Stemspan sfem 2

Manufactured by STEMCELL

Sourced in Canada

StemSpan SFEM II is a serum-free medium designed to support the expansion and maintenance of human and mouse hematopoietic stem and progenitor cells in vitro. It contains a proprietary blend of growth factors, cytokines, and other components to promote cell growth and differentiation.

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StemSpanTM SFEM II serum-free medium StemSpan SFEM II medium StemSpan-SFEM II with 100X StemSpan Erythroid Expansion Supplement StemSpan SFEM II expansion media StemSpan SFEM II media StemSpan™ Serum-Free Expansion Medium II (SFEM II) StemSpan SFEM II human hematopoietic stem cell expansion media StemSpan SFEM StemSpan II StemSpan

86 protocols using stemspan sfem 2

Erythroid Differentiation Protocol

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HEK293T cells, K562 cells, MEL cells, and human CD34⁺ primary erythroid progenitors from healthy donors were cultured as described previously (13 (link),29 (link)). Briefly, primary human CD34⁺ cells were isolated from healthy human adult peripheral blood (PB) mononuclear cells by magnetic sorting. Cells were cultured initially for expansion in StemSpan SFEM II medium supplied with 1× CC100 cytokine mix (StemCell Technologies, Inc.) and 2% penicillin/streptomycin for 6 days, and cells were then transferred into the same medium with SCF (20 ng/ml), EPO (1 IU/ml), IL-3 (5 ng/ml), dexamethasone (2 μM), β-estradiol (1 μM) and 2% penicillin/streptomycin. Cells were maintained at a density of 1 × 10⁶ cells per milliliter, and cultures were supplemented every other day with fresh medium during a 12-day differentiation stage. After differentiation, cell surface marker analysis with CD71 and glycophorin A (GPA) indicated that more than 95% of cultured cells were of the erythroid lineage.
HUDEP-2 cells (30 (link)) were expanded in StemSpan SFEM II (StemCell Technologies, Inc.) medium supplied with doxycycline (1 μg/ml), dexamethasone (10^–6 M), SCF (50 ng/ml), EPO (3 IU/ml), 1% l-glutamine and 2% penicillin/streptomycin.

Li X., Chen M., Liu B., Lu P., Lv X., Zhao X., Cui S., Xu P., Nakamura Y., Kurita R., Chen B., Huang D.C., Liu D.P., Liu M, & Zhao Q. (2021). Transcriptional silencing of fetal hemoglobin expression by NonO. Nucleic Acids Research, 49(17), 9711-9723.

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Expansion and Differentiation of CD34+ HPCs

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Human cord blood CD34⁺ HPCs were cultured in StemSpan SFEM II containing StemSpan CD34⁺ expansion supplement (STEMCELL Technologies). To differentiate them into MPCs, the HPCs were transferred to StemSpan SFEM II with StemSpan myeloid expansion supplement (STEMCELL Technologies). The medium was changed every 2 or 3 days, and the MPCs were used after 7 days of differentiation.

Nakazawa Y., Miyano M., Tsukamoto S., Kogai H., Yamamoto A., Iso K., Inoue S., Yamane Y., Yabe Y., Umihara H., Taguchi J., Akagi T., Yamaguchi A., Koga M., Toshimitsu K., Hirayama T., Mukai Y, & Machinaga A. (2024). Delivery of a BET protein degrader via a CEACAM6-targeted antibody–drug conjugate inhibits tumour growth in pancreatic cancer models. Nature Communications, 15, 2192.

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Culturing Cryopreserved Pediatric ALL Cells

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Cryo-preserved pediatric ALL patient bone marrow (BM) and PDX cells were thawed at 37 °C and cultured in serum-free medium designed to support viable culture of hematopoietic cells. B-ALL was cultured in StemSpan™ SFEM II (StemCell, cat no. 09655) medium supplemented with 1X Stemspan™ CC100 (StemCell, cat no. 02690). T-ALL was cultured in StemSpan™ SFEM II (StemCell, cat no. 09655) medium supplemented with 100 ng/mL interleukin 2, and 25 μl/mL ImmunoCult™ Human CD3/CD28/CD2 T-cell activator (StemCell, cat no. 10970). Complete media was freshly prepared according to manufacturer’s instructions.

Uzozie A.C., Ergin E.K., Rolf N., Tsui J., Lorentzian A., Weng S.S., Nierves L., Smith T.G., Lim C.J., Maxwell C.A., Reid G.S, & Lange P.F. (2021). PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways. Journal of Experimental & Clinical Cancer Research : CR, 40, 96.

