Stempro 34 sfm

Manufactured by Thermo Fisher Scientific

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StemPro-34 SFM is a serum-free medium specifically formulated for the expansion and maintenance of human hematopoietic stem and progenitor cells. It provides a defined, animal-component-free environment for the culture of these cell types.

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StemPro-34 (74 citations) StemPro-34 SFM medium (70 citations) StemPro-34 SFM complete Medium (5 citations)

82 protocols using stempro 34 sfm

Erythroblast differentiation regulation

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GFP⁺ lentivirus-transduced erythroblasts (CD71⁺/Ter119^-) were seeded at a density of 1 × 10⁶ cells/mL in StemPro-34 SFM (ThermoFisher) supplemented with l-glutamine (1%; ThermoFisher), Epo (Peprotech; 10 U/mL) and transferrin (Sigma-Aldrich; 1 mg/mL) as previously published^{36 (link)}. The GFP⁺ cells were cultured for 48 h in the presence and absence of ICG001 (Tocris) and JW74 (Tocris). Half media exchange was performed after 24 h, and after 48 h the cells were collected, stained with CD71 and Ter119 antibodies and analyzed using flow cytometry.
For experiments involving FL cells, FACS sorted CD71⁺Ter119^- FL cells were seeded at a density of 0.5 × 10⁶ cells/mL StemPro-34 SFM (ThermoFisher) supplemented with l-glutamine (1%; ThermoFisher), Epo (Peprotech; 10 U/mL) and transferrin (Sigma-Aldrich; 1 mg/mL). The FL cells were cultured for 48 h in the presence and absence of ICG001 (Tocris) and JW74 (Tocris). Half media exchange was performed after 24 h, and after 48 h the cells were collected, stained with CD71 and Ter119 antibodies and analyzed using flow cytometry.

Rothberg J.L., Maganti H.B., Jrade H., Porter C.J., Palidwor G.A., Cafariello C., Battaion H.L., Khan S.T., Perkins T.J., Paulson R.F., Ito C.Y, & Stanford W.L. (2018). Mtf2-PRC2 control of canonical Wnt signaling is required for definitive erythropoiesis. Cell Discovery, 4, 21.

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Mature Megakaryocyte Generation Protocol

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Bone marrow-derived mature megakaryocytes were generated as described [25 (link)]. Briefly, mouse femurs were flushed, and cells expressing Ly6G/Ly6C, CD11b, CD16/32, and B220 were depleted using magnetic beads (sheep anti-rat IgG Dynabeads, Thermo Fisher Scientific, Waltham, MA, USA) and the following antibodies: anti-mouse Ly6G/Ly6C (561103, BD Biosciences, Franklin Lakes, NJ, USA), anti-mouse CD11b (47-0112-82, Thermo Fisher Scientific), anti-mouse CD16/CD32 (553141, BD Biosciences), and anti-mouse B220 (BD Biosciences). Negatively selected cells were incubated in megakaryocyte medium (Stempro-34 SFM, Thermo Fisher Scientific) containing 2.6% nutrient supplement, 1% glutamine, 1% penicillin–streptomycin–fungizone, and 20 ng/mL stem cell factor (Peprotech EC Ltd., London, UK) for 2 d at 37 °C and 5% CO₂, followed by a 5-d incubation with additional 50 ng/mL thrombopoietin (TPO) (Peprotech EC Ltd.). Mature megakaryocytes were enriched with a gradient of 3%/1.5% BSA (PAA Laboratories, Fisher Scientific, Hampton, NH, USA) under gravity for 45 min at RT. Cells in the lower 25% of the gradient, representing mature megakaryocytes, were washed in PBS and harvested in TriFast™ (VWR, Radnor, PA, USA).

Goeritzer M., Schlager S., Kuentzel K.B., Vujić N., Korbelius M., Rainer S., Kolb D., Mussbacher M., Salzmann M., Schrottmaier W.C., Assinger A., Schlagenhauf A., Madreiter-Sokolowski C.T., Blass S., Eichmann T.O., Graier W.F, & Kratky D. (2022). Adipose Triglyceride Lipase Deficiency Attenuates In Vitro Thrombus Formation without Affecting Platelet Activation and Bleeding In Vivo. Cells, 11(5), 850.

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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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Generation of iPSC Lines from ALS Patients

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The SOD1^D90A and SOD1^D90D iPSC lines were purchased from WiCell (Madison, WI, USA). SOD1^G85R and sporadic ALS iPSC lines were generated from patients’ PBMCs with the approval of the Ethic Institutional Review Board in Hualien Tzu Chi Hospital (IRB105-131-A). Informed consent was obtained from patients. Patients’ PBMCs were cultured in StemPro 34 SFM (ThermoFisher Scientific, Waltham, MA, USA) supplemented with SCF, FLT-3, IL-3, and IL-6 (all Peprotech, Cranbury, NJ, USA). Reprogramming was processed with a CytoTune iPS 2.0 Sendai Reprogramming Kit [34 (link)] (ThermoFisher Scientific) according to the kit manual. Emerging colonies were transferred to 1% Geltrex (ThermoFisher Scientific)-coated culture dishes and expanded in TeSR-E8 medium (StemCell Technologies, Vancouver, BC, Canada) or Pluto human iPS/ES cell culture medium (DuoGenic Stem Cells, Taichung, Taiwan). The iPSCs were passaged for 3–5 days with Accutase (Merck-Millipore, Billerica, MA, USA) and then reseeded at 1:5 to 1:10 ratios. Culture media were refreshed daily.

