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Erythropoiesis pp 117-132 | Cite as

Functional Analysis of Erythroid Progenitors by Colony-Forming Assays

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1698)

Abstract

The capacity of erythroid-lineage progenitors to form colonies of maturing red blood cells in semisolid media has provided a functional assay for these progenitors and has greatly contributed to our understanding of erythropoiesis. Studies since the 1970s have led to the development of a model of the erythron, whereby the earliest erythroid-committed progenitor, the immature burst-forming unit erythroid (BFU-E), gives rise sequentially to late-stage BFU-E and to colony-forming units erythroid (CFU-E). CFU-E give rise, in turn, to maturing erythroblast precursors that hemoglobinize. It is these terminal cells that comprise the mature colonies of erythroid cells derived from the progenitors cultured in semisolid media. The in vitro generation of erythroid colonies requires cytokine support, most notably erythropoietin (EPO), which is critical for CFU-E survival and for promoting erythroblast maturation.

During mouse embryogenesis, a transient population of primitive erythroid colony-forming progenitors (EryP-CFC) emerges in the yolk sac and gives rise to a wave of maturing primitive erythroblasts in the fetal bloodstream. This wave of EryP-CFC is followed closely by a wave of BFU-E in the yolk sac that enter the bloodstream and seed the fetal liver to generate the first definitive red cells in the fetus. BFU-E in the fetal liver, unlike those in the adult bone marrow, can give rise to colonies in vitro when cultured with EPO alone and also are more sensitive to EPO levels. Here, we describe methods for the in vitro culture of murine embryonic (primitive) and fetal/adult (definitive) erythroid progenitors in semisolid media.

Key words

Erythropoiesis Progenitor Yolk sac Colony-forming cell Erythropoietin 

1 Introduction

Hematopoietic stem cells differentiate into multipotential progenitor cells that generate multiple lineage-restricted progenitors that, in turn, mature through progressive precursor stages into mature blood cells. We produce several million new cells every second to maintain steady-state levels of circulating red blood cells, platelets, neutrophils, monocytes, and lymphoid cells. Recent studies indicate that this massive output of mature blood cells is functionally maintained, not by hematopoietic stem cells , but by multipotent hematopoietic progenitor cells [1, 2]. The capacity of hematopoietic progenitors to form colonies of maturing blood cells in semisolid media provides a functional assay of these progenitors and has greatly contributed to our understanding of lineage relationships within the hematopoietic system. This colony-forming assay requires the placement of a single-cell suspension of cells in semisolid media to allow for the clonal growth of maturing progeny. Initially, soft agar was used to grow “colony-forming units in culture” (CFU-C), which gave rise to colonies of myeloid cells [3, 4]. This was followed by the use of plasma clots for the growth of colonies of erythroid cells [5, 6]. Currently, methylcellulose is the most commonly used semisolid medium because it supports the clonal differentiation of erythroid [7], as well as multiple myeloid progenitors.

The in vitro growth of colonies in semisolid media also requires the action of cytokines to support the survival and proliferative maturation of plated progenitors. While conditioned media from various cell types was the intial source of growth factors to support hematopoietic colony-forming assays, specific recombinant cytokines are now widely used to support colony formation. Erythropoietin (EPO ) , cloned in 1985, is the major cytokine that supports erythropoiesis by signaling through its specific receptor expressed on maturing erythroid cells to mediate their survival and multiple other aspects of erythroid maturation [8, 9, 10].

The first erythroid colonies recognized in semisolid media were small, compact colonies containing 16–64 late-stage hemoglobinizing erythroblasts (Fig. 1) [5, 11]. These small colonies are derived from colony-forming units erythroid (CFU-E) that peak in number at 2 and 7 days of culture, respectively, for mouse and human systems [5, 12, 13]. CFU-E require optimal concentrations of EPO, as well as the presence of reducing agents, to generate colonies in vitro [11, 14].
Fig. 1

Examples of colonies derived from murine definitive and primitive erythroid progenitors . Immature BFU-E-derived colonies, counted at day 7 of culture, typically consist of one or more large colonies surrounded by smaller colonies, which constitute a “burst.” Late-stage BFU-E-derived colonies, counted at day 3 of culture, consist of smaller single colonies. CFU-E-derived colonies, counted at day 2 of culture, consist of a single small colony containing 16–64 hemoglobinizing cells. Primitive erythroid progenitors (EryP-CFC ) derived from the mouse embryo or from differentiating mouse embryonic stem cells generate single compact grape-like colonies of large primitive erythroblasts at 5 days of in vitro culture. d day

