CD34+ hematopoietic stem and progenitor cells were obtained from the Fred Hutchinson Hematopoietic Cell Processing and Repository (Seattle, USA) or StemCell Technologies. The CD34+ samples were de-identified and approval for use of these samples for research purposes was provided by the Institutional Review Board and Biosafety Committees at Boston Children’s Hospital. Healthy donor peripheral blood mononuclear cells were obtained from StemCell Technologies. CD34+ cells were thawed and cultured in StemSpan II with 1x CC100 (StemCell Technologies, Inc.) at 37°C and 5% CO2. At indicated time points, these cells were seeded in media supporting the differentiation into monocytic and erythroid cells57 ,58 . Briefly, cells were cultured at a density of 105 - 106 cells per milliliter (ml) in IMDM supplemented with 2% human AB plasma, 3% human AB serum, 1% penicillin/streptomycin, 3 IU/ml heparin, 10 mg/ml insulin, 200 mg/ml holo-transferrin, 1 IU erythropoietin (Epo), 10 ng/ml stem cell factor (SCF) and 1 ng/ml IL-3 and incubated at 37°C and 5% CO2. For mtscATAC-seq processing at indicated time points and when additional cells were to be maintained to enable sampling of cells at a later time, ⅓ of the cultured cells were maintained and ⅔ of the cells were forwarded to single cell sequencing as described below.
Stemspan 2
Manufactured by STEMCELL
Sourced in United States
StemSpan II is a serum-free, xeno-free cell culture medium designed to support the expansion and maintenance of human hematopoietic stem and progenitor cells (HSPCs).
Automatically generated - may contain errors
Lab products found in correlation
Cc100, by STEMCELL (3 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (2 mentions)
Stemmacs hsc expansion cocktail, by Miltenyi Biotec (1 mentions)
Stemmacs hsc expansion media xf, by Miltenyi Biotec (1 mentions)
Meg 01, by American Type Culture Collection (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Methocult h4034 optimum, by STEMCELL (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fugene, by Promega (1 mentions)
Facsaria 2, by BD (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Hpgm medium, by Lonza (1 mentions)
Anti human gpa pe cd235a hir2, by BD (1 mentions)
Myelocult h5100, by STEMCELL (1 mentions)
Cd38 pecy7 hb7, by BD (1 mentions)
Anti human cd34 apc 8g12, by BD (1 mentions)
9 protocols using stemspan 2
1
Differentiation of CD34+ Hematopoietic Cells
2
Culturing Patient-Derived AML Cells
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Patient-derived AML cells for CRISPR experiments were cultured with Stemspan II (STEMCELL Technologies), 1% PS, completed with 100 ng/mL of human FLT3L and SCF, 50 ng/mL of human TPO, IL3, and IL6 (BioLegend), and 750 nmol/L of SR1 (Cayman Chemical). For the ex vivo cultures, AML and/or MDS cells were cultured on StemMACS HSC Expansion Media XF supplemented with StemMACS HSC Expansion Cocktail (Miltenyi Biotec).
3
Megakaryocyte Differentiation from Cord Blood CD34+ Cells
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Purified cord blood (CB) CD34+ hematopoietic stem cells were purchased from StemCell Technologies and cultured in StemSpan II media with the MK supplemental cytokines (StemSpan II, Stemcell Technologies, Inc.), according to the culture and differentiation protocols from Stemcell Technologies (Stemcell Technologies Inc. Cambridge, MA). These CD34+-derived MKs are referred to as SC-MKs throughout the paper. A megakaryoblastic cell line (Meg-01) was purchased from ATCC (American Type Culture Collection, Manassas, VA) and cultured in RPMI with 10% FBS, according to ATCC’s standard culture protocols. Meg-01 cells were used for experiments for both optimization of protocols prior to repetition with the SC CD34 cell line or when a high concentration of cells was needed for multiple replicates, such as the chemotaxis assays. Because Meg-01 cells are from a megakaryoblastic cell line and therefore may not exhibit the same behavior or receptors as the fully-mature, non-eternal MK cells, CD34+-derived MK cells were used as an additional MK model to further support our findings, although these cells may also not display the same behaviors as circulating MKs (48 (link), 49 (link)).
4
Hematopoietic Stem Cell Isolation and Culture
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BMMNC were isolated as above and cKit enrichment was performed using magnetic activated cell sorting (MACS)-conjugated cKit antibodies (Miltenyi Biotec) and an AutoMACS cell sorter according to the manufacturer’s protocol. Enriched cells were stained with antibodies and sorted to StemSpanII (StemCell Technologies) containing stem cell factor (SCF, 20 ng/mL, Peprotech) and THPO (20 ng/mL, Peprotech), or SCF, THPO, interleukin-3 (IL3, 20 ng/mL, Peprotech), interleukin-6 (IL6, 20 ng/mL, Peprotech) and erythropoietin (EPO, 10 ng/mL, R&D Systems). On day 10 of culture colonies were stained with CD41, CD16/32, CD71 (C2, BD Biosciences), Ter119, CD11b, Ly6C/G and propidium iodide was added to identify live cells. Lineages were gated as shown in Online Supplementary Figure S1 and colonies were defined by the presence of >20 cells of any one of each lineage.
