FBS (fetal bovine serum) and murine IL-3 (10 ng/mL).
      1. Alternatively, use home-made IL-3. To produce home-made IL-3, transduce HEK293 cells with IL-3 expressing vector and collect IL-3 containing supernatant²².
         NOTE: Antibiotics, such as penicillin G (100 IU/mL), streptomycin (100 µg/mL), ....

Results

Proliferation and differentiation of 32D/G-CSF-R cells

To assess proliferation of 32D/G-CSF-R cells under pro-proliferative and pro-differentiation conditions, 32D/G-CSF-R cells were cultured in media containing IL-3 and G-CSF, respectively. It was observed that cells cultured in IL-3 containing medium (10 ng/mL) divide approximately every 24 h (Figure 2A). Upon replacement of IL-3 with G-CSF (100 ng/m.......

Discussion

The choice of an experimental model is one of the main issues in research. Though primary animal and human cells are believed to produce the most biologically relevant data, these models may involve ethical concerns and are often associated with expensive and/or sophisticated isolation/culturing procedures. Primary cells are limited in numbers and it is hard to genetically manipulate them. In addition, primary cells represent a heterogeneous population composed of various cell types that may complicate data interpretatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640 powder medium	Merck, Kenilworth, NJ, USA	T 121-10	without NaHCO3, with L-glutamine
DMEM	Thermo Fisher Scientific, Waltham, MA, USA	15028
Opti-MEM I Reduced Serum Medium	Thermo Fisher Scientific, Waltham, MA, USA	31985-047	L-Glutamine, Phenol Red
Fetal bovine serum (FBS)	PAA Laboratories (GE Healthcare,Chicago, IL, USA)	MT35011CV	For differentiation of 32D/G-CSF-R cells
Fetal bovine serum (FBS)	Thermo Fisher Scientific, Waltham, MA, USA	10270	Used for culturing HEK293T, NIH3T3, BOSC23 cells
Penicillin	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	P3032
Streptomycin	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	S9137	Streptomycin sulfate salt powder
Gentamicin	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	G1914
murine IL-3	PeproTech, Rocky Hill, NJ, USA	213-13
human G-CSF	PeproTech, Rocky Hill, NJ, USA	300-23
Polyethylenimine	Polyscience, Warrington, PA, USA	23966	Linear, MW 25,000 (PEI 25000)
Polybrene	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	H9268
Trypsin	VWR Chemicals, Radnor, PA, USA	0458
EDTA	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	E5134
Crystal violet	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	C0775
Trypan blue	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	T6146
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich (Merck, Kenilworth, NJ, USA)	D2650
May-Grünwald Giemsa	DiaPath, Martinengo, BG, Italy	10802