Soon after the discovery of CFU-E, immature “burst-forming units capable of erythroid colony formation” (BFU-E ) were recognized that generated much larger colonies consisting of 500–10,000 erythroblasts that peak in maturation at 7–10 and 14–20 days of culture, respectively, for mouse and human systems [7, 12, 15]. BFU-E-derived colonies have variable morphology, but typically consist of a collection of one or more large colonies surrounded by smaller colonies that comprise a “burst” (Fig. 1). An intermediate late-stage BFU-E has also been identified that produces intermediate sized erythroid colonies at 3–4 and 10–12 days of culture, respectively, in mouse and human systems (Fig. 1) [12, 14]. The correlation between colony size (immature BFU-E > late stage BFU-E > CFU-E) and the time it takes for the colony to peak in culture, i.e., when it is comprised of terminal, hemoglobinized erythroid cells, supports the concept that in vivo immature BFU-E generate late-stage BFU-E that lie 1–2 cell divisions upstream of CFU-E that, in turn, generate maturing erythroblasts that undergo terminal maturation (Fig. 2).
Fig. 2

Schematic overview of definitive erythropoiesis, which consists of the sequential formation of erythroid progenitors, as defined by their potential to form distinctive colonies of erythroid cells in vitro, a wave of hemoglobinizing erythroblasts, defined by morphological criteria, and mature red blood cells . While CFU-E require EPO alone for cytokine support, immature and late-stage BFU-E require both EPO , as well as some form of BPA (e.g., IL-3 , IL-6, SCF ) to form colonies in vitro. BFU-E burst-forming unit erythroid, CFU-E colony-forming unit erythroid, EPO erythropoietin, BPA burst-promoting activity, RBC red blood cells, HSC hematopoietic stem cells

Unlike CFU-E that only require EPO for cytokine support, the optimal in vitro growth of BFU-E-derived colonies requires a combination of two cytokines—EPO as well as some form of “burst-promoting activity” (BPA). It was subsequently recognized that interleukin 3 (IL-3 ), GM-CSF , IL-6, or stem cell factor can function as BPA, with IL-3 being the most potent of these [16, 17, 18]. CFU-E do not respond to these BPA-containing factors. Interestingly, immature BFU-E numbers in the bone marrow, unlike the numbers of late-stage BFU-E and CFU-E, do not change in response to changes in serum EPO levels [19] or to exogenously administered EPO [20]. Taken together, these findings suggest that immature BFU-E sequentially require burst-promoting activity followed by EPO to generate erythroid colonies in vitro (Fig. 2). These findings also indicate that the initial cellular commitment to the erythroid lineage (i.e., immature BFU-E) occurs prior to the development of EPO -responsive erythroid progenitors (late-stage BFU-E and CFU-E ). Small numbers of immature BFU-E, but not CFU-E, normally circulate in the bloodstream [21, 22].

During embryogenesis, the yolk sac is the first site of blood cell development, which is characterized by the appearance of pools of erythroblasts in blood islands. Studies of the spatial and temporal emergence of hematopoietic progenitors in the mouse embryo indicate that the initial commitment to hematopoietic fates occurs soon after the start of gastrulation with two overlapping waves of erythroid progenitors in the yolk sac [23, 24]. The first wave consists of primitive erythroid progenitors (EryP-CFC) , which give rise to the embryo’s first circulating primitive erythroblasts. When cultured in vitro, murine EryP-CFC generate an intermediate sized colony of large erythroblasts that peaks at day 5 of culture (Fig. 1). The primitive erythroid cells comprising these colonies are large, round cells that express predominantly embryonic hemoglobins [24, 25].

Subsequently, a wave of definitive erythroid progenitors (BFU-E) also emerges in the yolk sac of the mouse embryo prior to the emergence of hematopoietic stem cells from large arterial vessels [23, 24]. Yolk sac-derived BFU-E rapidly enter the newly formed bloodstream and seed the liver where they give rise to large numbers of CFU-E and the first circulting definitive red blood cells in the fetus [24, 26, 27]. CFU-E in the murine fetal liver are more sensitive to EPO than CFU-E in the adult bone marrow [28]. Unlike adult marrow-derived BFU-E that require burst-promoting activity and EPO , fetal liver-derived BFU-E can generate colonies when cultured in EPO alone [29, 30]. Interestingly, in response to acute anemia, adult BFU-E can also give rise to colonies in vitro in the presence of EPO alone [31], consistent with other findings that stress erythropoiesis share features associated with fetal erythropoiesis [32]. Closer examination of erythroid progenitors in the murine fetal liver has led to the identification of two populations of BFU-E, one population that require burst-promoting activity and another population, like adult stress BFU-E, that require EPO alone [33].

This chapter contains detailed materials and methods to assay embryonic, fetal, and adult erythroid lineage-specific hematopoietic progenitors by their in vitro colony-forming potential. While the focus is on the in vitro culture of murine erythroid progenitors, information for the culture of human erythroid progenitors is also provided.

2 Materials

2.1 Mice

For studies of erythroid progenitors during embryogenesis, we primarily use outbred strains of mice (ICR or CD-1), since they have higher fecundity and increased litter size compared to inbred strains (see Note 1). Timed pregnant mice can be obtained from all major mouse suppliers. Alternatively, in-house colonies of mice can be maintained to generate timed pregnancies (see Subheading 3.2 below).

2.2 Laboratory Equipment

  1. 1.

