诱导的人多能干细胞（iPS细胞）和胚胎干细胞（ES细胞）向树突状细胞（DC）亚型的分化

Stephanie Sontag; Malrun Förster; Kristin Seré; Martin Zenke

doi:10.21769/BioProtoc.2419

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Peer-reviewed

Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets

诱导的人多能干细胞（iPS细胞）和胚胎干细胞（ES细胞）向树突状细胞（DC）亚型的分化

SS Stephanie Sontag

MF Malrun Förster

KS Kristin Seré

Martin Zenke email

发布: 2017年08月05日第7卷第15期 DOI: 10.21769/BioProtoc.2419 浏览次数: 15034

评审: Xiao LiAnonymous reviewer(s)

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Abstract

Induced pluripotent stem cells (iPS cells) are engineered stem cells, which exhibit properties very similar to embryonic stem cells (ES cells; Takahashi and Yamanaka, 2016). Both iPS cells and ES cells have an extraordinary self-renewal capacity and can differentiate into all cell types of our body, including hematopoietic stem/progenitor cells and dendritic cells (DC) derived thereof. This makes iPS cells particularly well suited for studying molecular mechanisms of diseases, drug discovery and regenerative therapy (Grskovic et al., 2011; Bellin et al., 2012; Robinton and Daley, 2012).

DC are the major antigen presenting cells of the immune system and thus they are key players in modulating and directing immune responses (Merad et al., 2013). DC patrol peripheral and interface tissues (e.g., lung, intestine and skin) to detect invading pathogens, and upon activation they migrate to lymph nodes to activate and prime lymphocytes.

DC comprise a phenotypically heterogeneous family with functionally specialized subsets (Schlitzer and Ginhoux, 2014). Generally, classical DC (cDC) and plasmacytoid DC (pDC) are distinguished, exhibiting a classical and plasma cell-like DC morphology, respectively. cDC recognize a multitude of pathogens and secrete proinflammatory cytokines upon activation, while pDC are specialized to detect intracellular pathogens and secrete type I interferons (Merad et al., 2013; Schlitzer and Ginhoux, 2014). cDC are further divided into cross-presenting cDC1 and conventional cDC2, in the human system referred to as CD141⁺ Clec9a⁺ cDC1 and CD1c⁺ CD14^- cDC2. Human pDC are characterized as CD303⁺ CD304⁺ (Jongbloed et al., 2010; Joffre et al., 2012; Swiecki and Colonna, 2015).

To investigate subset specification and function of human DC, we established a protocol to generate cDC1, cDC2 and pDC in vitro from human iPS cells (or ES cells) (Sontag et al., 2017). Therefore, we differentiated iPS cells (or ES cells), via mesoderm commitment and hemato-endothelial specification, into CD43⁺ CD31⁺ hematopoietic progenitors. Subsequently, those were seeded onto inactivated OP9 stromal cells with FLT3L, SCF, GM-CSF and IL-4 or FLT3L, SCF and GM-CSF to specify cDC1 and cDC2, or cDC1 and pDC, respectively.

Keywords: iPS cells (iPS细胞)

ES cells (ES细胞)

Hematopoiesis (造血)

Hematopoietic differentiation (造血分化)

Human dendritic cells (人树突状细胞)

Dendritic cell differentiation (树突状细胞分化)

Background

DC and their development have mostly been studied in mice (Belz and Nutt, 2012; Merad et al., 2013; Schlitzer and Ginhoux, 2014). Owing to their rarity in non-lymphoid tissues and the restricted access to human lymphoid tissues, studying DC in humans is challenging (Jongbloed et al., 2010; Villadangos and Shortman, 2010). Yet, understanding developmental pathways, origins and mechanisms during DC subset specification is important in order to generate autologous DC subsets in vitro for therapeutic applications (e.g., anti-tumor agents). iPS cells (and ES cells) with their unlimited self-renewal and differentiation potential raise hopes that DC can be generated and modified (e.g., loaded with patient and disease specific antigens) in high numbers and quality in vitro to study DC development and function and to improve DC immunotherapies (Sontag et al., 2017).

