Stemspan

Manufactured by STEMCELL

Sourced in Canada, United States, China, United Kingdom

StemSpan is a serum-free, animal component-free medium designed for the expansion and maintenance of human and non-human progenitor and stem cells in vitro. It provides the necessary components to support the growth and proliferation of various types of stem and progenitor cells.

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StemSpan medium (104 citations) StemSpan SFEM medium (90 citations) StemSpan SFEM II medium (49 citations) StemSpan™ Serum-Free Expansion Medium II (10 citations) StemSpan Myeloid Expansion Supplement (5 citations) StemSpanTM SFEMII (4 citations) StemSpan SFEM II human hematopoietic stem cell expansion media (2 citations) MethoCult GF 3434 StemSpan media (2 citations) StemSpan CC110 cocktail (2 citations) StemSpan T Cell Progenitor Expansion Supplement (2 citations)

104 protocols using stemspan

Culturing Human and Murine Hematopoietic Cells

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Human leukemia cell lines U937 (male, CALM10-AFF10) and MOLM13 (male, MLL-AF9, FLT3-ITD) were gifts from Dr. Ali Shilatifard at Northwestern University. Jurkat (male) cells were a gift from Dr. Panos Ntziachristos at Northwestern University. These cell lines were authenticated by short tandem repeat testing (IDEXX) prior to use, and cultured in RPMI-1640 (Gibco) supplemented with 10% FBS (Gibco), Penicillin/Streptomycin (Gibco), and LGlutamine (Gibco). CD34⁺ cells were obtained from Fred Hutchinson Cancer Center and cultured in StemSpan (StemCell Technologies) supplemented with 50 ng/ml SCF (StemCell), 10 ng/mL each of IL3, IL6, and Flt3L (StemCell Technologies). Primary murine hematopoietic stem and progenitor cells (HSPCs) were selected from flushed bone marrow of the hindlimbs using a CD117⁺ selection kit according to manufacturer’s protocol (StemCell Technologies). All primary HSPCs were maintained in StemSpan (StemCell Technologies) supplemented with 50 ng/mL recombinant murine SCF, 10 ng/mL recombinant murine IL3, 10 ng/mL recombinant murine IL6, and 1:200 human LDL. MLL-AF9 cells were maintained in RPMI-1640 (Gibco) supplemented with 10% FBS, L-glutamine, 100 ng/mL recombinant murine SCF, 50 ng/mL recombinant murine IL6, and 20 ng/mL recombinant murine IL3. All cells were cultured in 6-well flat-bottom plates at 37C with 5% CO₂.

Volk A., Liang K., Suraneni P., Li X., Zhao J., Bulic M., Marshall S., Pulakanti K., Malinge S., Taub J., Ge Y., Rao S., Bartom E., Shilatifard A, & Crispino J.D. (2018). A CHAF1B-dependent molecular switch in hematopoiesis and leukemia pathogenesis. Cancer cell, 34(5), 707-723.e7.

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Hematopoietic Progenitor Expansion and Electroporation

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Patient-derived PB and/or BM MNC were first treated with ammonium chloride to deplete mature, non-nucleated red blood cells, washed in RPMI, and then plated into methylcellulose (see Hematopoietic progenitor colony forming assays for further details) to allow for progenitor expansion. Cells were liberated from methylcellulose by solubilizing them with PBS warmed to 37 °C. Cells were washed with RPMI before being re-suspended in Lonza electroporation solution from Kit C (Lonza, Basel, Switzerland) and electroporated using program W-001 using the Amaxa nucleofector (Lonza, Basel, Switzerland). For every one million cells transfected, 2 µg of plasmid constructs or 60 pg of siRNA was used. Electroporation efficiency was monitored by transfecting an eGFP construct as a control. After electroporation, cells were maintained in RPMI supplemented with StemSpan (StemCell Technologies #02691) for 72 h before experiments were conducted.