    Dissection forceps (#5 straight forceps with fine tips). Forceps can be sharpened using an Arkansas oil stone and mineral oil.

     
  2. 2.

    Dissection stereomicroscopes with external fiber-optic light source are available from many manufacturers, including Leica, Nikon, and Olympus.

     
  3. 3.

    Microscopes.

     
  4. 4.

    Laminar flow hood.

     
  5. 5.

    Microcentrifuge and centrifuge for 15 ml conical tubes.

     
  6. 6.

    Vortexor.

     
  7. 7.

    37 °C incubator with 5% CO2.

     

2.3 Plastic Ware

2.3.1 Dishes

The 35 mm dishes must be non-tissue culture treated for methylcellulose cultures, but the other dishes can be either treated or non-treated.
  1. 1.

    35 mm non-tissue culture treated (see Note 2).

     
  2. 2.

    60 mm.

     
  3. 3.

    60 mm gridded.

     
  4. 4.

    245 × 245 square.

     

2.3.2 Tubes

  1. 1.

    1.5 ml microcentrifuge.

     
  2. 2.

    15 ml conical centrifuge.

     
  3. 3.

    50 ml conical centrifuge.

     
  4. 4.

    5 ml round-bottom snap cap.

     

2.3.3 Syringes

  1. 1.

    3 ml.

     
  2. 2.

    5 ml.

     
  3. 3.

    10 ml.

     
  4. 4.

    16 gauge needles.

     
  5. 5.

    22 gauge needles.

     

2.4 Buffers, Media, and Supplements

  1. 1.

    Protein Free Hybridoma Medium II (PFHM II; GibcoBRL) (see Note 3).

     
  2. 2.

    Glutamax (GibcoBRL).

     
  3. 3.

    MTG (1-monothioglycerol).

     
  4. 4.

    2ME (2-mercaptoethanol, 55 mM stock).

     
  5. 5.

    PB-2 solution: Dulbecco’s phosphate-buffered saline (DPBS) with calcium chloride, 1.0 g Glucose, 3.0 g Bovine serum albumin (BSA, fraction V-Cohn), tissue culture grade water to 1 l. Sterile filter through a 0.2 μm filter and store at 4 °C.

     
  6. 6.

    2× Iscove’s Modified Dulbecco’s Medium (2× IMDM): IMDM powder for 1 l, 10 ml Penicillin/Streptomycin, 3.025 g NaHCO3, 500 ml of tissue culture grade water. Sterile filter through a 0.2 μm filter.

     
  7. 7.

    1× IMDM: Dilute 2× IMDM with tissue culture grade water.

     
  8. 8.

    1× IMDM/10% PDS (Platelet-poor Plasma Derived Serum).

     
  9. 9.

    PBS, calcium and magnesium free/1 mM EDTA.

     
  10. 10.

    0.25% trypsin and 1 mM EDTA solution in calcium and magnesium-free PBS (see Note 4). Sterile filter and store at 4 °C.

     
  11. 11.

    0.25% Collagenase Type 1 diluted 1:1 with PB2 or 1× IMDM/10% PDS.

     
  12. 12.
    The final concentrations of specific cytokines used for the culture of mouse and human erythroid progenitors are included in Tables 1 and 2, respectively.
    Table 1

    Media, supplements, and cytokines for the growth of primitive and definitive murine erythroid progenitor-derived colonies in methylcellulose (see Note 6)

     

    Definitive

    Primitive

    Components

    BFU-E

    CFU-E

    EryP-CFC

    IMDM (1×)

    To final volume

    To final volume

    To final volume

    BIT

    20%

    20%

    PDS

    10%

    10%

    10%

    PFHM II

    5%

    5%

    5%

    GlutaMAX

    IL-6

    20 ng/ml

    IL-3

    20 ng/ml

    EPO

    2 U/ml

    0.3 U/ml

    2 U/ml

    SCF

    120 ng/ml

    MTG

    0.15 mM

    2-ME

    55 μM

    55 μM

    Methylcellulose stock solution

    55–65%

    55–65%

    55–65%

    Table 2

    Media, supplements, and cytokines for the growth of human erythroid progenitor-derived colonies in methylcellulose

    Components

    BFU-E/CFU-E

    IMDM (1×)

    To final volume

    BIT

    20%

    PDS

    10%

    PFHM II

    5%

    GlutaMAX

    IL-6

    5 ng/ml

    IL-3

    40 ng/ml

    EPO

    2 U/ml

    SCF

    100 ng/ml

    IGF-1

    25 ng/ml

    2-ME

    55 μM

    Methylcellulose stock solution

    55–65%

     

2.5 Sera and Serum Substitutes

Different lots of serum must be tested to ensure appropriate biological activity, especially for primitive erythroid progenitors [23]. Heat inactivation of sera is not necessary.
  1. 1.

    Stem Cell Accelerator-PDS (Platelet-poor Plasma Derived Serum) (Animal Technologies). PDS supports the growth of murine primitive erythroid progenitors [24] (see Note 5).

