Human stem cell factor

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Human stem cell factor is a recombinant protein that acts as a growth and differentiation factor for hematopoietic stem cells and progenitor cells. It promotes the survival, proliferation, and differentiation of these cells.

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Recombinant human stem cell factor (24 citations) Recombinant human stem cell factor (rhSCF; (3 citations) Recombinant human (rh) stem-cell factor (SCF, (2 citations) Human stem cell factor (hSCF; (2 citations)

27 protocols using human stem cell factor

Generation and Characterization of Human Mast Cells

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hMCs were generated, as previously described.21 (link), 22 (link), 23 (link) Briefly, CD117⁺CD34⁺ cells were purified from buffy coat blood mononuclear cells by using a positive selection kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in serum-free StemSpan medium (STEMCELL Technologies, Vancouver, British Columbia, Canada) supplemented with 100 U/mL penicillin (Invitrogen, Carlsbad, Calif), 100 μg/mL streptomycin (Invitrogen), human IL-6 (50 ng/mL; PeproTech, Rocky Hill, NJ), human IL-3 (10 ng/mL; PeproTech), human stem cell factor (100 ng/mL; PeproTech), and 10 μg/mL human low-density lipoprotein (STEMCELL Technologies). After 30 days, the cells were transferred progressively to culture medium containing Iscove modified Dulbecco medium with GlutaMAX-I, 50 μmol/L β₂-mercaptoethanol, 0.5% BSA, 1% Insulin-Transferrin-Selenium (Life Technologies, Grand Island, NY), 100 U/mL penicillin, 100 μg/mL streptomycin, human IL-6 (50 ng/mL), and human stem cell factor (100 ng/mL). After 8 to 10 weeks of culture, the cells were tested for maturity and found to be greater than 90% CD117⁺ and FcεRIa⁺ cells.
We used immunocytochemistry to characterize hMCs generated from peripheral blood precursors (details are provided in the Methods section in this article's Online Repository at www.jacionline.org).

Bahri R., Custovic A., Korosec P., Tsoumani M., Barron M., Wu J., Sayers R., Weimann A., Ruiz-Garcia M., Patel N., Robb A., Shamji M.H., Fontanella S., Silar M., Mills E.N., Simpson A., Turner P.J, & Bulfone-Paus S. (2018). Mast cell activation test in the diagnosis of allergic disease and anaphylaxis. The Journal of Allergy and Clinical Immunology, 142(2), 485-496.e16.

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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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Differentiation of Human Mast Cells from PBMCs

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Human peripheral blood mononuclear cell-derived mast cells were generated as previously described by Gaudenzio et al.(73 (link)). Briefly, peripheral blood mononuclear cells were obtained from buffy coats of healthy blood donors and CD34+ precursor cells were isolated using the EasySep Human CD34 Positive Selection Kit (STEMCELL Technologies). CD34+ cells were maintained for 4 weeks under serum-free conditions using StemSpan medium (STEMCELL Technologies) supplemented with recombinant human IL-6 (50 ng/ml; Peprotech), human IL-3 (10 ng/ml; Peprotech) and human Stem Cell Factor (100 ng/mL Peprotech, Rocky Hill, NJ). Thereafter, the cells were maintained in IMDM Glutamax I, sodium pyruvate, 2-mercaptoethanol, 0.5% BSA, insulin- 175 transferrin selenium (all from Invitrogen), ciprofloxacin (10 ug/ml; Sigma-Aldrich), IL-6 (50 ng/ml) and human Stem Cell Factor (100 ng/mL Peprotech, Rocky Hill, NJ). After 8-12 weeks, PBCMCs were tested for maturity by Giemsa or toluidine blue staining and beta-hexosaminidase release assays (see below).

Folkerts J., Redegeld F., Folkerts G., Blokhuis B., van den Berg M.P., de Bruijn M.J., van IJcken W.F., Junt T., Tam S.Y., Galli S.J., Hendriks R.W., Stadhouders R, & Maurer M. (2020). Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling. Allergy, 75(8), 1966-1978.