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Expansion of CD34+ Hematopoietic Stem Cells

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CD34⁺ HSPCs derived from mobilized peripheral blood were purchased from AllCells, and CD34⁺ HSPCs derived from cord blood were provided from donors under informed consent via the Binns Program for Cord Blood Research at Stanford University. CD34⁺ donors were either used fresh or frozen immediately after isolation for later use. All CD34⁺ HSPCs were cultured in StemSpan SFEM II (StemCell Technologies), supplemented with TPO (100 ng/mL), SCF (100 ng/mL), Flt 3 ligand (100 ng/mL), interleukin-6 (IL-6) (100 ng/mL), Stem Regenin 1 (0.75 μM; Cellagen Tech), UM171 (35 nM; StemCell Technologies), streptomycin (20 U/mL), and penicillin (20 U/mL). K562 cells were cultured in RPMI 1640 (HyClone) supplemented with 10% bovine growth serum, 100 U/mL streptomycin, 100 units/mL penicillin, and 2 mM L-glutamine. All cells were cultured at 37°C with 5% CO₂, K562 cells were cultured at ambient O₂, and CD34⁺ HSPCs were cultured at 5% O_2. Cells were cultured at densities ranging from 1 × 10⁵–1 × 10⁶ cells/mL.

Charlesworth C.T., Camarena J., Cromer M.K., Vaidyanathan S., Bak R.O., Carte J.M., Potter J., Dever D.P, & Porteus M.H. (2018). Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting. Molecular Therapy. Nucleic Acids, 12, 89-104.

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CRISPR Gene Editing of Mobilized HSPCs

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Mobilized human CD34⁺ HSPCs (AllCells, Alameda, CA, USA) were thawed and cultured for 48 h at a density of 2.5 × 10⁵ cells/mL in StemSpan SFEM II (StemCell Technologies, Vancouver, Canada), supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin (Biological Industries, Beit Haemek, Israel), stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3), and interleukin (IL)-6 (100 ng/mL each; PeproTech, Rocky Hill, NJ, USA). Cells were cultured at 37°C, 5% CO₂, and 5% O₂. 0.5 × 10⁵ CD34⁺ HSPCs were reconstituted in P3 Primary Cell electroporation solution, according to the manufacturer’s instructions (Lonza, Basel, Switzerland) and mixed with RNP complexes at a final concentration of 1 or 4 μM. Cells were then supplemented with either 3.85 μM Alt-R EE (IDT, Coralville, IA, USA)¹⁴ (link) or an equivalent volume of PBS. The supplemented cell solution (final cell concentration of 2 × 10⁶ /mL) was transferred into the Lonza 4D-Nucleofector and electroporated using the DZ-100 program. Recovered cells were cultured for 48–72 h prior to gDNA extraction with QuickExtract (Lucigen, Middleton, WI, USA). RNP complexes were formed and delivered in the same manner with the Alt-R HiFi Cas9 variant (IDT, Coralville, IA, USA).

Shapiro J., Iancu O., Jacobi A.M., McNeill M.S., Turk R., Rettig G.R., Amit I., Tovin-Recht A., Yakhini Z., Behlke M.A, & Hendel A. (2020). Increasing CRISPR Efficiency and Measuring Its Specificity in HSPCs Using a Clinically Relevant System. Molecular Therapy. Methods & Clinical Development, 17, 1097-1107.

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Nucleofection of Hematopoietic Stem Cells

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Hematopoietic stem and progenitor cells were counted after two days and centrifuged at 300 g for 10 minutes. T-cells were counted after 4–6 days in culture. Supernatant was removed and cells were resuspended in P3 Primary Cell Nucleofector^® Solution with Supplement 1 (Lonza, V4XP-3032) at 5–15 million cell/mL. Cell mixture was transferred to Lonza strip at 20 μL per well and electroporated using program DZ100 with solution setting P3. 80 μL of StemSpan^™ SFEM II supplemented with StemSpan^™ CD34+ Expansion Supplement (10X) (StemCell Technologies, Catalog #09720) was added to each well. AAV was added after electroporation, if necessary, at 10⁴ vg/cell. HSPCs were transferred to 24-well non-treated plates at a density of 100,000 cells/mL and incubated at 37°C for 2–3 days. T-cells were cultured at densities of 500,000–1,000,000 cells/mL.

Kanke K.L., Rayner R.E., Abel E., Venugopalan A., Suu M., Stack J.T., Nouri R., Guo G., Vetter T.A., Cormet-Boyaka E., Hester M.E, & Vaidyanathan S. (2024). Single-Stranded DNA with Internal Base Modifications Mediates Highly Efficient Gene Insertion in Primary Cells. bioRxiv.