     
  2. 2.

    BIT 9500 Serum Substitute (Stem Cell Technologies): BSA, insulin and transferrin.

     

2.6 Preparation of Methylcellulose Stock Solution (See Note 6)

  1. 1.

    Autoclave a 2 l Erlenmeyer flask containing a large stir bar and 27 g of methylcellulose powder (Sigma). Use two layers of aluminum foil over top of flask. Do not use a bottle with a screw cap.

     
  2. 2.

    While the methylcellulose is still warm, in a laminar flow hood, add 500 ml 37 °C pre-warmed sterile tissue culture grade water. While stirring the methylcellulose mix, carefully bring mixture to a boil. Take care not to burn the methylcellulose on the bottom of the flask. Immediately reduce the heat and stir for 2–4 h. Repeat the boiling process once more and then stir for 2–4 h. Do not allow the methylcellulose to boil over thereby compromising sterility.

     
  3. 3.

    Pre-warm the 500 ml of sterile filtered 2× IMDM/NaHCO3/Pen/Strep (Subheading 2.4, item 6) to 37 °C and add it to the warm methylcellulose mix (37 °C) in the laminar flow hood. Stir at room temperature for 1 h. This results in a 2.6% methylcellulose stock solution.

     
  4. 4.

    Transfer to the cold room and stir overnight. The cloudy solution will be clear by morning.

     
  5. 5.

    In a laminar flow hood, sterily pour 30–40 ml of the methylcellulose into 50 ml conical tubes and freeze at −20 °C. Methylcellulose must be frozen and thawed before use.

     
  6. 6.

    Thaw the methylcellulose stock solution in the refrigerator overnight before use. Never thaw methylcellulose at room temperature or 37 °C. Frozen methylcellulose stock solution is good for up to 1 year in a nonfrost free freezer.

     

2.7 Cell Enumeration and Staining

  1. 1.

    Hemocytometer or cell counter.

     
  2. 2.

    Trypan blue stain.

     
  3. 3.

    Benzidine stock: 2 mg/ml benzidine dihydrochloride in 0.5% acetic acid. This stock solution can be stored in the dark at room temperature for several months. Care must be taken when handling benzidine since it is a carcinogen.

     
  4. 4.

    Working solution for benzidine staining: 1 ml of the benzidine stock with 3 μl of 50% hydrogen peroxide. The final concentration of hydrogen peroxide is 0.15%. This working solution is best made daily but can be stored for up to 1 week in a dark tube at 4 °C.

     

3 Methods

Studies of the spatial and temporal distribution of erythroid progenitors and their response to various stimuli and perterbations have been carried out most extensively in the mouse [10]. During embryogenesis, erythroid progenitors first emerge in the yolk sac and are subsequently found in the fetal liver. In the adult mouse, the bone marrow is the predominant site of red cell synthesis with the spleen playing a secondary role in steady state and an expanded role following the induction of acute anemia. Specific hematopoietic tissues are isolated and dissociated into single cells, and the single cells are plated in semisolid media with serum and cytokine(s). Hematopoietic progenitors generate colonies of maturing blood cells that are identified and enumerated on an inverted microscope at specific days of in vitro culture.

3.1 Isolation of Bone Marrow Cells

  1. 1.

    Mice are euthanized as per institutional protocols.

     
  2. 2.

    Dissect the femur and remove the skin and muscle.

     
  3. 3.

    Cut off both ends of the femur and flush out the marrow using 4 ml of PB2 with a 22 gauge needle, collecting the cells in a 15 ml tube.

     
  4. 4.

    Since bone marrow cells will be clumped together, pipette up and down several times through a 1 ml pipette tip to generate a single-cell suspension.

     
  5. 5.

    To determine the total number of erythroid progenitors per femur, collect and count the marrow cells prior to plating a known number of cells.

     

3.2 Generation of Timed Mouse Pregnancies

  1. 1.

    Mice are mated overnight by placing one male into a cage with 3–4 females. Mating is assumed to occur at the mid-point of the dark cycle. Vaginal plugs are checked the next morning, with noon of that day defined as embryonic day 0.5 (E0.5) if the dark cycle runs from 6 PM to 6 AM.

     
  2. 2.

    Staged embryos are derived from timed pregnant mice. At specific times, pregnant mice are euthanized by approved animal care protocols.

     
  3. 3.

    The uteri are removed from the peritoneum and washed with several changes of PB-2.

     

3.3 Embryo Dissection and Staging

The dissection strategy depends on the age/stage of the mouse embryos [34].
  1. 1.

    For E6.5–E8.5 conceptuses, the uterine muscle is removed using #5 dissecting forceps and the decidual tissue is split into two halves starting from the mesometrial side. Subsequently, a tear is carefully made in Reichert’s membrane and the embryos are freed from the decidual tissue. The conceptuses are transferred with a pipetteman to a fresh 35 mm dish containing 2–3 ml of PB-2 and completely freed of Reichert’s membrane.

     
  2. 2.