Ting H.C., Yang H.I., Harn H.J., Chiu I.M., Su H.L., Li X., Chen M.F., Ho T.J., Liu C.A., Tsai Y.J., Chiou T.W., Lin S.Z, & Chang C.Y. (2021). Coactivation of GSK3β and IGF-1 Attenuates Amyotrophic Lateral Sclerosis Nerve Fiber Cytopathies in SOD1 Mutant Patient-Derived Motor Neurons. Cells, 10(10), 2773.

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Generation and Expansion of Stem Cell Lines

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The H9 hESC, SOD1^D90A, and SOD1^D90D iPSC lines were purchased from WiCell (Madison, WI, USA) [6 (link)]. TW1 hESCs were kindly provided by Lee Women’s Hospital (Taichung, Taiwan) [31 (link)]. SOD1^G85R and sporadic ALS iPSC lines were generated from patients’ peripheral blood mononuclear cells (PBMCs) with the approval of the Ethical Institutional Review Board at Hualien Tzu Chi Hospital (IRB105-131-A), and SOD1^G85G iPSCs were generated from SOD1^G85R iPSCs [24 (link)]. Informed consent was obtained from all the patients. The iPSC properties were previously identified. Briefly, PBMCs were cultured in StemPro-34 SFM (ThermoFisher Scientific) and supplemented with SCF, FLT-3, IL-3, and IL-6 (Peprotech, Cranbury, NJ, USA). Reprogramming was performed using CytoTune iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) as per the manufacturer’s instructions [40 (link)]. All hESC and iPSC lines were transferred onto 1% Geltrex (ThermoFisher Scientific)-coated culture dishes and expanded in the TeSR-E8 media (StemCell Technologies, Vancouver, BC, Canada) or DuoESy pluripotent stem cell culture media (Without Geltrex coating, DuoGenic Stem Cells, Taichung, Taiwan). The hESCs and iPSCs were passaged for 3–5 days using Accutase (Merck-Millipore, Billerica, MA, USA) and then reseeded at 1:5–1:10 ratios. Culture media were refreshed daily.

Ting H.C., Su H.L., Chen M.F., Harn H.J., Lin S.Z., Chiou T.W, & Chang C.Y. (2022). Robust Generation of Ready-to-Use Cryopreserved Motor Neurons from Human Pluripotent Stem Cells for Disease Modeling. International Journal of Molecular Sciences, 23(21), 13462.

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Expansion and Transduction of CD34+ Cells

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Human CD34⁺ cells were cultured in StemPro34 SFM (serum free medium, Thermo Fisher Scientific) supplemented with the human cytokines fms-related tyrosine kinase 3 ligand (hFlt3L), Stem Cell Factor (hSCF), and interleukin-3 (hIL-3) (at 50, 50, and 10 ng/mL, respectively; all from Miltenyi Biotech) in the presence of 20% serum substitute (BIT 9500, STEMCELL Technologies). After 7 days, erythropoietin (EPO) at 3 IU/mL was used to replace FLt3L, SCF, and IL-3 (medium 1). Alternatively, CD34⁺ cells were cultured in Iscove's modified Dubelcco's medium (IMDM) supplemented with SCF (100 ng/mL), IL-3 (5 ng/mL), and EPO (3 U/mL) from day 0, supplemented with SCF and EPO from day 8, and with EPO only from day 11 (medium 2). When indicated, CD34⁺ cells were transduced 24 h after the beginning of culture in medium containing protamine sulfate (8 μg/mL) at MOIs between 2 and 10.

Nualkaew T., Sii-Felice K., Giorgi M., McColl B., Gouzil J., Glaser A., Voon H.P., Tee H.Y., Grigoriadis G., Svasti S., Fucharoen S., Hongeng S., Leboulch P., Payen E, & Vadolas J. (2021). Coordinated β-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for β-thalassemia. Molecular Therapy, 29(9), 2841-2853.