Carr R.M., Vorobyev D., Lasho T., Marks D.L., Tolosa E.J., Vedder A., Almada L.L., Yurcheko A., Padioleau I., Alver B., Coltro G., Binder M., Safgren S.L., Horn I., You X., Solary E., Balasis M.E., Berger K., Hiebert J., Witzig T., Buradkar A., Graf T., Valent P., Mangaonkar A.A., Robertson K.D., Howard M.T., Kaufmann S.H., Pin C., Fernandez-Zapico M.E., Geissler K., Droin N., Padron E., Zhang J., Nikolaev S, & Patnaik M.M. (2021). RAS mutations drive proliferative chronic myelomonocytic leukemia via a KMT2A-PLK1 axis. Nature Communications, 12, 2901.

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Hematopoietic Colony-Forming Assays

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HB-CFCs were detected using a semisolid colony-forming serum-free medium (CF-SFM) containing 40% ES-Cult M3120 methylcellulose (2.5% solution in IMDM, Stem Cell Technologies), 25% StemSpan serum-free expansion medium (SFEM, Stem Cell Technologies), 25% human endothelial serum-free medium (ESFM, ThermoFisher), 10% BIT 9500 supplement (Stem Cell Technologies), GlutaMAX (1/100 dilution, ThermoFisher), Ex-Cyte (1/1000 dilution, Millipore), 100 μM MTG, 50 μg/ml ascorbic acid and 20 ng/ml FGF (Peprotech) (Vodyanik et al., 2010 (link)). Hematopoietic CFCs were detected using serum containing H4435 MethoCult with FGF, SCF, IL-3, IL-6 and EPO (Stem Cell Technologies) following plating 1000 CD43⁺ cells/dish in duplicates. CFCs numbers recalculated per 10⁵ cells.

Jung H.S., Uenishi G., Park M.A., Liu P., Suknuntha K., Raymond M., Choi Y.J., Thomson J.A., Ong I.M, & Slukvin I.I. (2021). SOX17 integrates HOXA and arterial programs in hemogenic endothelium to drive definitive lympho-myeloid hematopoiesis. Cell reports, 34(7), 108758.

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Single-cell bipotent erythroid-megakaryocyte culture

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Single cells were directly sorted into 96-well plates with erythroid/megakaryocyte bipotential medium for erythroid and megakaryocytic output (Psaila et al., 2016 (link)). StemSpan (StemCell Technologies, Inc.) was supplemented with thrombopoietin (TPO; 100 ng/ml), erythropoietin (EPO; 1 IU/ml), stem cell factor (SCF; 100 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml; all from Peprotech), and human low-density lipoprotein (40 mg/ml; StemCell Technologies, Inc.). Single cells were cultured at 37°C in 5% CO₂ for 2 wk, and colony readout was performed by direct light microscopy and flow cytometry using CD41a APC (eBioscience; Clone: HIP8, Cat# 17–0419-42, RRID: AB_2573144), CD42b PE (eBioscience; Clone: HIP1, Cat# 12–0429042, RRID: AB_10852864), CD11b FITC (eBioscience; Clone: ICRF44, Cat# 11–0118-42, RRID: AB_1582242), CD14 FITC (eBioscience; Clone: 61D3, Cat# 11–0149-41, RRID: AB_10597445), CD34 APC–eFluor 780 (Clone: 4H11, Cat# 47–0349-42, RRID: AB_2573956), and CD235a PerCp Cy5.5 (Biolegend; Clone: HIR2, Cat# 306614, RRID: AB_10683170). CD41a and CD42b double-positive cells were used to identify the megakaryocytic output.

Louka E., Povinelli B., Rodriguez-Meira A., Buck G., Wen W.X., Wang G., Sousos N., Ashley N., Hamblin A., Booth C.A., Roy A., Elliott N., Iskander D., de la Fuente J., Fordham N., O’Byrne S., Inglott S., Norfo R., Salio M., Thongjuea S., Rao A., Roberts I, & Mead A.J. (2021). Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia. The Journal of Experimental Medicine, 218(2), e20180853.

