Human interleukin 3

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Human interleukin-3 is a recombinant protein that functions as a hematopoietic growth factor. It stimulates the growth and differentiation of hematopoietic progenitor cells.

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Recombinant human interleukin (IL)-3 (2 citations) Recombinant human interleukin-3 (2 citations)

4 protocols using human interleukin 3

Establishing and Maintaining AML and HEK293T Cell Lines

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AML cell lines and HEK293T cells were maintained under standard conditions with up to 0.01% DMSO. Cell line identity was verified using the Multiplex Cell Authentication Test (Multiplexion, Friedrichshafen, Germany) or the Human Cell Line Authentication Service (Eurofins Genomics GmbH, Ebertsberg, Germany). All cell lines were routinely tested for mycoplasma contamination. AML PDX cells were isolated from the spleen of leukemic mice as described previously^{56 (link)} and cultured in StemPro34 SFM (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with provided nutrient supplement, 2% FBS, 1% penicillin/streptomycin and human FLT3 ligand, human thrombopoietin, human interleukin 3, and human stem cell factor (PeproTech, Princeton, New Jersey, USA; 10 ng/mL each). Murine cell lines were a gift from Stephen M. Sykes and were generated and cultured as described previously^{57 (link)} (liquid culture) or in methylcellulose medium (MethoCult GF M3434, Stem Cell Technologies, Vancouver, Canada). Palbociclib (PD-0332991) and LIMKi3 (CAS number 1338247-35-0) were obtained from Selleck (Munich, Germany) or Merck Millipore (Burlington, Massachusetts, USA), respectively.

Jensen P., Carlet M., Schlenk R.F., Weber A., Kress J., Brunner I., Słabicki M., Grill G., Weisemann S., Cheng Y.Y., Jeremias I., Scholl C, & Fröhling S. (2020). Requirement for LIM kinases in acute myeloid leukemia. Leukemia, 34(12), 3173-3185.

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Hematopoietic Colony Forming Assay

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After 14-day hPSC-to-hematopoietic differentiation, the differentiated hematopoietic cells were plated (1 × 10⁴ cells/9.5 cm²) in Methocult H4230 (STEMCELL Technologies) supplemented with 2.5 μg/ml human Stem Cell Factor, 2.5 μg/ml human interleukin-3, 5 μg/ml human granulocyte-macrophage colony-stimulating factor, and 500 U/ml erythropoietin, all recombinant human cytokines from PeproTech. For cells treated with forskolin or IBMX, or a combination of both, similar treatment was continued in the CFU assay (chemicals added to the CFU medium on the first day of CFU assay), and after 12 days hematopoietic colonies were scored microscopically to evaluate various CFU phenotypes.

Saxena S., Rönn R.E., Guibentif C., Moraghebi R, & Woods N.B. (2016). Cyclic AMP Signaling through Epac Axis Modulates Human Hemogenic Endothelium and Enhances Hematopoietic Cell Generation. Stem Cell Reports, 6(5), 692-703.

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Lentiviral Expression Analysis in K562 and HSPCs

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For lentiviral expression analysis, K562 cells were plated in 24-well plates at 5 × 10⁴ cells/well and treated with a range of vector doses to obtain populations with 10% transduction or lower, thus ensuring that the majority of cells received only single integrations. Cells were cultured for 1–2 weeks before flow cytometric analysis to dilute out non-integrated vector and to allow fluorescent protein levels to reach steady state.
For expression analysis in primary human CD34+ HSPCs from mobilized peripheral blood, cryopreserved cells were thawed and prestimulated overnight in X-VIVO 15 medium (Lonza) supplemented with 50 ng/ml human fms-like tyrosine kinase 3 (FLT-3) ligand, 50 ng/ml human stem cell factor and 50 ng/ml human thrombopoietin (PeproTech, Rocky Hill, NJ, USA). Viral vector was then added in an equal volume of the same medium to achieve a final vector concentration of 3 × 10⁵ transducing units/ml, as determined by transduction of K562 cells. This vector dose yielded ∼10% transduction. Twenty-four hours after vector addition, 2 ml of myeloid differentiation medium was added, composed of IMDM supplemented with 20% foetal bovine serum, 0.5% bovine serum albumin, 5 ng/ml human interleukin-3, 10 ng/ml human interleukin-6 and 25 ng/ml human stem cell factor (PeproTech).

Cooper A.R., Lill G.R., Gschweng E.H, & Kohn D.B. (2014). Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter. Nucleic Acids Research, 43(1), 682-690.

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Isolation and Transduction of CD34+ HSPCs

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All BM aspirates were obtained from voluntary healthy donors supplied by AllCells (Alameda, CA, USA). BM mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. CD34⁺ HSPCs were enriched using a CD34⁺ MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Enriched CD34⁺ HSPCs were cryopreserved in fetal bovine serum supplemented with 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) in liquid nitrogen. Cells were thawed and plated on non-tissue culture-treated six-well plates pre-coated with RectroNectin (20 μg/mL, Takara Shuzo, Otsu, Japan) at 1 × 10⁶ cells/mL. Cells were pre-stimulated for 16–24 hours in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with 1× glutamine, penicillin, and streptomycin (Gemini Bio-Products, Sacramento, CA, USA), human SCF (50 ng/mL), human Flt-3 ligand (50 ng/mL), human thrombopoietin (50 ng/mL), and human interleukin-3 (20 ng/mL; all cytokines were acquired from PeproTech, Rocky Hill, NJ, USA). Concentrated viral supernatants were used at various MOIs to transduce CD34⁺ HSPCs for 24 h. These cells were washed, re-plated, and cultured under myeloid or erythroid culture conditions, as described by Romero et al.⁶⁶ On day 14 of culture, gDNA and/or mRNA was extracted from transduced cells.

Morgan R.A., Ma F., Unti M.J., Brown D., Ayoub P.G., Tam C., Lathrop L., Aleshe B., Kurita R., Nakamura Y., Senadheera S., Wong R.L., Hollis R.P., Pellegrini M, & Kohn D.B. (2020). Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences. Molecular Therapy. Methods & Clinical Development, 17, 999-1013.

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