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Cd34 expansion supplement

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The CD34+ Expansion Supplement is a cell culture medium component designed to support the proliferation and maintenance of CD34+ cells, which are hematopoietic stem and progenitor cells. The supplement contains a proprietary blend of cytokines, growth factors, and other nutrients that promote the expansion of CD34+ cells in vitro.

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Lab products found in correlation

Ultra low attachment 6 well plate, by Corning (2 mentions) Nucleofector 2b, by Lonza (2 mentions) Mtesr1, by STEMCELL (2 mentions) Stemspan sfem 2 expansion media, by STEMCELL (2 mentions) Stemspan sfem 2 media, by STEMCELL (2 mentions) Um171, by STEMCELL (2 mentions) Human cord blood cd34 positive selection kit 2, by STEMCELL (2 mentions) Clinmacs cd34 microbead kit, by Miltenyi Biotec (2 mentions) Sfem 2 medium, by STEMCELL (2 mentions) Dmpge2, by Cayman Chemical (2 mentions) Poloxamer 407, by BASF (2 mentions) Methocult h4434, by STEMCELL (1 mentions) Nsg mice, by Jackson ImmunoResearch (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions) X vivo 15, by Lonza (1 mentions) Stemspan sfem media, by STEMCELL (1 mentions)

Close spelling variants of this product

StemSpan CD34+ expansion supplement (18 citations) StemSpan™ CD34+ Expansion Supplement (10X) (4 citations) STEMSPAN II plus StemSpan™ CD34+ Expansion Supplement (2 citations) StemSpan with CD34 expansion supplement (2 citations)

10 protocols using cd34 expansion supplement

1

Reprogramming CD34+ Cells to iPSCs

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PBMC aliquots were thawed and resuspended in StemSpan SFEM II expansion media (StemCell Technologies, Cat. # 09605) with CD34+ expansion supplement (StemCell Technologies, Cat. # 02691). Cells were seeded into an Ultra-Low Attachment 6-well plate (Corning, Cat. # 3471). After 6 days, the CD34+ cells were counted and up to 1 million cells were reprogrammed using a combination of plasmids purchased from Addgene encoding for POU5F1, and shRNA for TP53 (ID: 27077) SOX2, and KLF4 (ID: 27078), L-MYC, and LIN28(ID: 27080), and EBNA1(ID: 37624) (Okita et al., 2013 (link)). Cells were transfected using program U-008 on the Lonza Nucleofector 2B device (Lonza, Cat. # AAB-1001), seeded onto mitomycin C treated SNL feeder cells (Applied StemCel,l Cat. # ASF-1327) in SFEM II media plus CD34+ Expansion supplement, and fed every other day with mTeSR1 (StemCell Technologies, Cat. # 85850) until colony establishment.
Kuang Y.L., Munoz A., Nalula G., Santostefano K.E., Sanghez V., Sanchez G., Terada N., Mattis A.N., Iacovino M., Iribarren C., Krauss R.M, & Medina M.W. (2019). Evaluation of commonly used ectoderm markers in iPSC trilineage differentiation. Stem cell research, 37, 101434.
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2

Reprogramming CD34+ Cells to iPSCs

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PBMC aliquots were thawed and resuspended in StemSpan SFEM II expansion media (StemCell Technologies, Cat. # 09605) with CD34+ expansion supplement (StemCell Technologies, Cat. # 02691). Cells were seeded into an Ultra-Low Attachment 6-well plate (Corning, Cat. # 3471). After 6 days, the CD34+ cells were counted and up to 1 million cells were reprogrammed using a combination of plasmids purchased from Addgene encoding for POU5F1, and shRNA for TP53 (ID: 27077) SOX2, and KLF4 (ID: 27078), L-MYC, and LIN28(ID: 27080), and EBNA1(ID: 37624) (Okita et al., 2013 (link)). Cells were transfected using program U-008 on the Lonza Nucleofector 2B device (Lonza, Cat. # AAB-1001), seeded onto mitomycin C treated SNL feeder cells (Applied StemCel,l Cat. # ASF-1327) in SFEM II media plus CD34+ Expansion supplement, and fed every other day with mTeSR1 (StemCell Technologies, Cat. # 85850) until colony establishment.
Kuang Y.L., Munoz A., Nalula G., Santostefano K.E., Sanghez V., Sanchez G., Terada N., Mattis A.N., Iacovino M., Iribarren C., Krauss R.M, & Medina M.W. (2019). Evaluation of commonly used ectoderm markers in iPSC trilineage differentiation. Stem cell research, 37, 101434.
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3