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Multiparametric Flow Cytometry for AML Blasts

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We used multiparametric flow cytometry to identify and sort AML blasts (CD45^low CD33^midCD19^-CD3^-) and normal CB-CD34⁺ (CD34⁺CD3^-CD19^-). Post-sort analysis demonstrated > 98% purity. Sorted cells were cultured in StemSpan (Stem Cell Technologies) supplemented with human recombinant TPO, SCF, and Flt3-L (all 20 ng/ml, PeproTech) at a density of 5×10⁵/ml for 72 hours, and supernatant was collected to create a cell-free conditioned medium.

Zhang T.Y., Dutta R., Benard B., Zhao F., Yin R, & Majeti R. (2020). IL-6 Blockade Reverses Bone Marrow Failure Induced by Human Acute Myeloid Leukemia. Science translational medicine, 12(538), eaax5104.

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Erythroid Differentiation Co-culture Assay

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Erythroid differentiation co-cultures were carried out using a 24-well plate and semipermeable transwells (0.4 μM pores, Sigma-Aldrich). Flow-purified AML blasts were plated (5×10⁵/ml) on the bottom of the transwell where indicated and cultured for 72 hours in StemSpan supplemented with Erythroid Expansion Supplement (StemCell Technologies). After 72 hours, normal CB-CD34⁺ cells (10⁴) were added to the top to allow for erythroid differentiation. A 50 μl aliquot was removed from the top of the transwell at various time points to quantitate erythroid progenitors immunophenotypically using CD71 and CD235a/GPA. For IL-6 blocking experiments, neutralizing antibodies for human IL-6 and an isotype control antibody (both 1 μg /ml, ThermoFisher) were added at the beginning of the culture and replaced every 72 hours during medium change. For cell cycle analysis, EDU was added to erythroid differentiation cultures for 12 h for incorporation during S/G2 phase of the cell cycle. EDU stain was performed according to manufacturer’s instructions (Click-iT EdU Cell Proliferation Kit, ThermoFisher) and analyzed by flow cytometry. EDU-positive cells indicated S/G2 phase of the cell consistent with actively proliferating cells. PI was used to distinguish live/dead cells.

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Culturing Hematopoietic Stem Cells

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Pigtailed macaque steady-state bone marrow, primed bone marrow and umbilical cord blood, rhesus macaque steady-state bone marrow as well as human CD34⁺ cells were harvested, enriched and cultured as previously described (11 (link), 36 (link)). For more details on the CD34⁺ enrichment see Supplemental Methods. Enriched CD34⁺ cells were cultured in StemSpan (Stemcell Technologies, Vancouver, British Columbia, Canada) supplemented with 100 U/ml penicillin streptomycin (Gibco by Life Technologies, Waltham, MA) and either stem cell factor (Peprotech, Rocky Hill, NJ), TPO (Thrombopoietin, Peprotech) and FLT3-L (Fms-related tyrosine kinase 3 ligand, Miltenyi Biotec), or stem cell factor and IL-3 (Interleukin 3, Peprotech) (100 ng/ml each).

Radtke S., Adair J.E., Giese M.A., Chan Y.Y., Norgaard Z.K., Enstrom M., Haworth K.G., Schefter L.E, & Kiem H.P. (2017). A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates. Science translational medicine, 9(414), eaan1145.

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Functional Characterization of Leukemic Blasts and Residual HSPCs

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Blasts (SSC^low, CD45^int, CD99^high) and residual HSPC (SSC^low, CD45^int, CD34⁺, CD38^-, CD99^low) from primary AML specimens were double FACS–sorted to > 95% purity and seeded in methylcellulose (MethoCult H4435, Stem Cell Technologies). After 14 days, colonies were counted under an Olympus BX41 microscope. For liquid differentiation assays, sorted cells were plated at 1×10⁶/mL in Stem Span (Stem Cell Technologies) media, supplemented with human cytokines that support monocyte/macrophage differentiation: SCF (50 ng/mL), IL-3 (10 ng/mL), Fms-like tyrosine kinase-3–ligand (FLT3-L; 50 ng/mL), G-CSF (10 ng/mL) (Peprotech). After 7 days, immunophenotypic analysis was performed by flow cytometry for CD14 expression.

