Name: proliferation and differentiation of murine myeloid precursor 32d/g-csf-r cells
Uploaded: 2018-02-21T14:00:00
Description: Watch this Scientific Journal Video about Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells at JoVE.com

The authors thank Prof. Ruud Delwel and Prof. Ivo Touw for providing us with the 32D/G-CSF-R cell line, and Prof. Daniel G. Tenen for providing us with the Bosc23 cell line. This work was supported by grants of the Grant Agency of the Czech Republic (GACR 15-03796S and GACR 17-02177S) to MA-J, support from the Institute of Molecular Genetics of the Czech Academy of Sciences (RVO 68378050) to MA-J, a GA UK fellowship (project No. 341015) from Charles University in Prague to MK, and a GA UK fellowship (project No. 1278217) from Charles University in Prague to PD.

NOTE: Steps describing expansion, differentiation, and transduction of 32D/G-CSF-R cells are presented below.
1. Preparation
<ol>
	<li>Media preparation
	<ol>
		<li>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).
		<ol>
			<li>Alternatively, use home-made IL-3. To produce home-made IL-3, transduce HEK293 cells with IL-3 expressing vector and collec...

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...

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 conditions1,2. In addition, alterations in myeloid lineage development may also be associated with hematological malignancies, such as acute myeloid leukemia (AML)3. Alterations in myelopoiesis may occur due to various reasons, such as defects in cell surface receptors4, altered expressions of transcription factors5, impaired signaling pathways6, mutations resulting in formation/activation of oncogenes7, or inactivation of tumor suppressor genes8. 
Various methods have been developed to study myeloid development, and assess the effect of specific genetic alterations in this process. Common approaches used to study myelopoiesis involve primary cells and transgenic mice. Though these models allow acquisition of biologically relevant data, they have certain limitations. The use of primary cells encounters a limited number of cells and a restricted period of culture, narrowing the possibilities to alter gene expression and subsequent biological or biochemical analysis. Transgenic mice are costly and require a reasonable degree of biological justification. In addition, working with in vivo models adds a degree of complexity into understanding the role of a gene of interest in a given process. Therefore, alternative approaches to circumvent these limitations are needed. Cell lines have indisputable advantages: (1) they possess unlimited proliferation capacity that allows generating enough material for biochemical and biological studies, (2) they are susceptible to genetic manipulations (knockdown, knockout, overexpression), (3) the cost is relatively low, and (4) they allow a degree of biological simplification required in certain experimental approaches. 
The parental IL-3 (Interleukin-3) dependent 32D cell line was established in 1983 by Greenberger and colleagues by infection of bone marrow cells from C3H/HeJ mice with Friend murine leukemia virus9. Several 32D clones were described in literature: cl-239, cl-310, and cl-1011. The 32D cl-3 cells were shown to proliferate in IL-3 and undergo neutrophilic differentiation upon treatment with granulocyte-colony stimulation factor (G-CSF)10. On the contrary, 32D cl-10 cells, while being IL-3 dependent, originally were not differentiating in response to G-CSF treatment. In 1995 the group of Dr. Ivo Touw retrovirally transduced 32D cl-10 cells with wild type and mutant forms of G-CSF receptor (G-CSF-R), in order to identify functionally important regions of this receptor11. This study resulted in generation of the 32D/G-CSF-R cells, which are similarly dependent on IL-3, but within 6 to 10 days after replacement of IL-3 with G-CSF, cells stop to proliferate and irreversibly differentiate into mature neutrophils. These properties make 32D cl-3 and 32D/G-CSF-R cells simplified models of murine neutrophilic differentiation that can be modulated by two well-defined growth and differentiation factors - IL-3 and G-CSF. During the last decades multiple groups have used 32D/G-CSF-R cells to study the role of particular genes in proliferation and differentiation of myeloid cells in culture12,13,14,15,16, and to study G-CSF signaling17,18. Importantly, the results obtained using this cell line correlated with data obtained with primary cells and transgenic mice16,19,20,21. Consequently, we believe that 32D/G-CSF-R cells, being a widely used and well-established model, represent a valuable system to study myeloid differentiation which can be used in parallel with other approaches addressing this question. 
Here, detailed protocols describing handling of the 32D/G-CSF-R cell line, which cover expansion, differentiation, and assessment of proliferation and differentiation of these cells is presented. Detailed information for genetic modification of 32D/G-CSF-R cells, either by retroviral or lentiviral transduction, as well as protocols for virus titration are provided. In addition, several representative results that demonstrate potential applications of 32D/G-CSF-R cells are provided.

<table>
	<tbody>
		<tr>
			<td>RPMI 1640 powder medium</td>
			<td>Merck, Kenilworth, NJ, USA</td>
			<td>T 121-10</td>
			<td>without NaHCO3, with L-glutamine</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>15028</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>31985-047</td>
			<td>L-Glutamine, Phenol Red</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>PAA Laboratories (GE Healthcare,Chicago, IL, USA)</td>
			<td>MT35011CV</td>
			<td>For differentiation of 32D/G-CSF-R cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>10270</td>
			<td>Used for culturing HEK293T, NIH3T3, BOSC23 cells</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>S9137</td>
			<td>Streptomycin sulfate salt powder</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>G1914</td>
			<td></td>
		</tr>
		<tr>
			<td>murine IL-3</td>
			<td>PeproTech, Rocky Hill, NJ, USA</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>human G-CSF</td>
			<td>PeproTech, Rocky Hill, NJ, USA</td>
			<td>300-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polyscience, Warrington, PA, USA</td>
			<td>23966</td>
			<td>Linear, MW 25,000 (PEI 25000)</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR Chemicals, Radnor, PA, USA</td>
			<td>0458</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>C0775</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>T6146</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>May-Gr&#252;nwald Giemsa</td>
			<td>DiaPath, Martinengo, BG, Italy</td>
			<td>10802</td>
			<td></td>
		</tr>
	</tbody>
</table>

proliferation and differentiation of murine myeloid precursor 32d/g-csf-r cells

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.

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...

Watch this Scientific Journal Video about Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells at JoVE.com

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

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.

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

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