Several groups have generated monocyte derived DC from iPS cells (and ES cells) with conventional granulocyte macrophage colony stimulating factor (GM-CSF)/interleukin 4 (IL-4) protocols, but such DC represent inflammatory DC and are not subset specific (Senju et al., 2007; Tseng et al., 2009; Choi et al., 2011; Senju et al., 2011; Belz and Nutt, 2012; Rossi et al., 2012; Yanagimachi et al., 2013; Li et al., 2014). From mouse studies, it is known that fms-like tyrosine kinase ligand (FLT3L) is the key cytokine for DC development and that the combination of FLT3L and GM-CSF signaling specifies DC subsets in vivo (Gilliet et al., 2002; Schmid et al., 2010). Recently, human cDC1, cDC2 and pDC were generated from cord blood (CB) with FLT3L, stem cell factor (SCF) and GM-CSF on MS5 stromal cells (Lee et al., 2015). Additionally, Poulin et al. (2010) reported on the generation of cDC1 from CB with FLT3L, SCF, GM-CSF and IL-4 in a feeder-free environment (Poulin et al., 2010). In contrast, Silk et al. (2012) described the differentiation of cDC1 in a feeder-free GM-CSF/IL-4 system (Silk et al., 2012). However, recent genome wide transcriptional profiling studies highlight the impact of microenvironmental cues during DC development, indicating that feeder support is important (Lundberg et al., 2013). Therefore, we used FLT3L, SCF, GM-CSF and IL-4 (referred to as FSG4) and FLT3L, SCF and GM-CSF (referred to as FSG) in combination with OP9 stromal cells to generate cDC1, cDC2 and pDC from iPS cell (or ES cell) derived hematopoietic progenitors.

Here we describe a two-step protocol: First, human iPS cells (or ES cells) are differentiated into hematopoietic progenitors (adapted from Kennedy et al., 2012). Second, these hematopoietic stem/progenitor cells are then further differentiated into cDC1, cDC2 and pDC (Sontag et al., 2017).