    Fetal livers can be dissected away from the remaining embryo proper starting at E10.5.

     
  3. 3.

    Because the mouse embryo develops extremely rapidly and there can be wide intra- and inter-litter variation, it is important to determine the stage of the dissected embryos using morphologic criteria (see Note 7).

     

3.4 Digestion and Single-Cell Preparation

Progenitor assays depend on plating a single-cell suspension in semisolid media to ensure that each colony is clonal in nature. Collagenase or trypsin each can provide effective cell separation of E6.5–E11.5 embryos with equivalent high cell viabilities and hematopoietic progenitor plating efficiencies (see Note 8). Embryos older than E11.5 must be dissociated with collagenase. However, as the embryo ages and organogenesis proceeds, it becomes increasingly difficult to obtain complete cell dissociation with good viabilities. The volume of trypsin or collagenase depends on the amount of tissue being dissociated. It is important to have as little of the dissection media present during the digestion with trypsin.

3.4.1 Dissociation of Whole Embryos and Yolk Sacs with Trypsin

  1. 1.

    Embryo proper and yolk sac tissues are treated in 0.25% trypsin/1× PBS/1 mM EDTA at 37 °C for 5–7 min (see Note 4). 5–15 yolk sacs from E7.5 embryos are dissociated in 0.2 ml of trypsin/EDTA.

     
  2. 2.

    After 3 min the tissue is triturated with a pipette to aid in digestion and checked microscopically to determine if the tissues are completely dissociated.

     
  3. 3.

    If dissociation is not complete, the dishes are incubated at 37 °C for another 2–5 min and again triturated.

     
  4. 4.

    Once dissociated, one half volume of IMDM/10% PDS is added to stop the action of the trypsin.

     
  5. 5.

    The cells are pelleted by centrifugation, resuspended and live cells counted after staining with trypan blue.

     

3.4.2 Dissociation of Fetal Livers with Trypsin

  1. 1.

    Fetal livers from E10.5–E12.5 mouse embryos are incubated for 2–3 min in 0.25% trypsin/EDTA and then mechanically disrupted with a 1 ml pipet.

     
  2. 2.

    The fetal livers of E14.5 or later mouse embryos do not require enzymatic digestion, since mechanical trituration in PBS/EDTA with a 1 ml pipet tip is sufficient to generate a single-cell suspension.

     
  3. 3.

    Once dissociated, one half volume of IMDM/10% PDS is added to stop the action of the trypsin.

     
  4. 4.

    The cells are pelleted by centrifugation, resuspended, and live counted after staining with trypan blue.

     

3.4.3 Dissociation with Collagenase

  1. 1.

    Tissues are incubated in 2.5 mg/ml collagenase Type 1/PB2 at 37 °C for 10 min. 5–15 yolk sacs or embryo propers from E7.5 embryos can be dissociated in 1–2 ml of collagenase/PB2.

     
  2. 2.

    Add 1 ml PBS/1 mM EDTA, pipette 10× with a 1 ml pipette tip and return to the incubator for another 10 min.

     
  3. 3.

    Pipette 10× with a 1 ml pipette tip.

     
  4. 4.

    Pellet by centrifugation, resuspend and count cells after staining with trypan blue.

     

3.5 Methylcellulose Plating

  1. 1.

    Add reagents for BFU-E, CFU-E or EryP-CFC colony assays in the order listed in Table 1 or 2 to a 50 ml tube (i.e., methylcellulose will be added last with a needleless syringe, see step 2). Vortex on high for 1 min for complete mixing of the reagents with the viscous methylcellulose. Keep on ice or at 4 °C until use. Allow the bubbles to surface for 15–20 min. before plating.

     
  2. 2.

    For each sample to be plated, aliquot 1.4 ml of the methylcellulose into a 5 ml tube, using a syringe with a 16-gauge needle since the methylcellulose is very viscous. Keep on ice or at 4 °C until use.

     
  3. 3.

    It is necessary to optimize the number of cells plated per ml of semisolid media (see Note 9). Plate enough cells to count between 25–200 CFU-E colonies and 20–50 BFU-E colonies per 35 mm dish. Add cells in 0.14 ml of Pb2 or IMDM/PDS at the appropriate concentration of cells per ml (see Note 10).

     
  4. 4.

    Vortex vigorously and allow the bubbles to surface for 15–20 min on ice. This ensures even mixing of the cells in the methylcellulose.

     
  5. 5.

    Use a 3 ml syringe with a 16 gauge needle to plate 1 ml into each 35 mm culture dish.

     
  6. 6.

    Swirl the dishes enough to distribute the methylcellulose evenly.

     
  7. 7.

    Place the 35 mm dishes in the 245 × 245 square dishes with a 60 mm dish filled with sterile water. The increase in humidity helps to prevent desiccation of the methylcellulose.

     
  8. 8.

    Cultures are incubated at 37 °C, 5% CO2, for 2–14 days for mouse cultures and 7–20 days for human cultures as needed to enumerate colony numbers (see Subheading 3.6).

