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. 2018 May 5;8(9):e2824.
doi: 10.21769/BioProtoc.2824.

Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells

Surya Amarachintha  1 , Qishen Pang  2
Affiliations

Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells

Surya Amarachintha et al. Bio Protoc. .

Abstract

Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution CAFC assay.

Keywords: Co-culture assay; Cobblestone area-forming cell assay; Hematopoietic Stem Cells; Mesenchymal Stromal Cells.

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Figures

Figure 1.
Figure 1.. Mouse Bone Marrow Mesenchymal stromal cells.
Plastic adhered MSCs were shown in phase contrast image. Scale bar represents 50 µm.
Figure 2.
Figure 2.. Representative images of Mouse BM MSCs stained with antibodies Fabp4, Collagen II, and Osteopontin to identify adipocytes, chondroblasts, and osteoblasts lineage cells respectively present in bone marrow derived mesenchymal stromal cells.
Scale bar represents 10 µm.
Figure 3.
Figure 3.. Gating strategy to identify and isolate Hematopoietic stem cells from Mouse Bone Marrow.
Bone Marrow cells flushed from mouse femurs were subjected to Ficoll-Paque PLUS separation to isolate Bone Marrow Mono Nuclear Cells and then stained with HSC cell surface markers to identify and cell-sort using the flow cytometer.
Figure 4.
Figure 4.. Representative image of Cobblestone area forming cell assay.
HSCs are cultured on a confluent layer of MSCs to form the cobblestone areas. Yellow arrow–phase dull cells (true stem cells growing under the MSCs layer), Blue arrow–phase bright cells (stem progenitor cells suspended or loosely attached to MSCs), and Red arrow–MSCs (Confluent cell layer adhered to the plastic dish). Scale bar represents 50 µm.
Figure 5.
Figure 5.. CAFC Limited dilution assay.
A. CAFC assay performed in a 96-well flat-bottom plate. Flow sorted LSK cells were counted and plated on confluent MSCs in a flat-bottom 96-well plate. Each assay was performed in triplicates. Representative images of the cobblestones formed in wells with no LSK cells, 90 and 270 LSK cells. Scale bars represent 100 µm. A portion of the cobblestone area was magnified; Scale bar represents 50 µm. Phase dull cells were identified in a dotted circle while phase bright cells were identified in a solid circle. Both phase dull and phase bright cells need to be present to call it a true cobblestone area. B. Phase dull cell areas were measured in each well as shown in above panel. Percentage of the well covered with cobblestone areas was counted and plotted against the no of LSK cells plated in each well. Cultures were maintained for two weeks and the areas were counted at the end of each week.
See this image and copyright information in PMC

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