Materials and Reagents

6-well tissue culture plates (TPP Techno Plastic Products, catalog number: 92006 )
10 cm microbiology grade Petri dishes (SARSTEDT, catalog number: 82.1473.001 )
50 ml Falcon tubes (Corning, Falcon^®, catalog number: 352070 )
70 µm cell strainer (Greiner Bio One International, catalog number: 542070 )
40 µm cell strainer (Greiner Bio One International, catalog number: 542040 )
Top bottle filter (TPP Techno Plastic Products, catalog number: 99505 )
5 ml Serological pipettes (Corning, Falcon^®, catalog number: 357543 )
10 ml Serological pipettes (Corning, Falcon^®, catalog number: 357551 )
25 ml Serological pipettes (Corning, Falcon^®, catalog number: 357525 )
Pipette tips 10 µl (STARLAB INTERNATIONAL, catalog number: S1180-3810 )
Pipette tips 20 µl (STARLAB INTERNATIONAL, catalog number: S1120-1810 )
Pipette tips 200 µl (STARLAB INTERNATIONAL, catalog number: S1120-8810 )
Pipette tips 1,000 µl (STARLAB INTERNATIONAL, catalog number: S1126-7810 )
Collagenase IV (Thermo Fisher Scientific, Gibco^TM, catalog number: 17104019 )
Knockout-Dulbecco’s modified Eagle medium (KO-DMEM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10829018 )
Gelatin (Sigma-Aldrich, catalog number: G1890 )
1-Thiogylcerol (Sigma-Aldrich, catalog number: M1753 )
L-ascorbic acid (Sigma-Aldrich, catalog number: A4403 )
Bovine serum albumin (BSA) low endotoxin (PAA, catalog number: K31-011 )
1x Dulbecco’s phosphate buffered saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190094 )
HumanKine bone morphogenic protein 4 (BMP4) research grade (Miltenyi Biotec, catalog number: 130-110-921 )
Recombinant human basic fibroblast growth factor (bFGF) (PeproTech, catalog number: 100-18B )
Recombinant human FLT3L (PeproTech, catalog number: 300-19 )
Recombinant human GM-CSF (PeproTech, catalog number: 300-03 )
Recombinant human insulin-like growth factor 1 (IGF1) (PeproTech, catalog number: 100-11 )
Human interleukin 3 (IL-3) research grade (Miltenyi Biotec, catalog number: 130-094-193 )
Human IL-4 research grade (Miltenyi Biotec, catalog number: 130-095-373 )
Human SCF research grade (Miltenyi Biotec, catalog number: 130-096-693 )
Human thrombopoietin (TPO) research grade (Miltenyi Biotec, catalog number: 130-094-013 )
Recombinant human vascular endothelial growth factor (VEGF) (PeproTech, catalog number: 100-20 )
1x StemPro-34 SFM (Thermo Fisher Scientific, catalog number: 10639011 )
Penicillin-streptomycin 10,000 U/ml (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
L-glutamine 200 mM (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081 )
Hyper-interleukin 6 (IL-6) (kindly provided by S. Rose John, Institute of Biochemistry, Medical Faculty, Christian-Albrechts-University, Kiel, Germany, [Fischer et al., 1997], see also Notes)
α-Minimal essential medium (α-MEM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 12571063 )
β-mercaptoethanol (50 mM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 31350010 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10500064 )
Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 11875093 )
Collagenase IV solution (1 mg/ml) (see Recipes)
0.1% gelatin solution (see Recipes)
1-Thioglycerol stock solution (100 mM) (see Recipes)
L-ascorbic acid stock solution (50 mg/ml) (see Recipes)
0.1% BSA solution (see Recipes)
BMP4 stock solution (25 µg/ml) (see Recipes)
bFGF stock solution (100 µg/ml) (see Recipes)
FLT3L stock solution (25 µg/ml) (see Recipes)
GM-CSF stock solution (100 µg/ml) (see Recipes)
IGF1 stock solution (40 µg/ml) (see Recipes)
IL-3 stock solution (150 µg/ml) (see Recipes)
IL-4 stock solution (20 µg/ml) (see Recipes)
SCF stock solution (100 µg/ml) (see Recipes)
TPO stock solution (20 µg/ml) (see Recipes)
VEGF stock solution (100 µg/ml) (see Recipes)
Hematopoietic progenitor basal differentiation medium (see Recipes)
Induction medium d0 (see Recipes)
Induction medium d1 (see Recipes)
Induction medium d2 (see Recipes)
Induction medium d4 (see Recipes)
Induction medium d6 (see Recipes)
OP9 culture medium (see Recipes)
DC differentiation basal medium (see Recipes)
DC differentiation FSG4 medium (see Recipes)
DC differentiation FSG medium (see Recipes)

Equipment

1,000 µl pipette (Gilson, catalog number: F123602 )
200 µl pipette (Gilson, catalog number: F123601 )
20 µl pipette (Gilson, catalog number: F123600 )
10 µl pipette (Gilson, catalog number: F144802 )
Pipetboy (INTEGRA Biosciences, catalog number: 155000 )
Water bath (JULABO, model: SW22 )
Inverted light microscope (Leica Microsystems, model: Leica DM IL LED )
Autoclave
Centrifuge (Thermo Fisher Scientific, model: Heraeus^TM Multifuge 3 L )
Flow hood (Heraeus)
Automatic CO₂ incubator with nitrogen supply and O₂ sensor (Thermo Fisher Scientific, Thermo Scientific^TM, model: Heraeus^TM240i , catalog number: 51026331)
Vacuum pump (INTEGRA Biosciences, catalog number: 158300 )
Standard fridge
Standard non-defrosting freezer

Procedure

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Sontag, S., Förster, M., Seré, K. and Zenke, M. (2017). Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets. Bio-protocol 7(15): e2419. DOI: 10.21769/BioProtoc.2419.