Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Summary

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Abstract

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

Introduction

Erythropoiesis is the process of differentiation of multipotent hematopoietic stem cells to mature erythrocytes. This stepwise process includes the formation of committed erythroid burst forming units (BFU-Es), the rapidly dividing erythroid colony forming units (CFU-Es), and morphologically recognizable erythroblasts^1,2. Terminal erythropoiesis from CFU-E progenitor cells involves sequential erythropoietin-dependent and independent stages^2,3. In the early stage of terminal erythropoiesis, Erythropoietin (Epo) binds to its receptor on the cell surface and induces a series of downstream signaling pathways that prevent cell apoptosis and promote rapid cell divisions and gene expression^1,4. In the late stage of terminal erythropoiesis, erythroblasts undergo terminal cell cycle exit, chromatin and nucleus condensation, and extrusion of the highly condensed nuclei⁵.

Our understanding of terminal erythropoiesis has greatly improved in the last few decades, which is largely due to the successful use of several in vitro and in vivo mouse models^6-9. Among these models, in vitro culture of mouse fetal liver erythroblasts provides many advantages including the ease of cell purification, fast proliferation and differentiation, and a closer mimic to human erythropoiesis^10,11. In this system, large numbers of erythroid progenitor cells from mouse fetal livers can be easily isolated by the single step purification of TER119 (a marker for the mature erythroid cells) negative erythroblasts. During the two-day culture of the erythroblasts, the differentiation of these cells can be monitored by a flow cytometric analysis based on surface expression of the transferrin receptor (CD71) and the TER119 antigen¹². In addition, enucleation of the terminally differentiation erythroblasts can be detected by a DNA maker (Hoechst 33342)¹³. Furthermore, the purified progenitors can be genetically modified by exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest, which facilitates the mechanistic studies of the functions of gene expression on erythropoiesis^11,13,14.

On the other hand, the fast cell growth rate can be a double-edged sword since it is difficult to characterize gene functions in different stages of terminal erythropoiesis. In most cases, it is difficult to determine whether a specific gene functions in the early stage of terminal erythropoiesis since by the time the cDNAs or shRNAs expressed, the cells already passed the early stage. To solve this problem, we developed a unique system to dissect the early and late stages of terminal erythropoiesis. For the early stage of terminal erythropoiesis, genetically modified TER119 negative erythroblasts were cultured in Epo-free medium but containing stem cell factor (SCF), IL-6 and FLT3 ligand to maintain their progenitor status and allow the transduced cDNAs or shRNA to expression¹³. The cells were changed to Epo containing medium after 12 hr to induce cell proliferation and differentiation. In this way, when the cells started to differentiate, the transduced cDNAs or shRNAs were already expressed. For the late stage of terminal erythropoiesis, TER119 negative erythroblasts were cultured in Epo containing medium immediately after transduction. Therefore, one can analyze the functions of the genes of interest in the late stage of terminal erythropoiesis. In summary, a broad application of this system would help dissect gene functions in different stages of terminal erythropoiesis.

Protocol

The experiments described in this protocol were performed in accordance with the guidelines and regulations set forth by Northwestern University Institutional Animal Care and Use Committee. 1. Preparation of Culture Medium Prepare fibronectin solution. Add 1 ml of water to one vial of human fibronectin (1 mg). Leave solution in the tissue culture hood for 30 min without agitation. Transfer the total liquid to 50 ml of PBS to make a final concentration of 20 µg/ml and gently mi…

Representative Results

Figure 1 outlines the experimental strategies. The protocol consists of two independent conditions for targeting the functions of the signaling molecules in the early and late stages of terminal erythropoiesis. TER119 negative fetal liver erythroblasts were purified from E13.5 mouse fetus. Flow cytometric analysis of fetal liver erythroid cells before and after purification demonstrated that the purification was efficient (Figures 2A and 2B). For the early stage of termi…

Discussion

Here we present a unique system to chronologically analyze mouse fetal liver terminal erythropoiesis. Through the application of different culture conditions, we successfully dissected terminal erythropoiesis in early and late stages. This is particularly important to determine the mechanisms of genes with multiple functions. For example, Rac GTPases play important roles in different stages of terminal erythropoiesis. Inhibition of Rac GTPases in the early stage of terminal erythropoiesis influences cell differentiation …

Materials

Iscove's Modified Dulbecco's MediumIMDM (IMDM)	Gibco	12440-053
Fetal bovine serum (FBS)	GEMINI Bio-product	700-102P
b-mecaptoethanol	Sigma	M6250	0.1 M in IMDM,  4℃stock
Penicillin-Streptomycin solution	Hyclone	SV30010	100 X
L-Glutamine	Hyclone	SH30034.01	200 mM
Stem cell factor (SCF)	STEMCELL TECH	2630	100 ng/ul, -20℃ stock
Mouse FIT3 Ligand (Flt-3L)	BD Biosciences	14-8001-80
Mouse Recombinant Interleukin-6 (IL-6)	GEMINI Bio-product	300-327P
fibronectin	BD Biosciences	354008
Bovine Serum Albumin (BSA)	STEMCELL TECH	9300	10% stock in IMDM
Insulin solution, human	Sigma	I9278	4℃ stock
holo-transferrin human	Sigma	T0665	50 mg/ml in Water -20℃ stock
Erythropoietin(Epo)	PROCRIT	NOC 59676-303-00	3,000 U/ml stock
Streptavidin particles Plus	BD Pharmingen	557812
EasySep Magnet	STEMCELL TECH	18000
Polypropylene Round-Bottom Tube, 5 ml	FALCON	352063