. Author manuscript; available in PMC: 2023 Dec 8.

Published in final edited form as: Eur J Immunol. 2022 Oct 27;53(11):e2249816. doi: 10.1002/eji.202249816

Table 12.

Selected protocols for in vitro simultaneous generation of human cDC1, cDC2, pDC, and eventually DC3 from CD34⁺ HSC

References	Culture protocol	Generated cell types	Yields^a) thereof	Remarks and recommended use
Proietto et al. 2012 [92]	M: Yssel’s + 10% human AB serum C: huFLT3-L (100 ng/ml) + huTPO (50 ng/ml) No feeder cells Time: 21 d (medium exchange every 5 d)	cDC1: CD11c^intHLA− DR⁺CLEC9A⁺CD14⁻CD172a^−/lowCD11b⁻XCR1^−b) cDC2: CD11c⁺HLA-DR⁺CD1c⁺CD14⁻CD172a⁺CD11b +pDC: CD11c⁻HLA-DR⁺CD123⁺CD14⁻CD172a^low/+ CD11b⁻ monocytes: CD14⁺HLA-DR⁺	2×10⁴ 3×10⁴ 3×10⁴ 30×10⁴	• Differentiation analysis
Breton et al. 2015 [83]	M: α-MEM 1 with 10% FCS C: huFLT3-L (200 ng/ml), huGM-CSF, huSCF (40 ng/ml each) F: MS5 Time: 7 d	cDC1: CD11c⁺HLA-DR⁺CD141⁺CLEC9A⁺CD1c⁻CD14⁻CD123^low cDC2: CD11c⁺HLA-DR⁺CD1c⁺CD141⁻CD14^−/lowCD123^low pDC: CD11c⁻HLA-DR⁺CD123⁺CD303⁺CD14⁻CD1c⁻ monocytes: CD45⁺CD14⁺CD16⁻CD66b⁻	0.3×10⁴ 9×10⁴ 5×10⁴ 14×10⁴	• Differentiation analysis for DC types and several other hematopoietic lineages
Balan et al. 2018 [95]	M: a-MEM with 10% FCS C: expansion: huFLT3-L (25 ng/ml), huTPO (5 ng/ml), huSCF (2.5 ng/ml), hIL7 (5 ng/ml), hGM-CSF (1 ng/ml); differentiation: hFLT3L (15 ng/ml), hIL7 (5 ng/ml) hTPO (2.5 ng/ml), huGM-CSF (1 ng/ml) F: OP9 : OP9_DLL1 – 3:1 Time: 14–21 d (expansion 7 d + differentiation 7–14 d)	cDC1: CD11c⁺CD141⁺CLEC9A⁺CADM1⁺XCR1⁺CD1c⁺CD1a +cDC2: lin-HLA-DR⁺; CLEC10A⁺CX3CR1⁺CD1A +pDC: CD123⁺CD303⁺LILRA4⁺BTLA⁺CLEC9A⁻CADM1⁻XCR1⁻ monocytes: CD45⁺CD14⁺CD16⁻CD66b⁻	3×10⁵ 5×10⁵ 10×10⁵ 1×10⁵	• High frequency^c) and yields of pDC & cDC1 • in vitro-generated DC subsets match with their natural PB counterparts based on single cell RNA sequencing, phenotype and TLR responses • Differentiation analysis for the cDC1 and pDC lineages • Functional assays, including to evaluate impact of the genetic manipulation of amplified HSC
Kirkling et al. 2018 [96]	M: a-MEM with 10% FCS C: huFLT3-L (100 ng/ml), huGM-CSF, huSCF (20 ng/ml each) F: OP9_DL1 Time: 14–21 d (medium exchange every 7 d)	cDC1: lin-HLA-DR⁺CD141⁺CLEC9A⁺CD14⁻ cDC2: lin-HLA-DR⁺CD11c⁺CD1c⁺CD141^+/−CLEC9A⁻CD14⁻ pDC: lin-HLA-DR⁺CD11c⁻CD123⁺CD14⁻ monocytes: lin-HLA-DR⁺CD14⁺	5×10⁴ 2×10⁴ 2×10³ 8×10³	• Predominantly cDC1 • Differentiation analysis
