Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Summary

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Abstract

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Protocol

1. PREPARATION OF REAGENTS

1. Prepare sterile 1000x stock solutions of Fms-related tyrosine kinase 3 (FLT-3) ligand, granulocyte/macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), thrombopoietin (TPO), interleukin (IL)-3, and IL-6 according to manufacturer's instructions. Prepare sterile Retronectin stock solution (1mg/ml) also according to manufacturer's instructions. Divide these stock solutions into small aliquots and keep at -20 °C to avoid repeated freeze-thaw.
2. Prepare 2% FBS in IMDM.
3. Prepare complete IMDM medium with final concentrations of 20% FBS, 2 mM Glutamine, 100 units/ml Penicillin/Streptomycin (PS). ....

Discussion

The CFC assay has been used extensively to determine the proliferation and differentiation patterns of hematopoietic progenitors and to study the effects of oncogenes (4, 5). It has the advantage over liquid cultures of being a clonal assay, such that the colonies represent the progeny of a single progenitor and can be individually removed for further analysis. The limitation of the CFC assay is that it is not adequate for the detection of more immature progenitors or hematopoietic stem cells; such cells are detected u.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IMDM	Life Technologies	12440
FBS	Stem Cell Technologies	06150
L-Glutamine	Life Technologies	25030
Penicillin/Streptomycin (PS)	Life Technologies	15140
FLT-3 ligand	PeproTech Inc	300-19
GM-CSF	PeproTech Inc	300-03
SCF	PeproTech Inc	300-07
TPO	PeproTech Inc	300-18
IL3	PeproTech Inc	200-03
IL6	PeproTech Inc	200-06
Bovine Serum Albumin	Sigma-Aldrich	A7030
EDTA	Fisher Scientific	BP118
Retronectin	Takara Bio Inc	T100A
HBSS	Life Technologies	14175
PBS	Life Technologies	14200
Trypan Blue solution (0.4%)	Sigma-Aldrich	T8154
Methocult GF+ H4435	Stem Cell Technologies	04445
Human Methylcellulose Enriched Media	R&D Systems	HSC005
Wright/ Giemsa stain	Harleco	64571
Phosphate Buffer Solution, pH 6.4 - Giordano formula	Ricca Chemical Company	1450
Methanol	Fisher Scientific	A412-4
Cytoseal 60	Thermo Fisher Scientific, Inc.	8310
Normal Mouse Serum	Rockland Immunochemicals	D208
Anti-Human CD11b phyc–rythrin-conjugated	BD Biosciences	555388
Anti-Human CD33 allophycocyanin-conjugated	BD Biosciences	551378
Anti-Human CD45 phyc–rythrin-Cy7-congjugated	BD Biosciences	557748
Anti-Human CD71 phyc–rythrin-conjugated	BD Biosciences	555537
Anti-Human CD235a allophycocyanin-conjugated	BD Biosciences	551336
Anti-Human CD45 phyc–rythrin-Cy7-congjugated	BD Biosciences	557748
24-well non-treated tissue culture plates	BD Biosciences	35-1147
30 mm non-treated dish	Stem Cell Technologies	27150
100 mm tissue culture dish	Fisher Scientific	08-757-12
Gridded scoring dishes	Stem Cell Technologies	27500
15 ml centrifuge tubes	BD Biosciences	35-2097
50 ml centrifuge tubes	BD Biosciences	35-2070
Syringes 3 ml	Stem Cell Technologies	28240
16 gauge blunt-end, 1½ inch needle	Stem Cell Technologies	28110
50 μm CellTrics cell filter	Partec	04-004-2327
Hemocytometer	Fisher Scientific	0267110
TPX sample chambers	Thermo Fisher Scientific, Inc.	A78710018
Fisherbrand Superfrost/Plus Microscope Slides, Precleaned	Fisher Scientific	12-550-15
Shandon filter cards	Thermo Fisher Scientific, Inc.	5991022
Shandon cytospin slide holder	Thermo Fisher Scientific, Inc.	59920063
Shandon Complete Staining Assembly 100	Thermo Fisher Scientific, Inc.	100
Kimwipes	Kimberly-Clark Corporation	34155
1 μm filter paper	VWR international	28307-134
Inverted microscope	Nikon Instruments	Diaphot
Microscope camera	Nikon Instruments	DS-F11
Microscope	Olympus Corporation	BX51
Microscope camera	Olympus Corporation	DP71
Scanner	Microtek	Scanmaker 4
Vortex mixer	Fisher Scientific	12-812
Tissue culture incubator	Sanyo	MCO-18AIC
Cytospin	Shandon, Inc.	Cytospin 2
Bench-top centrifuge	Eppendorf	5810-R
Water purification system	Barnstead	Nanopure-Diamond