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Human Melanoma and Melanocyte Cell Culture Protocols

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SK2 and SK5 human melanoma cell lines were obtained from Lombardi Comprehensive Cancer Center Tissue Culture Shared Resource (Georgetown University, Washington, DC). UACC930, C81-61, and C8161 melanoma cells were generously provided by Dr. Suzie Chen (Rutgers University, Piscataway, NJ). HERMES 2 immortalized human melanocytes were purchased from the Wellcome Trust Functional Genomics Cell Bank (University of London, London, United Kingdom). Cells were cultured in 6% CO₂ at 37 °C on 60 mm dishes from MidSci (St. Louis, MO). Melanoma cells were cultured in DMEM (high glucose) containing 10% fetal bovine serum, 2 mM glutamine and antibiotic–antimycotic. Melanocytes were cultured in RPMI 1640 growth media supplemented with antibiotic–antimycotic,10 mM HCl, 200 nM TPA, 300 µM IBMX, 10 nM endothelin 1, 10 ng/ml human stem cell factor, 10% fetal bovine serum, and 2 mM glutamine (Life Technologies).

Gelb T., Pshenichkin S., Rodriguez O.C., Hathaway H.A., Grajkowska E., DiRaddo J.O., Wroblewska B., Yasuda R., Albanese C., Wolfe B.B, & Wroblewski J.T. (2014). Metabotropic Glutamate Receptor 1 acts as a Dependence Receptor Creating a Requirement for Glutamate to Sustain the Viability and Growth of Human Melanomas. Oncogene, 34(21), 2711-2720.

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Human Melanoma and Melanocyte Cell Culture Protocols

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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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Isolation and Expansion of CD34+ Cells

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Human cord blood samples were obtained from the maternity wards of Helsingborg General Hospital and Skåne University Hospital in Lund and Malmö, Sweden, after informed, written consent according to guidelines approved by the regional ethical committee. Mononuclear cells were separated through densitygradient centrifugation. CD34⁺ cells were magnetically isolated according to the manufacturer’s description (Milteny Biotec, cat. n. 130-046-702). Cells were cultured in serum-free expansion medium (Stem Cell Technologies), supplemented with human stem cell factor, thrombopoietin, and FLT3-ligand at 100 ng/mL from Peprotech. Full descriptions of transduction and erythroid differentiation are provided in the Online Supplementary Methods.

Liu Y., Dahl M., Debnath S., Rothe M., Smith E.M., Grahn T.H., Warsi S., Chen J., Flygare J., Schambach A, & Karlsson S. (2021). Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector. Haematologica, 107(2), 446-456.

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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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Differentiation of Human Mast Cells

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CD34⁺ progenitor stem cells were isolated from buffy coats (Sanquin Blood Bank, The Netherlands) to generate hMCs which has been described previously.^[²¹, ²²^] Stem cells were isolated according to the manufacturer's protocol (EasySep, Stemcell technologies). Stem cells were cultured in StemSpan medium (StemCell technologies) supplemented with 10 µg mL⁻¹ ciprofloxacin (Sigma‐Aldrich, St Louis, MO), human IL‐6 (50 ng mL⁻¹), human IL‐3 (10 ng mL⁻¹), and human stem cell factor (100 ng mL⁻¹) (Peprotech, Rocky Hill, NJ) in a humidified incubator (37 °C, 5% CO₂). After 30 days, the cells were progressively transferred to culture medium consisting of Iscove's modified Dulbeccos medium with GlutaMAX‐I, 50 µm 2‐mercaptoethanol (Life Technologies, Carlsbad, CA), 0.5% AlbuMAX (Gibco), 1% insulin‐transferrin‐selenium (Life Technologies, Carlsbad, CA), 10 µg mL⁻¹ ciprofloxacin, 50 ng mL⁻¹ human IL‐6, and 3% supernatant of Chinese hamster ovary transfectants secreting murine stem cell factor. Maturation of hMCs was confirmed by the presence of CD117 and FcεRIa on the cell membrane.

Smits M., Nooijen I., Redegeld F., de Jong A., Le T., Knulst A., Houben G, & Verhoeckx K. (2021). Digestion and Transport across the Intestinal Epithelium Affects the Allergenicity of Ara h 1 and 3 but Not of Ara h 2 and 6. Molecular Nutrition & Food Research, 65(6), 2000712.

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Primary Cell Culture Protocol

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Primary cells were maintained in RPMI-1640 supplemented with 20% fetal calf serum (FCS), 2mM l-glutamine, 100 U/mL penicillin/streptomycin, and human stem cell factor (5 ng/mL; Peprotech, Rocky Hill, NJ).

El Khawanky N., Hughes A., Yu W., Myburgh R., Matschulla T., Taromi S., Aumann K., Clarson J., Vinnakota J.M., Shoumariyeh K., Miething C., Lopez A.F., Brown M.P., Duyster J., Hein L., Manz M.G., Hughes T.P., White D.L., Yong A.S, & Zeiser R. (2021). Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia. Nature Communications, 12, 6436.

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