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Culturing the LAD2 Mast Cell Line

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LAD2 cell line was kindly provided by Dr. Kirshenbaum of the National Institutes of Health (Bethesda, MD, USA). LAD2 cells were cultured in StemPro-34 SFM (Thermo Fisher Scientific, MA, USA) with StemPro-34 nutrient supplement (2.5%, Thermo Fisher Scientific, MA, USA), l-glutamine (2 mM, Gibco, CA, USA), penicillin/streptomycin (1%, Hyclone, UT, USA), and recombinant human SCF (100 ng/mL, R&D Systems, MN, USA). Half of the medium was replaced weekly by adding an equal volume of fresh medium containing SCF. The cell density was maintained at 2–5 × 10⁵ cells/mL. The cells were incubated at 37 °C in 5% CO₂ incubator.

Kim K.H., Kim J.O, & Park S.G. (2022). A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells. Molecular and Cellular Biochemistry, 478(4), 861-873.

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Directed Differentiation of γδT-iPSCs into Hematopoietic Cells

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Prior to differentiation, single dissociated undifferentiated human γδT‐iPSCs were seeded onto a Laminin511‐E8‐coated 6‐well plate at a density of 2 × 10³ cells and cultured in Stem Fit medium. When individual colonies grew to around 500 µm in diameter, the medium was replaced by Stem Fit medium supplemented with 4 µM of CHIR99021 (Tocris Bioscience, Bristol, UK), 80 ng/ml of rhBMP4 (R&D Systems) and 80 ng/ml of rhVEGF (R&D Systems). This day was defined as day 0 of our protocol. On day 2, the medium was refreshed. On day 4, the medium was replaced by Essential 6 medium (ThermoFisher Scientific) supplemented with 4 µM of SB43152 (WAKO), 80 ng/ml of rhVEGF, 50 ng/ml of rh‐bFGF (Reprocell) and 50 ng/ml of rhSCF (R&D Systems). On day 6, the medium was replaced by StemPro‐34SFM (Thermo Fisher Scientific) supplemented with 20 ng/ml of rhVEGF, 50 ng/ml of rhSCF, 50 ng/ml of rhIL‐3 (R&D Systems), 50 ng/ml of hIL‐6 (Roche, Basel, Switzerland), 50 ng/ml of rhFlt3 ligand (R&D Systems) and 10 IU/ml of recombinant human Erythropoietin α (EPO) (Kyowa Hakko Kirin, Tokyo, Japan). On day 8, the medium was replaced by StemPro‐34SFM supplemented with 50 ng/ml of rhSCF, 50 ng/ml of hIL‐6 (Roche) and 10 IU/ml of EPO. On day 10, the medium was refreshed. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO_2.

Watanabe D., Koyanagi‐Aoi M., Taniguchi‐Ikeda M., Yoshida Y., Azuma T, & Aoi T. (2017). The Generation of Human γδT Cell‐Derived Induced Pluripotent Stem Cells from Whole Peripheral Blood Mononuclear Cell Culture. Stem Cells Translational Medicine, 7(1), 34-44.

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Directed Differentiation of h-iPSCs to Endothelial Cells

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h-iPSCs were dissociated into single cells with TrypLE Select and plated on Matrigel at a density of 50,000 cells/cm² in mTeSR1 medium with 10 μM Y27632. After 24 hours, the medium was changed to basal medium supplemented with 1 μM CP21R7 (Selleckchem, catalog no. S7954) and BMP4 (20 ng/ml). Basal medium was prepared by adding 1× B27 supplement (Thermo Fisher Scientific, catalog no. 17504044) and 1× N2 (Thermo Fisher Scientific, catalog no. 17502048) into DMEM/F12 (Thermo Fisher Scientific, catalog no. 11330032). After 72 hours, the differentiation medium was changed to S2 medium for 48 hours. S2 medium consisted of StemPro-34 SFM (Thermo Fisher Scientific, catalog no. 10639011) supplemented with VEGF-A (50 ng/ml), and 10 μM DAPT (Selleckchem, catalog no. S2215). Medium was changed every day throughout this protocol. This protocol is adapted from Sahara et al. (19 (link)).

Wang K., Lin R.Z., Hong X., Ng A.H., Lee C.N., Neumeyer J., Wang G., Wang X., Ma M., Pu W.T., Church G.M, & Melero-Martin J.M. (2020). Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA. Science Advances, 6(30), eaba7606.

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Erythroblast Cell Line 15.4 Induction

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The erythroblast cell line 15.4 was derived from p53-deficient mouse fetal livers as previously described [22 (link)], and cultured in Stempro-34 SFM (Thermo Fisher), containing penicilin-streptavidin, L-glutamin, Erythropoietin (1U/ml), Stem Cell Factor (supernatant CHO cells) and 1μM Dexamethasone (Sigma) [23 (link)]. For ER stress induction, cells were treated with 2.5μg/ml Tunicamycin (Tm) (Sigma) for 1.5h or left untreated.

Paolini N.A., Moore K.S., di Summa F.M., Fokkema I.F., ‘t Hoen P.A, & von Lindern M. (2018). Ribosome profiling uncovers selective mRNA translation associated with eIF2 phosphorylation in erythroid progenitors. PLoS ONE, 13(4), e0193790.

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