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Cell Culture Media Protocols

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Airway cells: PneumaCult Ex Plus medium (StemCell Technologies #05040) was used for culturing ABCs. Differentiation media was purchased from the University of North Carolina core.
HSPC: StemSpan^™ SFEM II supplemented with StemSpan^™ CD34+ Expansion Supplement (10X) (StemCell Technologies, Catalog # 09720). IPSC: StemFlex^™ media (ThermoFisher Scientific, A3349401). HUVEC: EGM^™−2 Endothelial Cell Growth Medium-2 BulletKit^™ (Lonza, CC-3162). T-cells: X-Vivo 15 media (Lonza; 02-060Q)

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CD34+ Cell Expansion and Culturing

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G-CSF Mobilized Human Peripheral Blood CD34+ Cells were purchased from StemCell Technologies (Catalog: 70060.1). Cells were thawed by resuspending in 10 mL of RPMI without FBS. The tube was centrifuged at 300 g for 10 minutes and the supernatant removed. Cells were resuspended in StemSpan^™ SFEM II supplemented with StemSpan^™ CD34+ Expansion Supplement (10X) (StemCell Technologies, Catalog # 09720). Cells were plated on 24-well non-treated plates at a density of 100,000 cells/mL and incubated at 37°C for 2–3 days.

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Generation of hiPSCs from PBMCs

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PBMCs were reprogrammed to hiPSCs as previously described (Rovina, Castiglioni et al. 2022 ). Briefly, the protocol consisted of electroporation with the reprogramming vectors of the Epi5 Episomal iPSC Reprogramming Kit (ThermoFisher Scientific) at day 0 (1650 V, 10 s, 3 pulse), followed by a gradual transition of the culture medium from StemSpanSFEM II (STEMCELL) to ReproTeSR (STEMCELL) for promoting iPSC colony maturation. Starting from day 7, the medium was refreshed daily until the colonies were ready to be picked. After 14–21 days of reprogramming, clones were manually picked and cultured in StemFlex medium (GIBCO) on Vitronectin-coated plates (37 °C, 5% CO₂). Non-enzymatically passages with ReLeSR™ (STEMCELL) were performed every 3–4 days. Mature hiPSCs were cryopreserved in CryoStor® (Sigma-Aldrich).

Rabino M., Rurali E., Zamboni C., Rovina D., Mallia S., Cauteruccio M., Baggiano A., Giacari C.M., Bellin M, & Pompilio G. (2023). Generation of four human induced pluripotent stem cell lines from COVID-19 hospitalized patients with increased levels of cardiac Troponin in the acute infection phase developing or not myocarditis. Stem Cell Research, 67, 103018.

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Lentiviral Transduction of CD34+ HSPCs

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De-identified UCB was obtained from New York Blood Center. CD34 + HSPCs were isolated by density gradient centrifugation (Lymphoprep, SepMate-50 tubes, Stemcell Tech), followed by magnetic separation on an AutoMACS Pro using microbeads conjugated with anti-CD34 antibodies (Miltenyi). CD34 + cells were cultured overnight in StemSpan SFEM II (Stemcell Tech.) supplemented with 10% fetal bovine serum, penicillin/streptomycin, L-glutamine, 2-mercaptoethanol, 1 μM SR-1 (Stemcell Tech.), 100 ng/mL SCF, 40 ng/mL FLT3L, 50 ng/mL TPO, 20 ng/mL IL3, 20 ng/mL IL6, and 15 ng/mL GM-CSF (Peprotech Inc). Subsequently, cells were sequentially transduced by spinoculation at 1500 RPM at 37 C for 90 min on retronectin-coated plates loaded with pTRIP-MND-GFP (first transduction) or pCL20.MSCV.mir30.PGK.mCherry (second transduction) lentiviral particles in culture media in the presence of 10 μg/mL polybrene. Subsequently, mCherry + GFP + cells were sorted and plated onto semi-solid methylcellulose media (Methocult H4230, Stemcell Tech) supplemented with 5 U/mL EPO, 10 ng/mL IL-3, 5 ng/mL SCF, 5 ng/mL GM-CSF. Colonies were enumerated 12–14 days after plating.

Balcerek J., Jiang J., Li Y., Jiang Q., Holdreith N., Singh B., Chandra V., Lv K., Ren J.G., Rozenova K., Li W., Greenberg R.A, & Tong W. (2018). Lnk/Sh2b3 deficiency restores hematopoietic stem cell function and genome integrity in Fancd2 deficient Fanconi anemia. Nature Communications, 9, 3915.

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