Isolation and Colony Formation of Murine Bone and Bone Marrow Cells

Abstract

Adult homeostasis is dependent on normal Wt1 expression. Loss of Wt1 expression in adult mice causes rapid loss of the mesenchymal tissues, fat and bone, amongst other phenotypes. Bone and bone marrow mesenchymal stromal cells can be studied by cell isolation and expansion. The stemness of these cells can then be characterized by carrying out a colony-forming unit-fibroblast assay and observing clonogenic capabilities.

1 Introduction

Wt1 is a major regulator of adult homeostasis. The tamoxifen -inducible ubiquitous knockout of Wt1 in adult mice results in multiple organ failure within 9 days following tamoxifen-induced deletion [1]. The phenotypes include rapid fat and bone loss; trabecular bone volume is decreased by 30 % and the inner surface of the bone is ragged compared to controls [1]. Adult bone mass is a balance between synthesis from osteoblasts (arising from the mesenchymal stem/progenitors ) and turnover by osteoclasts (arising from the hematopoietic progenitors). Increased osteoclast numbers are observed within the mutant bone marrow [1]. In addition, preliminary experiments show that mutant mesenchymal cells are less capable of differentiating into osteoblasts. The size of fat pads and fat vacuoles are also decreased following Wt1 deletion, as well as adipocyte numbers in bone marrow [1]. Osteoblasts and adipocytes, along with chondrocytes, all differentiate from stromal mesenchymal stem cells (MSCs ) found in the bone [2]. Interestingly, around 10 % of Wilms’ tumors contain heterologous factors that include fat, bone, and cartilage [3]. A recent study looking at the relationship between MSCs and the mesenchymal properties of Wilms’ tumors with WT1 mutations showed that a Wilms’ tumor cell line expresses surface proteins specific to human MSCs including CD105 , CD90 , and CD73 and that are able to differentiate into chondrocytes, osteoblasts, and adipocytes [4]. These findings support the involvement of Wt1 in MSC biology.

There are compelling parallels between different Wt1-expressing mesenchymal cell populations that suggest links with mesenchymal stem/progenitor cells. Wt1-expressing mesothelial cells in the developing liver are stellate cell precursors [5–7], while Wt1 is also expressed in the pancreatic mesothelium and pancreatic stellate cells [1]. The parallels between pancreatic and hepatic stellate cells suggest that pancreatic stellate cells may also be arising from the mesothelium, via epithelial to mesenchymal transition [1, 7, 8]. Stellate cells have spindle-shaped bodies similar in appearance to bone marrow cells cultured under MSC -favoring conditions [9] and are also positive for nestin [10], linking them with the nestin-expressing MSCs [11]. Stellate cells may originate from both the mesothelium - and bone marrow-derived HSCs and could possibly be another subset of the Wt1-expressing cells [6, 12]. Hepatic stellate cells share striking similarities with pericytes [13, 14]. A FACS -sorted pericyte population from the bone marrow was shown to include cells with MSC properties [15]. In the kidney , podocytes , which similarly to pericytes and stellate cells also have a spindle-shaped appearance due to their long processes or “feet”, express both Wt1, which is a podocyte regulator [16], and nestin [17]. In the developing kidney and heart , nestin and Wt1 expressions overlap. Nestin is potentially regulated by Wt1 as its expression is reduced drastically following the loss of Wt1 in these tissues [18]. Wt1 is required for mesenchymal to epithelial transition (MET) in the developing kidney as Wt1 deletion in kidney mesenchyme leads to failure of MET and nephron formation [19]. The opposite is seen in heart development where Wt1 is required for epithelial to mesenchymal transition (EMT) [20]. Mesenchymal progenitor cells are formed when the epicardial cells undergo EMT. An epicardial-specific Wt1 knockout resulted in a loss of these mesenchymal progenitor cells [20].

However, the most striking phenotype to support Wt1’s involvement with mesenchymal lineages is the effect of Wt1 deletion [1]. Bone and fat are both mesenchymal derivatives, strongly suggesting that Wt1 is playing a functional role in the genesis and homeostasis of these mesenchymal tissues, and may even act as a functional marker for a subset of mesenchymal stem or progenitor cells.

There is considerable topical interest in tissue stem and progenitor cells, particularly with regard to potential uses in therapy. Bone marrow is largely split into two halves: the hematopoietic and the non-hematopoietic. The hematopoietic component consists of HSCs, hematopoietic progenitor cells, and their derivatives. The non-hematopoietic component is made up mainly of endothelial and mesenchymal stem cells (MSC ) plus their derivatives, including adipocytes, osteoblasts , and chondrocytes [21]. It has been much more difficult to define mesenchymal stem and progenitor cells for different lineages than it has been for the hematopoietic compartment.