     

3.6 Colony Identification and Enumeration

  1. 1.

    The identification of hematopoietic colonies in semisolid media is often difficult, though erythroid colonies are easily recognized by the presence of hemoglobinizing cells (see Note 11). Murine CFU-E-derived colonies are extremely small with faint red color because of the small number of cells comprising the colonies (Fig. 1). Mouse CFU-E-derived colonies are best enumerated at 2–3 days of culture using a 10× objective. Late-stage mouse BFU-E-derived colonies are larger (Fig. 1) and are enumerated at 3 days of culture. Immature mouse BFU-E-derived colonies typically, but not always, have a distinctive burst morphology and are enumerated at 7–10 days of culture with a 4× objective (Fig. 1). Mouse EryP-CFC-derived colonies are composed of larger primitive erythroblasts and have a more orange-red color (Fig. 1). Murine EryP-CFC-derived colonies are enumerated at 5 days of culture.

     
  2. 2.

    Place the 35 mm dishes in 60 mm gridded dishes to aid in colony counts by the use of the grid lines.

     
  3. 3.

    Benzidine staining can be used to identify erythroid colonies or the erythroid component of mixed colonies in methylcellulose [35]. This approach is helpful in enumerating CFU-E-derived colonies and for trouble shooting plates that do not contain red colonies (see Note 12). Layer 0.5 ml of the benzidine working solution onto the methylcellulose dish. Dark blue-brown erythroid colonies are evident after 5–20 min of incubation at room temperature.

     

4 Notes

  1. 1.

    Strains of mice differ both in hematopoietic progenitor numbers and in progenitor kinetics during embryogenesis. Individual variability in progenitor numbers can be mitigated by pooling tissues from several similarly staged embryos prior to plating. Because the total number of cells in the embryo increases exponentially, we have found it more useful to compare total hematopoietic progenitor numbers per tissue rather than their frequency [24].

     
  2. 2.

    Non-tissue culture treated 35 mm dishes (Corning 430588). It is necessary to use non-tissue culture treated 35 mm dishes for plating of hematopoietic progenitors in methylcellulose. The plated cells will stick to the bottom of the wells of tissue culture treated dishes and fail to form colonies.

     
  3. 3.

    PFHM II: In our experience this reagent has a shelf life of 3–4 months after opening the bottle. We have not been able to aliquot and freeze PFHM II due to a persistent yellow precipitate that forms after freezing. It is pH sensitive and once it has turned pink it should be discarded. We do not aliquot PFHM II into smaller tubes but just access it from the 500 ml original bottle.

     
  4. 4.

    Each lot of trypsin must be tested to determine the optimum concentration to maximize dissociation and minimize cell death. We test serial dilutions (1:2–1:64) of the 0.25% trypsin in PBS/1 mM EDTA. We have also found that some lots of trypsin can interfere with colony growth, particularly of primitive erythroid progenitors. Optimum concentrations of trypsin provide for effective cell dissociation and produce minimal cell death.

     
  5. 5.

    PDS will form a white precipitate upon thawing that can be removed by filtration or letting the particulate matter settle after mixing. The particulate matter does not affect the assay.

     
  6. 6.

    Each batch of methylcellulose must be tested to determine the final concentration for optimal colony growth. We have found that final methylcellulose concentrations between 1.1 and 1.5% (i.e., 50–60% of the stock solution) work best. Growth factors, serum, and other reagents listed below are added to the stock methylcellulose solution. Premade preparations are now readily available and supplied in a 2.2% concentration with IMDM. Premade preparations that contain only methylcellulose and medium without additional cytokines, supplements or reducing agents can be used to facilitate supplementation to support the growth of specific hematopoietic progenitors. If using a premade stock, add 1× Pen/strep to the mix. Stem Cell Technologies has two products that can be used for human and mouse cultures, M3134 and H4100, respectively.

     
  7. 7.

    There are established gestational “times” at which specific stages of mouse embryos are typically found. However, there is significant variation in the stage of embryos both within a litter and between litters. We use the criteria of Downs and Davies [36] for presomite stage embryos (E6.5–E8.25), somite counts between E8.25–E10.5, and external morphological criteria between E10.5 and birth [36, 37].

     
  8. 8.

    We have found that mechanical dissociation with syringes and other non-enzymatic approaches, such as the use of glycine, have led to high levels of cell death when dissociating mouse embryos.

     
  9. 9.

    If plating density is too high, it becomes difficult to distinguish one colony from another and nutrients can be depleted before the colonies are scored. If plating density is too low, statistically meaningful data becomes difficult to generate. The number of cells plated will vary depending on (1) the colony types to be assayed, (2) the stage of the embryo, and (3) the tissue source of the cells. Plating serial dilutions of the cells is helpful in establishing an optimum cell number per dish. A good starting range is 5 × 103 cells/ml to 1 × 105 cells/ml. It is often useful to plate more than one concentration and to count the dishes with appropriate numbers of colonies.

     
  10. 10.