Xenograft Modeling of AML Cell Lines

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HEK293T, PHOENIX-Amphos, HL-60, KASUMI-1, THP-1, and MOLM-13 cells were purchased from ATCC and DSMZ. Human AML cells (Cureline Translational Cro) were cultured in StemSpan SFEM II media with CD34+ Expansion Supplement (StemCell Technologies). The shRNA plasmids were purchased from Sigma-Aldrich. FBXO21 and ∆FBXO21 were subcloned from pCDNA (kind gift from Dr. Yukiko Yoshida, Tokyo Metropolitan Institute of Medical Science) into pMIGR1 with tandem Strep and Flag tags, and retrovirus was produced as previously described [8 (link), 42 ]. Lentivirus was produced according to manufacturer instructions. Cells were treated with 1 μg/ml of puromycin 48 h post infection. For cytarabine treatment, cells were treated for 48 h with 50 nM cytarabine, and for MG132 treatment, cells were treated for 4 h with 20 µM MG132. For CFC assay cells were plated 100 cells/well of a 24-well plate in Methocult (H4434, StemCellTechnologies, Vancouver, BC, Canada), and counted between day 7-10.
For transplants, 5 × 105 MOLM-13 cells were transplanted into sub-lethally (250 cGy) irradiated 6–10 week old NSG mice (#005557, Jackson Labs). All experiments performed were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center in accordance with NIH guidelines.
Dobish K.K., Wittorf K.J., Swenson S.A., Bean D.C., Gavile C.M., Woods N.T., Ghosal G., Hyde R.K, & Buckley S.M. (2023). FBXO21 mediated degradation of p85α regulates proliferation and survival of acute myeloid leukemia. Leukemia, 37(11), 2197-2208.
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4

Cell Culture Conditions for Diverse Cell Lines

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MOLM-13, THP-1, Kasumi-1, K562, NB4, KCL22, Ku812 and U266B1 cell lines were cultured in RPMI1640 with 10% FBS and 1% Penicillin-Streptomycin (all from ThermoFisher, Waltham, MA). HL-60 cells were cultured in IMDM with 20% FBS and 1% Penicillin-Streptomycin. OCI-AML2 cells were cultured in αMEM with 10% FBS and 1% Penicillin-Streptomycin. HEK293T and HeLa cell lines were cultured in DMEM with 10% FBS and 1% Penicillin-Streptomycin (all from ThermoFisher, Waltham, MA). PDX and cord blood cells were cultured in StemSpan SFEM media supplemented with CD34+ Expansion Supplement (both from Stemcell Technologies, Cambridge, MA). Mouse AML cells and cKit+ bone marrow cells were sorted and cultured in in X-Vivo 15 (Lonza, Allendale, NJ) supplemented with 50 ng/ml SCF, 50 ng/ml TPO, 10 ng/ml IL-3 and 10 ng/ml IL-6 (all from Peprotech, Rocky Hill, NJ).
Eagle K., Jiang Y., Shi X., Li M., Obholzer N.P., Hu T., Perez M.W., Koren J.V., Kitano A., Yi J.S., Lin C.Y, & Nakada D. (2022). An oncogenic enhancer encodes selective selenium dependency in AML. Cell stem cell, 29(3), 386-399.e7.
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5