The following protocols detail how to isolate bone and marrow cells and how to carry out colony-forming unit-fibroblast assays to characterize the mesenchymal stromal compartment.

2 Protocols

2.1 Isolation of Murine Bone Marrow and Bone Mesenchymal Progenitors

2.1.1 Introduction

Embryonic bone development takes place when a core of prechondrogenic cells is surrounded by stacked osteogenic progenitor cells, all of which are avascular. This central core and surrounding stacked cells differentiate into cartilage to give a cartilage rod or “model”. The stacked cells then give rise to osteoblasts [22, 23]. Osteoblasts are located in close proximity to blood vessels with the “back” of the cell toward the capillary and the “front” of the cell secreting osteoid: a collagen type I-rich premineral layer [22, 23]. Vasculature is the dominant factor for the position and survival of the osteoblast cells, unlike chondrogenesis which actively inhibits vascularization [24, 25]. For this reason bone does not replace cartilage, but cartilage is invaded by vascular cells to give the resulting marrow cavity [23], and bone forms independent of the cartilage [24, 25]. In mice, bone development begins with mesenchyme condensing at embryonic day 9.5 (e9.5). By e12 the commitment of mesenchymal cells to the osteogenic lineage occurs, followed by commitment to the chondrogenic lineage on day 13, finishing with vasculature and marrow invasion of the cartilage occurring on days 16–17 of development [25, 26].

Primitive marrow is formed when the chondrocyte core is invaded by vasculature to form the marrow cavity [23]. As previously mentioned, postnatal bone marrow is an organ of two halves: the hematopoietic cells, including the hematopoietic stem cells (HSCs), and the non-hematopoietic cells [27]. The non-hematopoietic cells are also known as the bone marrow stroma, or associated supporting stroma, and this is where the mesenchymal stem cell resides [27]. The bone marrow is the only known organ where two separate stem cells, MSCs and HSCs, with distinct lineage pathways are located and found to interact together to coexist [27].

2.1.2 Materials

• 1. Culture medium:

  DMEM containing 10 % (v/v) fetal calf serum, 1 % (v/v) penicillin/streptomycin, 0.5 % (v/v) glutamine, and 0.5 % (v/v) sodium pyruvate.

• 2. Sterile dissection kit.

• 3. 25-gauge needle.

• 4. 21-gauge needle.

• 5. 10 mL Syringe.

• 6. 10 cm Culture dish.

• 7. Pestle and mortar.

• 8. Collagenase B (Roche).

• 9. Rocking chamber heated to 37 °C.

• 10. 70 micron cell strainer (Falcon).

• 11. Phosphate-buffered saline (PBS), pH 7.4.

• 12. Bench centrifuge with swinging bucket rotor.

• 13. Sterile cell culture plastic pipettes individually wrapped (5, 10, 25 mL).

• 14. Pipette aid.

• 15. Sterile conical centrifuge tubes (15 and 50 mL).

• 16. Cell aspirator.

2.1.3 Methods

2.1.3.1 2.1.3.1 Isolation of Bone Marrow Cells

• 1. Euthanize mice by cervical dislocation and dissect out the bilateral femur and tibia.

• 2. In a sterile culture hood and with a sterile dissection kit remove each end of the long bones and set aside. Carry this out in a 10 cm culture dish. Add a small amount of culture medium to prevent the tissue and bone drying out.

• 3. Using 8 mL of culture medium flush out the bone marrow from the long bones using the 10 mL syringe and 25-gauge needle.

• 4. Dissociate the flushed marrow cells using a 21-gauge needle.

2.1.3.2 2.1.3.2 Isolation of Bone Cells

• 1. Prepare the collagenase B at a concentration of 3 mg/mL dissolved in culture medium and kept on ice.

• 2. Crush the pre-flushed long bones and bone ends by pestle and mortar in 10 mL collagenase B until a pulp-like consistency is reached.

• 3. Transfer the bone pulp and 10 mL collagenase B to a sterile 15 mL tube and incubate at 37 °C for 90 min in a rocking chamber to enable enzymatic digestion.

• 4. Pass the bone cells through a 70 micron cell strainer into a sterile 50 mL tube.

• 5. Centrifuge the tube at 300 rcf for 5 min at room temperature in a swinging bucket rotor. Remove the enzyme-containing supernatant by vacuum aspiration and resuspend the cell pellet in pre-warmed culture medium.