Cimmino L., Dolgalev I., Wang Y., Yoshimi A., Martin G.H., Wang J., Ng V., Xia B., Witkowski M.T., Mitchell-Flack M., Grillo I., Bakogianni S., Ndiaye-Lobry D., Martín M.T., Guillamot M., Banh R.S., Xu M., Figueroa M.E., Dickins R.A., Abdel-Wahab O., Park C.Y., Tsirigos A., Neel B.G, & Aifantis I. (2017). Restoration of TET2 Function Blocks Aberrant Self-Renewal and Leukemia Progression. Cell, 170(6), 1079-1095.e20.

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Expansion of Human CB CD34+ Cells

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Samples of human CB were collected by the Beijing Yuhe Chinese and Western Medicine Integrative Rehabilitation Hospital. After density gradient centrifugation and MACs sorting (anti-CD34 microbeads, Miltenyi Biotec, Westphalia, Gladbach, Germany), a total of 5 × 10⁴ CD34⁺ cells were cultured in StemSpan (STEMCELL Technologies, Shanghai, China) supplemented with 50 ng/mL of SCF, TPO, Flt-3 L (PeproTech, Rocky Hill, NJ, USA), with hFLSECs-E4orf1 (plus cytokines) or feeder-free (cytokines alone). Additionally, we also performed a notch signaling blocking experiment in hCB cells cocultured with hFLSECs-E4orf1 by adding Compound E (200 nM; CpE; MERCK, Darmstadt, Germany), which is a notch signal inhibitor. CpE was added every other day and equivalent dose of DMSO was added as a control. Passage should be performed if the concentration of expanded cells was reached 2 × 10⁶ cells/well. After 14 days, amplified cells were harvested and analyzed by cell enumerated, FACS, colony-forming unit (CFU) assays, cell cycle analysis, and in vivo transplantation assays. All data are represented as mean ± SD.

Li H., Pei H., Xie X., Wang S., Jia Y., Zhang B., Fan Z., Liu Y., Bai Y., Han Y., He L., Nan X., Yue W, & Pei X. (2019). Liver Sinusoidal Endothelial Cells Promote the Expansion of Human Cord Blood Hematopoietic Stem and Progenitor Cells. International Journal of Molecular Sciences, 20(8), 1985.

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Murine Bone Marrow Megakaryocyte Isolation

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Hips, femurs, and tibias of 4–6 week-old mice were dissected and isolated. Bone marrow was obtained after crushing in ice-cold PBS containing 1% fetal bovine serum (FBS). Following centrifugation, red blood cell lysis (BD Pharmlyse, San Jose, CA) was performed. Next, cells were washed with PBS containing 2 mM EDTA and further cultured or stained for flow cytometry analysis. For MK differentiation, bone marrow cells were grown at 5x10⁶ cells/ml in StemSpan (StemCell Technologies, Vancouver, Canada) supplemented with 30% BIT9500 (StemCell Technologies) plus 20 ng/ml murine TPO for a total of 4 days before flow cytometry analysis.

Zou S., Teixeira A.M., Kostadima M., Astle W.J., Radhakrishnan A., Simon L.M., Truman L., Fang J.S., Hwa J., Zhang P.X., van der Harst P., Bray P.F., Ouwehand W.H., Frontini M, & Krause D.S. (2017). SNP in human ARHGEF3 promoter is associated with DNase hypersensitivity, transcript level and platelet function, and Arhgef3 KO mice have increased mean platelet volume. PLoS ONE, 12(5), e0178095.

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