Characterizing Venetoclax Sensitivity in AML

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HL-60, KASUMI-1, and MOLM-13 cells were purchased and cultured as recommended by ATCC and DSMZ, respectively. All cells were confirmed mycoplasma negative prior to use. Human CD34+ cells were cultured in StemSpan SFEM II media with CD34+ Expansion Supplement (StemCell Technologies) for 24 hours prior to viral infection. The pLKO.1 shRNA plasmid vectors were purchased from Sigma-Aldrich. Lentivirus was produced following manufacturer’s instructions. Cells were infected as previously described and treated with 1μg/ml of puromycin 48 hours post infection. Assays were performed 5 days after initial puromycin treatment. MOLM-13 cells were treated for 72 hours with 0.1–50nM venetoclax and analyzed by MTT assay, and for flow cytometry analysis cells were treated with 5nM venetoclax for 48 hours.
Caplan M., Wittorf K.J., Weber K.K., Swenson S.A., Gilbreath T.J., Willow Hynes-Smith R., Amador C., Hyde R.K, & Buckley S.M. (2022). Multi-omics reveals mitochondrial metabolism proteins susceptible for drug discovery in AML. Leukemia, 36(5), 1296-1305.
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6

Expanded CB CD34+ Cell Expansion

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Expanded CB CD34+ cells were cultured in StemSpan SFEM (Stemcell Technologies) supplemented with 1× CD34+ Expansion Supplement (Stemcell Technologies), 100 units/mL penicillin, and 100 μg/mL streptomycin. The cells were exposed to ENU at concentrations of 8.54, 85.4, and 854 µM, D-mannitol at 1,152.8 and 11,528 µM, or 20 µL ultrapure water (vehicle control) for 48 h. Single-cell suspensions from treated cells and vehicle control samples were harvested and stained for viability as described above. Single-cell sorts were also carried out as described above. PTA was performed and libraries were prepared as per the manufacturer’s instructions (startup).
Gonzalez-Pena V., Natarajan S., Xia Y., Klein D., Carter R., Pang Y., Shaner B., Annu K., Putnam D., Chen W., Connelly J., Pruett-Miller S., Chen X., Easton J, & Gawad C. (2021). Accurate genomic variant detection in single cells with primary template-directed amplification. Proceedings of the National Academy of Sciences of the United States of America, 118(24), e2024176118.
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7

Venetoclax Sensitivity in AML Cell Lines

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HL-60, KASUMI-1, and MOLM-13 cells were purchased and cultured as recommended by ATCC and DSMZ, respectively. All cells were confirmed mycoplasma negative prior to use. Human CD34+ cells were cultured in StemSpan SFEM II media with CD34+ Expansion Supplement (StemCell Technologies) for 24 h prior to viral infection. The pLKO.1 shRNA plasmid vectors were purchased from Sigma-Aldrich. Lentivirus was produced following manufacturer’s instructions. Cells were infected as previously described and treated with 1 μg/ml of puromycin 48 h post infection. Assays were performed 5 days after initial puromycin treatment. MOLM-13 cells were treated for 72 h with 0.1–50 nM venetoclax and analyzed by MTT assay, and for flow cytometry analysis cells were treated with 5 nM venetoclax for 48 h.
Caplan M., Wittorf K.J., Weber K.K., Swenson S.A., Gilbreath T.J., Willow Hynes-Smith R., Amador C., Hyde R.K, & Buckley S.M. (2022). Multi-omics reveals mitochondrial metabolism proteins susceptible for drug discovery in AML. Leukemia, 36(5), 1296-1305.
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8

Expansion and Proliferation of CD34+ Cells

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HHCC were propagated in H3000 (StemSpan media) with CD34+ expansion supplement (StemSpan media) (StemCell Technologies) and 10% of FBS (Thermo Fisher Scientific) in standard conditions. Proliferation properties of the CD34+ donor-derived cells, as well as the fused HHCC were assessed using CountBrightTM Absolute Counting Beads (Thermo Fisher Scientific). Briefly, cells were plated in a 24-well tissue culture plate (1×104 cells/well) and mixed with CountBrightTM absolute counting beads according to the manufacturer’s instructions. After adding the beads, cells were analyzed at days 0, 7, 14, 21 and 28 by FC (Gallios, Beckman Coulter) and the total number of cells was calculated based on the formula provided by the manufacturer. Kaluza software (Kaluza Analysis 2.1) was used to analyze the results.
Siemionow M., Brodowska S., Różczka K, & Roesler C. (2022). Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation. Stem Cell Investigation, 9, 11.
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9