5
CD34+ Stem Cell Culture and Assay
6
Expansion and Erythroid Differentiation of CD34+ Cells
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The G-CSF mobilized CD34+ cells were purchased from the Fred Hutchinson Cancer Center Co-Operative Center for Excellence in Hematology. The cells were quickly thawed in a 37C water bath followed by graduated osmotic equilibration by doubling the total volume with PBS + 0.5% BSA every 2 minutes for a total of 5 doubling. Following centrifugation, the cells were cultured in Stem Span II (Stem Cell Technologies) supplemented with 100 ng/mL each of TPO, IL-6, SCF and FLT-3 ligand. For all the nucleofection optimization experiments the cells were expanded for 3 days prior to nucleofection and cultured in this media for 3 days post nucleofection.
For the erythroid differentiation test the cells were initially expanded as described above. Following nucleofection, the cells were culture as described in Uchida et al. 2018 [23 (link)], in erythroid differentiation media consisting of IMDM supplemented with 20% Knockout Serum Replacement (Thermo Fisher Scientific) 2 U/mL EPO, 10 ng/mL SCF, 1ng/mL IL-3, 1 μM dexamethasone and 1 μM estradiol for 5 days. The cells were then transitioned to Erythroid maturation media containing in IMDM supplemented with 20% Knockout Serum Replacement, 2 U/mL EPO, 10 ng/mL insulin and 0.5 mg/mL holo-transferrin for 2 days followed by flow cytometry analysis. EPO was purchased from PeproTech and all other cytokines were purchased from Shenandoah Biotechnology.
For the erythroid differentiation test the cells were initially expanded as described above. Following nucleofection, the cells were culture as described in Uchida et al. 2018 [23 (link)], in erythroid differentiation media consisting of IMDM supplemented with 20% Knockout Serum Replacement (Thermo Fisher Scientific) 2 U/mL EPO, 10 ng/mL SCF, 1ng/mL IL-3, 1 μM dexamethasone and 1 μM estradiol for 5 days. The cells were then transitioned to Erythroid maturation media containing in IMDM supplemented with 20% Knockout Serum Replacement, 2 U/mL EPO, 10 ng/mL insulin and 0.5 mg/mL holo-transferrin for 2 days followed by flow cytometry analysis. EPO was purchased from PeproTech and all other cytokines were purchased from Shenandoah Biotechnology.
7
CDC42 Mutant Effects on Hematopoietic Stem Cells
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HEK293T and NIH-3T3 cell lines were obtained and maintained in
Dulbelcco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% of
fetal bovine serum (FBS; Atlanta Biologicals) and 1% of penicillin/streptomycin
(GIBCO). Human primary adult bone marrow-derived
CD34+ hematopoietic stem and progenitor cells
(HSPCs) were obtained from Fred Hutchinson Cancer Research Center and maintained
in StemSpan II and 100X CC100 (Stem Cell Technologies) with recombinant
thrombopoietin at 50 ng/mL (TPO; Peprotech).
CDC42 WT and R186C mutant lentiviral constructs were transfected
into HEK293T with transfection reagent FuGene (Promega) and helper plasmids, as
previously described [19 ,
26 (link)]. The primary HSPCs were
infected with virus during day 1 of culture. Primary HSPCs were then sorted for
GFP on day 3 and processed for assays. Multiple donors were used for downstream
assays and produced similar results. NIH-3T3 cells were infected with lentivirus
and 48 h later, used for further downstream applications.
Dulbelcco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% of
fetal bovine serum (FBS; Atlanta Biologicals) and 1% of penicillin/streptomycin
(GIBCO). Human primary adult bone marrow-derived
CD34+ hematopoietic stem and progenitor cells
(HSPCs) were obtained from Fred Hutchinson Cancer Research Center and maintained
in StemSpan II and 100X CC100 (Stem Cell Technologies) with recombinant
thrombopoietin at 50 ng/mL (TPO; Peprotech).
CDC42 WT and R186C mutant lentiviral constructs were transfected
into HEK293T with transfection reagent FuGene (Promega) and helper plasmids, as
previously described [19 ,
26 (link)]. The primary HSPCs were
infected with virus during day 1 of culture. Primary HSPCs were then sorted for
GFP on day 3 and processed for assays. Multiple donors were used for downstream
assays and produced similar results. NIH-3T3 cells were infected with lentivirus
and 48 h later, used for further downstream applications.
8
Differentiation of CD34+ Hematopoietic Cells
9
Lentiviral Transduction of CD34+ Cord Blood Cells
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