2.1.4 Notes

CFU-F assays and MSC isolation are usually carried out using marrow cells flushed from the bone. This protocol uses isolated cells from both marrow and bone which are crushed with a collagenase digest [28]. This extra bone digest step is included as MSCs are located near the endosteum of the bone, in the bone-lining endosteal region, where they interact with bone-lining osteoblasts [29]. Cells in this area are difficult to remove by flushing alone.

3 Colony-Forming Unit-Fibroblast Assay

3.1 Introduction

Colony-forming unit-fibroblast (CFU-F) assays are often used to characterize stromal marrow cells and assess the number of mesenchymal stem and progenitor cells [27, 30, 31]. The bone and fat loss phenotypes seen from the ubiquitous Wt1 deletion point to the involvement of mesenchymal stromal cells. This makes the CFU-F assay an appropriate tool to investigate its involvement. Adherent stromal cells form colonies which originate from a single cell: the CFU-F [27, 31–34]. There are several methods of obtaining material for CFU-F assays, most commonly the flushing of bone marrow, but also including the crushing of pre-flushed long bones followed by enzymatic digestion [27, 31–34]. The isolation of these cells is covered previously in this chapter.

Proliferation without terminal differentiation, a stem cell characteristic, is demonstrated using CFU-F assays [32], proving that a single cell can result in a colony of cells. Mesenchymal stem cells, or mesenchymal stromal cells (MSCs ), are commonly described as spindle-shaped multipotent cells able to self-replicate and generate progenitors that produce a variety of skeletal tissues: bone, cartilage, marrow stroma, fat, ligament, tendon, and connective tissue. MSCs are mainly found in the bone marrow; however they make up a very small proportion of the total number of marrow cells, between 0.01 and 0.001 % [35]. Bone marrow contains MSCs which can undergo chondrogenic, osteogenic, and adipogenic differentiation to form cartilage, bone, and fat, respectively [35, 36]. MSCs are also found in bone fragments which have been crushed and digested with a collagen enzyme. These cells are also able to differentiate into the three main mesenchymal lineages [37].

3.2 Materials

• 1. Hemocytometer.

• 2. Sterile 6-well culture plates.

• 3. MesenCult^® media.

• 4. 0.5 % (w/v) Cresyl violet acetate (Sigma) in methanol: Filter through 25 μm filter paper and store at room temperature.

• 5. Incubator.

3.3 Methods

• 1. Count the cells with a hemocytometer.

• 2. Plate 5×10⁵ cells per well of a 6-well culture plate with 2 mL of MesenCult^® media per well.

• 3. Incubate the plates for 48 h to allow adherent cells to attach.

• 4. Wash with pre-warmed PBS to remove media and nonadherent cells.

• 5. Add 2 mL pre-warmed MesenCult^® to each well and culture.

• 6. Every 3 days, remove the media and replace with fresh MesenCult^® for 10 days. Check that the colonies are not becoming too confluent.

• 7. Wash plates with PBS.

• 8. Remove the PBS and stain colonies with 0.5 % Cresyl violet acetate solution for 30 min at room temperature.

• 9. Wash plates with PBS three times, followed by a single wash with distilled H₂O.

• 10. Remove H₂O and leave to dry.

• 11. Count colonies with a diameter greater than 1 mm.

3.4 Notes

Higher numbers of colonies form in the bone CFU-F assays compared to the marrow [28, 33, 38]. The trabecular bone is an enriched source of mesenchymal progenitors which corroborates with higher numbers of bone colonies [31, 39].

Due to the bone loss phenotype, the effect of Wt1 on colony-forming abilities was investigated using mice with a tamoxifen -inducible Wt1 deletion (CAGG-CreER^T2 ; Wt1 ^loxP/loxP) [38]. It is difficult to determine whether Wt1 deletion has any effect on colony formation due to the Cre-only control showing the same trend, potentially masking any gene loss effect [38] (Fig. 1). The results suggest that, at least for colony-forming capabilities, bone and marrow cells are affected by the activated CreER recombinase, rather than Wt1 deletion. This raises a great concern as to whether activated CreER recombinase is the cause of other phenotypes seen in published studies using this construct and deletion method.

Fig. 1

Activated CreER^T2 recombinase negatively affects colony-forming capabilities

The appropriate Cre-only control should be included if using CFU-F assays in conjunction with the Cre-loxP system of gene knockout. It is important to control for not only Cre effects but also gender and tamoxifen [38].