Formation of granulocyte-macrophage colonies by purified cells supported with various factors
| Factors . | No. of GM colonies . | |||
|---|---|---|---|---|
| Patients (n = 5) . | Subjects (n = 9) . | |||
| Mean ± SD . | Range . | Mean ± SD . | Range . | |
| CD34+/Kit+/G-CSFR+cells | ||||
| G-CSF (1 ng/mL) | 0 ± 0 | 0-1 | 3 ± 1 | 2-6 |
| G-CSF (10 ng/mL) | 2 ± 1* | 1-5 | 16 ± 3 | 11-20 |
| G-CSF (100 ng/mL) | 7 ± 1* | 6-8 | 29 ± 6 | 21-35 |
| G-CSF (1000 ng/mL) | 8 ± 2* | 6-11 | 30 ± 7 | 21-40 |
| SCF, FL, IL-3 | 14 ± 3† | 11-16 | 30 ± 8 | 25-46 |
| SCF, FL, IL-3, G-CSF (100 ng/mL) | 28 ± 3* | 23-30 | 67 ± 8 | 56-76 |
| CD34+/Kit+/G-CSFR−cells | ||||
| G-CSF (10 ng/mL) | 1 ± 1 | 0-2 | 1 ± 1 | 0-2 |
| G-CSF (100 ng/mL) | 3 ± 2 | 1-5 | 2 ± 1 | 1-4 |
| G-CSF (1000 ng/mL) | 3 ± 1 | 1-4 | 4 ± 2 | 1-5 |
| SCF, FL, IL-3 | 23 ± 6 | 17-30 | 18 ± 6 | 14-29 |
| SCF, FL, IL-3, G-CSF (100 ng/mL) | 41 ± 8 | 28-48 | 36 ± 5 | 31-48 |
| Factors . | No. of GM colonies . | |||
|---|---|---|---|---|
| Patients (n = 5) . | Subjects (n = 9) . | |||
| Mean ± SD . | Range . | Mean ± SD . | Range . | |
| CD34+/Kit+/G-CSFR+cells | ||||
| G-CSF (1 ng/mL) | 0 ± 0 | 0-1 | 3 ± 1 | 2-6 |
| G-CSF (10 ng/mL) | 2 ± 1* | 1-5 | 16 ± 3 | 11-20 |
| G-CSF (100 ng/mL) | 7 ± 1* | 6-8 | 29 ± 6 | 21-35 |
| G-CSF (1000 ng/mL) | 8 ± 2* | 6-11 | 30 ± 7 | 21-40 |
| SCF, FL, IL-3 | 14 ± 3† | 11-16 | 30 ± 8 | 25-46 |
| SCF, FL, IL-3, G-CSF (100 ng/mL) | 28 ± 3* | 23-30 | 67 ± 8 | 56-76 |
| CD34+/Kit+/G-CSFR−cells | ||||
| G-CSF (10 ng/mL) | 1 ± 1 | 0-2 | 1 ± 1 | 0-2 |
| G-CSF (100 ng/mL) | 3 ± 2 | 1-5 | 2 ± 1 | 1-4 |
| G-CSF (1000 ng/mL) | 3 ± 1 | 1-4 | 4 ± 2 | 1-5 |
| SCF, FL, IL-3 | 23 ± 6 | 17-30 | 18 ± 6 | 14-29 |
| SCF, FL, IL-3, G-CSF (100 ng/mL) | 41 ± 8 | 28-48 | 36 ± 5 | 31-48 |
GM indicates granulocyte-macrophage, G-CSFR, granulocyte colony-stimulating factor receptor; SCF, stem cell factor; FL, flk2/flt3 ligand; IL-3, interleukin-3; SCN, severe congenital neutropenia.
Cultures were grown under serum-deprived conditions. One milliliter of the culture mixture contained 250 purified cells, 1% deionized crystallized bovine serum albumin, 300 μg/mL fully iron-saturated human transferrin, 10 μg/mL soybean lecithin, 6 μg/mL cholesterol, 1 × 10−7 mol/L sodium selenite, 10 μg/mL insulin, 4.5 mmol/L L-glutamine, 1.5 mmol/L glycine, 5 × 10−5mol/L 2-mercaptoethanol, 1.2% 1500 cP methylcellulose, and designated factors. Cultures were incubated for 14 days at 37°C in a humidified atmosphere with 5% CO2/95% air. The concentrations of SCF, FL, and IL-3 were 100 ng/mL, 50 ng/mL, and 100 U/mL, respectively. GM colonies included pure granulocyte colonies consisting primarily of neutrophils and their precursors and of mixed GM colonies consisting mainly of neutrophils, macrophages-monocytes, and their precursors. All assays were carried out in triplicate cultures. The CD34+/Kit+/G-CSFR+ and CD34+/Kit+/G-CSFR− cells were purified according to flow cytometric analysis based on the expression of Kit and G-CSFR on CD34 cells, as shown in Figure 1.
P < .001, compared with subjects without SCN.
P < .01, compared with subjects without SCN.