人造血干细胞和祖细胞在单细胞水平上的高维功能组学分析

Thomas Luh; Kimberly Lucero; Wenji Ma; Jaeyop Lee; Yu Jerry Zhou; Yufeng Shen; Kang  Liu

doi:10.21769/BioProtoc.2851

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Peer-reviewed

High Dimensional Functionomic Analysis of Human Hematopoietic Stem and Progenitor Cells at a Single Cell Level

人造血干细胞和祖细胞在单细胞水平上的高维功能组学分析

发布: 2018年05月20日第8卷第10期 DOI: 10.21769/BioProtoc.2851 浏览次数: 7691

评审: Ruth A. FranklinLokesh KalekarChris Tibbitt

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Cover of Nature Immunology, featuring study using the protocol.

Jul 2017

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Abstract

The ability to conduct investigation of cellular transcription, signaling, and function at the single-cell level has opened opportunities to examine heterogeneous populations at unprecedented resolutions. Although methods have been developed to evaluate high-dimensional transcriptomic and proteomic data (relating to cellular mRNA and protein), there has not been a method to evaluate corresponding high-dimensional functionomic data (relating to cellular functions) from single cells. Here, we present a protocol to quantitatively measure the differentiation potentials of single human hematopoietic stem and progenitor cells, and then cluster the cells according to these measurements. High dimensional functionomic analysis of cell potential allows cell function to be linked to molecular mechanisms within the same progenitor population.

Keywords: CD34⁺ hematopoietic stem and progenitor cells (CD34⁺造血干细胞和祖细胞)

Single cell culture (单细胞培养)

Differentiation (分化)

Quantitative clonal output (定量克隆输出)

Barnes-Hut t-SNE (Barnes-Hut t-SNE)

Dimension reduction (降维)

Background

The development of techniques for single-cell measurements of cell transcription, signaling, and function at the single-cell level, alongside preexisting technologies such as flow cytometry, has allowed new lenses to examine complex, heterogeneous populations. Such methods generate large amounts of data, which can be interpreted with aid from dimensionality reduction algorithms, as illustrated on single-cell RNA-Seq using Mpath, Monocole, PCA, Wishbone, or diffusion map algorithms (Paul et al., 2016; See et al., 2017), and on CyTOF using tSNE or PhenoGraph (Amir el et al., 2013; Levine et al., 2015).

We developed this protocol to allow functional analysis and subsequent dimension reduction of large-scale culture of hematopoietic progenitors in single-cell environments. In this protocol, we describe a method to culture single cells of human CD34⁺ hematopoietic stem and progenitor cells (HSPCs) in a stromal cell culture with cytokines, to enumerate the clonal outcome (functionomics) of six different lineages (i.e., granulocyte, monocyte, lymphocyte, CD141⁺ dendritic cell (conventional type 1 DC (cDC1)), CD1c⁺ dendritic cell (conventional type 2 DC (cDC2)), and plasmacytoid dendritic cells (pDC)) from each progenitor, and to cluster the progenitors according to function with a dimension reduction method. In our previous paper, we showed that this is feasible for a population of 2,247 progenitors; each progenitor can be individually plotted to form a two-dimensional ‘map’ of 2,247 data points (Lee et al., 2017). Our protocol allows for the functional clustering of single cells. Such high dimensional functionomic analysis aids in linking cell function to molecular mechanism of any given cell population.

Materials and Reagents

15 cm culture plates (Corning, Falcon^®, catalog number: 353025 )
15 ml Falcon tubes (Corning, Falcon^®, catalog number: 352097 )
96-well V-bottom plates (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 249570 )
Flat-bottom 96-well plates (Corning, Falcon^®, catalog number: 353072 )
50 ml Falcon tubes (Corning, Falcon^®, catalog number: 352098 )
Serological pipettes (Fisher Scientific, catalog number: 13-678-11 )
Opaque Eppendorf tubes (CELLTREAT Scientific Products, catalog number: 229437 )
Eppendorf tubes (Fisher Scientific, catalog number: 05-408-129 )
Pipette tips
Pasteur pipette (Fisher Scientific, catalog number: 13-678-8B )
100 μm cell strainer (Corning, Falcon^®, catalog number: 352360 )
0.20 μm filter (Corning, catalog number: 431229 )
MS5 (Lee et al., 2015a and 2015b)
OP9 (Lee et al., 2015a and 2017)
dH₂O (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10977023 )
Trypsin (Corning, catalog number: 25-052-CI )
Trypan blue (Thermo Fisher Scientific, Gibco^TM, catalog number: 15250061 )
70% ethanol
Ficoll-Paque Plus (GE Healthcare, catalog number: 17144002 )
Anti-human CD34 MACS microbeads (Miltenyi Biotec, catalog number: 130-046-702 )
Recombinant human Flt3L (Celldex Therapeutics, catalog number: CDX-301 )
Recombinant human SCF (PeproTech, catalog number: 300-07 )
Recombinant human GM-CSF (PeproTech, catalog number: 300-03 )
Antibodies (details of clone name, fluorochrome, manufacturer and dilution are listed in Tables 1 and 3)
MEMα with deoxyribonucleosides (Thermo Fisher Scientific, Gibco^TM, catalog number: 12571071 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10437028 )
Pen-Strep (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Mitomycin C (Sigma-Aldrich, catalog number: M4287-5X2MG )
Dulbecco’s phosphate-buffered saline (DPBS) (GE Healthcare, catalog number: SH30378.02 )
Ethylenediaminetetraacetic acid (EDTA) (Corning, catalog number: 46-034-CI )
Bovine serum albumin (BSA) (ThermoFisher, catalog number: BP1600-100 )
FcR blocking buffer (Miltenyi Biotec, catalog number: 130-059-901 )
CountBright Absolute Counting Beads (Thermo Fisher Scientific, Invitrogen^TM, catalog number: C36950 )
Complete MEMα medium (see Recipes)
Mitomycin C stock (1 mg/ml) (see Recipes)
FACS buffer (see Recipes)
Anti-CD34 microbeads/FcR blocking mix (see Recipes)

Equipment

Pipettes
Incubator (Forma Scientific, model: 3354 )
Centrifuge (Eppendorf, model: 5810 R , catalog number: 5811000320)
Vortex with universal holder (VWR, catalog number: 97043-562 )
Microscope
Flow cytometer (BD, models: BD LSRII or LSRFortessa^TM )

Procedure

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如何引用

Luh, T., Lucero, K., Ma, W., Lee, J., Zhou, Y. J., Shen, Y. and Liu, K. (2018). High Dimensional Functionomic Analysis of Human Hematopoietic Stem and Progenitor Cells at a Single Cell Level. Bio-protocol 8(10): e2851. DOI: 10.21769/BioProtoc.2851.