Expansion and Lentiviral Transduction of CD34+ Cells

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Cord blood (CB) CD34+ cells were purified with Human Cord Blood CD34 Positive Selection Kit II (StemCell Technologies, 17896) and cultured in SFEM II medium (StemCell Technologies, 09605) supplemented with CD34+ Expansion Supplement (StemCell Technologies, 02691). Bone marrow (BM) and mobilized peripheral blood (mPB) CD34+ cells were purified with ClinMACS CD34 MicroBead Kit (Miltenyi,130-046-702) and cultured in in SCGM (CellGenix, 20802-0500) supplemented with 100ng/ml recombinant human cytokines thrombopoietin (TPO) (PrimeGene, 102-06), 100ng/ml fms-related tyrosine kinase 3 ligand (FltL) (PrimeGene , 103-05), 100ng/ml stem cell factor (SCF) (PrimeGene, 102-01) and 25ng/ml IL3 (PrimeGene, 101-03). CD34+ cells for transduction were cultured at a concentration of 1×10 6 to 2×10 6 cells/ml at 37℃ in a humidified incubator of 5% CO2. For lentiviral transduction, CD34+ cells were pre-stimulated in culture medium for 24 hours, and then transduced with lentivirus for 24 hours. The enhancers of protamine (Sigma, P4020), dmPGE2 (Cayman, 39746-25-3), UM171 (StemCell Technologies, 72914) and Poloxamer407 (BASF, 9003-11-06) were added at indicated concentration immediately prior to the addition of lentivirus.
Ouyang W., Dong G., Zhao W., Li J., Zhou Z., Yang G., Liu R., Li Y., Zhang Q., Du X., Sun H., Gu Y., Lai Y., Liu S, & Liu C. (2021). Restoration of β-Globin Expression with Optimally Designed Lentiviral Vector for β-Thalassemia Treatment in Chinese Patients. Human gene therapy, 32(9-10).
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10

Expansion and Lentiviral Transduction of CD34+ Cells

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Cord blood (CB) CD34+ cells were purified with Human Cord Blood CD34 Positive Selection Kit II (StemCell Technologies, 17896) and cultured in SFEM II medium (StemCell Technologies, 09605) supplemented with CD34+ Expansion Supplement (StemCell Technologies, 02691). Bone marrow (BM) and mobilized peripheral blood (mPB) CD34+ cells were purified with ClinMACS CD34 MicroBead Kit (Miltenyi,130-046-702) and cultured in in SCGM (CellGenix, 20802-0500) supplemented with 100ng/ml recombinant human cytokines thrombopoietin (TPO) (PrimeGene, 102-06), 100ng/ml fms-related tyrosine kinase 3 ligand (FltL) (PrimeGene , 103-05), 100ng/ml stem cell factor (SCF) (PrimeGene, 102-01) and 25ng/ml IL3 (PrimeGene, 101-03). CD34+ cells for transduction were cultured at a concentration of 1×10 6 to 2×10 6 cells/ml at 37℃ in a humidified incubator of 5% CO2. For lentiviral transduction, CD34+ cells were pre-stimulated in culture medium for 24 hours, and then transduced with lentivirus for 24 hours. The enhancers of protamine (Sigma, P4020), dmPGE2 (Cayman, 39746-25-3), UM171 (StemCell Technologies, 72914) and Poloxamer407 (BASF, 9003-11-06) were added at indicated concentration immediately prior to the addition of lentivirus.
Ouyang W., Dong G., Zhao W., Li J., Zhou Z., Yang G., Liu R., Li Y., Zhang Q., Du X., Sun H., Gu Y., Lai Y., Liu S., & Liu C. (2020). Restoration of β-globin expression with optimally designed lentiviral vector for β-thalassemia treatment in Chinese patients.
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