    We have used an alternative plating strategy when cell numbers are limiting. 1 ml of the methylcellulose mix is plated into each 35 mm dish and the cells, resuspended in 0.1 ml of IMDM/10% PDS, are added directly to the dish. The tip of the pipette is used to mix the cells with the methylcellulose. This method leads to an uneven distribution of cells in the dishes, necessitating scoring of all the colonies in the dish. However, this plating method has the advantage of overcoming the loss of 30–40% of the cells because of the viscous nature of methylcellulose.

     
  11. 11.

    The identity of the cellular components of an individual colony can be confirmed by transferring the colony onto microscope slides to evaluate cell morphology. The colony is plucked out of methylcellulose, transferred into 0.2–0.3 ml of PBS/50% serum, dispersed by pipetting, and cytospun at 400 RPM for 3–5 min on a Shandon cytocentrifuge. After air drying, the cells on the slide are stained with May-Grunwald-Giemsa or with Wright’s stain and examined to determine the hematopoietic cell types present. Erythroid colonies should only contain maturing erythroblasts and some reticulocytes.

     
  12. 12.

    The growth of primitive erythroid and definitive erythroid colonies requires different reducing agents and differs in the need for BIT. In addition, EryP-CFC-derived cultures are particularly sensitive to several factors, including the trypsin used to dissociate the yolk sac, as well as the activity of the reducing agents and of PFHM II. If one or more of these reagents are not optimal then the primitive erythroblasts will not hemoglobinize well in vitro and the colonies will be a pale red color. We have tried several different suppliers of trypsin and find Worthington to work best for us. The standard reducing agent, beta mercaptoethanol does not support primitive erythroid colony growth.

     

Notes

Acknowledgments

We thank Gordon Keller and Marion Kennedy for sharing so generously of their knowledge and expertise of tissue culture and hematopoietic progenitor colony assays. Scott Peslak and Paul Kingsley photographed the erythroid colonies. This work has been supported by funds from the National Institutes of Health and from the Strong Children’s Research Center, University of Rochester, Rochester, NY.