Cytlak et al. 2020 [101]	M: a-MEM with 10% FCS C: huFLT3-L (100 ng/ml), huGM-CSF, huSCF (20 ng/ml each) F: OP9 Time: 14–21 d (medium exchange every 7 d)	cDC1: CD45⁺CD15⁻CD34⁻HLA-DR⁺CD141⁺CLEC9A +cDC2: CD45⁺CD15⁻CD34⁻HLA- DR⁺CD123⁻CD1c⁺CD2⁺CD14⁻CD5^+/− DC3: CD45⁺CD15⁻CD34⁻HLA-DR⁺CD123⁻CD1c⁺CD2⁺CD14⁺CD5⁻ pDC: CD45⁺CD15⁻CD34⁻HLA-DR⁺CD123⁺CD303/4 +monocytes: CD45⁺CD15⁻CD34⁻HLA-DR⁺CD123⁻CD1c⁻CD2⁻CD14⁺	1×10³ 4×10⁴ 2×10⁴ 1×10³ 2×10³	• Predominantly cDC2 & DC3 • Differentiation analysis of the cDC2 versus DC3 lineages
Thordardottir et al. 2014 [97]	M: Glycostem Basal Growth Medium C: huFLT3-L, huTPO, huSCF, IL-6 (100 ng/ml each) + 1μM StemRegenin 1 (SR1) Time: 21 d (medium exchange every 2–4 d)	cDC1: HLA-DR⁺CD141⁺CD1c⁻CD14⁻CD123^low cDC2: HLA-DR⁺CD1c⁺CD141⁻CD14⁻CD123^low pDC: CD11c⁻HLA-DR⁺CD123⁺CD303⁺ CD14⁻	1.2×10^{5 d)} 5.3×10⁵ 3.8×10⁵	• Functional assays • Clinical application
I Thordardottir et al. 2017 [98]	M: Cellgro DC medium (GMPr compliant) with 2% human serum, 50 μg/ml ascorbic acid, 1 μM SR1 C: huFLT3-L (100 ng/ml), huTPO, huSCF (100 ng/ml each for 7 d then 50 ng/ml) Time: 14–20 d	cDC1: HLA-DR⁺CD141⁺CLEC9A⁺CD1c⁻CD14⁻CD123^low cDC2: HLA-DR⁺CD1c⁺CD141⁻CD14⁻CD123^low pDC: CD11c⁻CD123⁺CD303⁺ CD14⁻	0.1×10⁵ 1.8×10⁵ 1.6×10⁵	• High frequency of pDC and cDC2 • GMPr-compliant clinical application
II Thordardottir et al. 2017 [98]	M: Cellgro DC medium (GMPr compliant) with 2% human serum, 50 μg/ml ascorbic acid, 1 μM SR1 C: expansion: huFLT3-L, huTPO, huSCF (100 ng/ml each); differentiation: huGM-CSF (800 IU/ml), huIL-4 (500 IU/ml) Time: 14–21 d (expansion 7–13 d + differentiation 7 d)	cDC1: HLA-DR⁺CD141⁺CD1c⁻CD14⁻CD123^low cDC2: HLA-DR⁺CD1c⁺CD141⁻CD14⁻CD123^low pDC: CD11c⁻CD123⁺CD303⁺ CD14⁻	0.1×10⁵ 1.8×10⁵ 0.2×10⁵	• High frequency of cDC2 • Increased expression of CD11c & HLA-DR on cDC1 &cDC2 • GMPr-compliant clinical application
van Eck van der Sluijs et al. 2021 [99]	M: Cellgro DC medium (GMPr compliant) with 2% human serum, 50 μg/ml ascorbic acid, 1μM SR1 C: expansion: huFLT3-L, huTPO, huSCF (100 ng/ml each); differentiation: huGM-CSF (800 IU/ml), huFLT3-L (100 ng/ml), huIFN-a (1000 IU/ml) Time: 14 d (expansion 7 d + differentiation 7 d)	cDC1: HLA-DR⁺CD141⁺CD1c⁻CD14⁻CD123^low cDC2: HLA-DR⁺CD1c⁺CD141⁻CD14⁻CD123^low pDC: CD11c⁻CD123⁺CD303⁺ CD14⁻	0.5×10⁵ 3×10⁵ 1.6×10⁵	• High frequency of cDC2 • Match in vitro-generated DC subsets with their natural counterparts from peripheral blood, based on transcriptome, phenotype and function • G-CSF mobilized CD34⁺ HPCs • GMPr-compliant clinical application

M: medium; C: cytokines; F: feeder cells.

^a)

Yield refers to 10⁴ CD34⁺ input HSC.

^b)

Italic letters refer to marker gene expression;

^c)

“high frequency” refers to more than 10% of all the cells present in the culture;

^d)

Overestimation since a significant fraction of the CD141⁺ DC are shown to be negative for CLEC9A