References

  1. 1.
    Sun J, Ramos A, Chapman B et al (2014) Clonal dynamics of native hematopoiesis. Nature 514:322–327CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Busch K, Klapproth K, Barile M et al (2015) Fundamental properties of unperturbed haematopoiesis from stem cells in vivo. Nature 518:542–546CrossRefPubMedGoogle Scholar
  3. 3.
    Pluznik DH, Sachs L (1965) The cloning of normal “mast” cells in tissue culture. J Cell Comp Physiol 66:319–324CrossRefGoogle Scholar
  4. 4.
    Bradley TR, Metcalf D (1966) The growth of mouse bone marrow cells in vitro. Aust J Exp Biol Med Sci 44:287–300CrossRefPubMedGoogle Scholar
  5. 5.
    Stephenson JR, Axelrad A, McLeod D, Shreeve M (1971) Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc Natl Acad Sci U S A 68:1542–1546CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Heath DS, Axelrod AA, McLeod DL, Shreeve MM (1976) Separation of the erythropoietin-responsive BFU-E and CFU-E in mouse bone marrow by unit gravity separation. Blood 47:777–792PubMedGoogle Scholar
  7. 7.
    Iscove NN, Sieber F (1975) Erythroid progenitors in mouse bone marrow detected by macroscopic colony formation in culture. Exp Hematol 3:32–43PubMedGoogle Scholar
  8. 8.
    Koury MJ, Bondurant MC (1990) Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells. Science 248:378–381CrossRefPubMedGoogle Scholar
  9. 9.
    Wojchowski DM, Sathyanarayana P, Dev A (2010) Erythropoietin receptor response circuits. Curr Opin Hematol 17:169–176Google Scholar
  10. 10.
    Koury MJ (2016) Tracking erythroid progenitor cells in times of need and times of plenty. Exp Hematol 44:653–663CrossRefPubMedGoogle Scholar
  11. 11.
    Cooper MC, Levy J, Cantor LN, Marks PA, Rifkind AR (1974) The effect of erythropoietin on colonial growth of erythroid precursor cells in vitro. Proc Natl Acad Sci U S A 71:1677–1680CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gregory CJ, Eaves AC (1977) Human marrow cells capable of erythropoietic differentiation in vitro: definition of three erythroid colony responses. Blood 49:855–864PubMedGoogle Scholar
  13. 13.
    Eliason JF, Van Zant G, Goldwasser E (1979) The relationship of hemoglobin synthesis to erythroid colony and burst formation. Blood 53:935–945PubMedGoogle Scholar
  14. 14.
    Gregory CJ (1976) Erythropoietin sensitivity as a differentiation marker in the hemopoietic system: studies of three erythropoietic colony responses in culture. J Cell Physiol 89:289–301CrossRefPubMedGoogle Scholar
  15. 15.
    Axelrad AA, McLeod DL, Shreeve MM, Heath DS (1974) Properties of cells that produce erythrocytic colonies in vitro. In: Robinson WA (ed) Proceedings of the Second international workshop on hemopoiesis in culture, DHEW publication no NIH 74-205. US Government Printing Office, Washington, DC, pp p226–p234Google Scholar
  16. 16.
    Sonoda Y, Yang YC, Wong GG et al (1988) Erythroid burst-promoting activity of purified recombinant human GM-CSF and interleukin-3: studies with anti-GM-CSF and anti-IL-3 sera and studies in serum-free cultures. Blood 72:1381–1386PubMedGoogle Scholar
  17. 17.
    Mitjavila MT, Natazawa M, Brignaschi P et al (1989) Effects of five recombinant hematopoietic growth factors on enriched human erythroid progenitors in serum-replaced cultures. J Cell Physiol 138:617–623CrossRefPubMedGoogle Scholar
  18. 18.
    Sonoda Y, Sakabe H, Ohmisono Y et al (1994) Synergistic actions of stem cell factor and other burst-promoting activities on proliferation of CD34+ highly purified blood progenitors expressing HLA-DR or different levels of c-kit protein. Blood 84:4099–4106PubMedGoogle Scholar
  19. 19.
    Gregory CJ, McCollough EA, Till TA (1973) Erythropoietic progenitors capable of colony formation in culture: state of differentiation. J Cell Physiol 81:411–420CrossRefPubMedGoogle Scholar
  20. 20.
    Peslak SA, Wenger J, Bemis JC et al (2012) EPO-mediated expansion of late-stage erythroid progenitors in the bone marrow initiates recovery from sublethal radiation stress. Blood 120:2501–2511CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Clarke BJ, Housman D (1977) Characterization of an erythroid precursor cell of high proliferative capacity in normal human peripheral blood. Proc Natl Acad Sci U S A 74:1105–1109Google Scholar
  22. 22.
    Hara H, Ogawa M (1977) Erythropoietic precursors in murine blood. Exp Hematol 5:161–165PubMedGoogle Scholar
  23. 23.
    Wong PMC, Chung SW, Reicheld SM, Chui DH (1986a) Hemoglobin switching during murine embryonic development: evidence for two populations of embryonic erythropoietic progenitor cells. Blood 67:716–721PubMedGoogle Scholar
  24. 24.
    Palis J, Robertson S, Kennedy M, Wall C, Keller G (1999) Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126:5073–5084PubMedGoogle Scholar
  25. 25.
    Wong PMC, Chung SW, Chui DHK, Eaves CJ (1986b) Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac. Proc Natl Acad Sci U S A 83:3851–3854CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lux C, Yoshimoto M, McGrath KE et al (2008) All primitive and definitive hematopoietic progenitor cells emerging prior to E10 in the mouse embryo are products of the yolk sac. Blood 111:3435–3438CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    McGrath KE, Frame JM, Fromm GJ, Koniski AD, Kingsley PD, Little J, Bulger M, Palis J (2011) A transient definitive erythroid lineage with unique regulation of the beta-globin locus in the mammalian embryo. Blood 117:4600–4608CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Zimmermann F, Rich IN (1996) The sensitivity of in vitro erythropoietic progenitor cells to different erythropoietin reagents during development and the role of cell death in culture. Exp Hematol 24:330–339PubMedGoogle Scholar
  29. 29.
    Emerson SG, Thomas S, Ferrara JL, Greenstein JL (1989) Developmental regulation of erythropoiesis by hematopoietic growth factors: analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver. Blood 74:49–55PubMedGoogle Scholar
  30. 30.
    Valtieri M, Gabbianelli M, Pelosi E et al (1989) Erythropoietin alone induces erythroid burst formation by human embryonic but not adult BFU-E in unicellular serum-free culture. Blood 74:460–470PubMedGoogle Scholar
  31. 31.
    Perry JM, Harandi OF, Paulson RF (2007) BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors. Blood 15:4494–4502CrossRefGoogle Scholar
  32. 32.
    Alter BP (1979) Fetal erythropoiesis in stress hematopoiesis. Exp Hematol 7(Suppl 5):200–209PubMedGoogle Scholar
  33. 33.
    Porayette P, Paulson RF (2008) BMP4/Smad5 dependent stress erythropoiesis is required for the expansion of erythroid progenitors during fetal development. Dev Biol 317:24–35CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Hogan BLM, Beddington RSP, Constantini F, Lacy E (1994) Manipulating the mouse embryo: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, PlainviewGoogle Scholar
  35. 35.
    Hara H, Ogawa M (1978) Murine hematopoietic colonies in culture containing normoblasts, macrophages, and megakaryocytes. Am J Hematol 4:23–34CrossRefPubMedGoogle Scholar
  36. 36.
    Downs KM, Davies T (1993) Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope. Development 118:1255–1266PubMedGoogle Scholar
  37. 37.
    Kaufman MH (1992) The atlas of mouse development. Academic, New YorkGoogle Scholar

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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Pediatrics, Center for Pediatric Biomedical ResearchUniversity of Rochester Medical CenterRochesterUSA

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