Developmental Biology Cell Culture

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Authors: Fang Ni, Rui Sun, Binqing Fu, Fuyan Wang, Chuang Guo, Zhigang Tian & Haiming Wei

Abstract

A number of different cytokine combinations have been tested for their ability to support the proliferation and differentiation of human CD34+ hematopoietic stem cells (HSC) into mature NK cells. Here, we describe an improved protocol based on multiple cytokines (SCF, Flt3-L, IL-15 and IGF-1) culture for obtaining a high number of lytic NK cells from human umbilical cord blood CD34+ cells.More than 6×107 highly lytic NK cells can be obtained from 1×105 fresh CD34+ HSC using this protocol. This facilitates the use of the above culture system as a tool for researchers to study the development, properties and functions of NK cells in vitro, and this procedure also facilitates cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with highly cytotoxic potential.

Introduction

NK cells are important effectors of the innate immune system, and the cytotoxic function of NK cells is crucial to many processes such as defending against pathogens and tumors (1, 2). In vitro, human NK cells can differentiate from bone marrow- or cord blood-derived hematopoietic progenitor cells in appropriate culture conditions. In early studies, NK-cell development from purified HSC was shown to be stromal-cell dependent (3). It has later been demonstrated that the stromal-cell requirements may be replaced by the provision of combined cytokines such as SCF, Flt3-L and IL-15 to the cultures (4). In particular, SCF and Flt3-L directly induce the expression of IL-2 receptor-βchain on HSC, thereby rendering them susceptible to the NK-cell commitment induced by IL-15 (5). Recently, a number of different cytokine combinations have been tested for their ability to support the proliferation and differentiation of human CD34+ hematopoietic stem cells (HSCs) into mature NK cells. However, a cytokine that has not been extensively studied in this regard is insulin-like growth factor (IGF)-1. IGF-1, also known as somatomedin C, is one of the main endocrine mediators of growth and development under physiological conditions (6). In addition to this important function, IGF-1 also regulates the hematopoiesis and direct effector functions of diverse aspects of cells of the innate and acquired immune systems. In this study, we found that IGF-1 may foster the IL-15-induced NK differentiation of human umbilical cord blood (UCB) CD34+ cells.

Here, we described an improved protocol based on combined cytokines (SCF, Flt3-L, IL-15 and IGF-1) culture for ex vivo expansion of highly lytic NK cells from human UCB/CD34+ cells.

Reagents

  1. Human CD34 Microbead Kit (Miltenyi Biotec, 130-046-702)
  2. Fluorescently labelled anti-CD34, anti-CD3, anti-CD56, anti-perforin and anti-CD107a (BD Pharmingen).
  3. GMP Serum-free Stem Cell Growth Medium (SCGM, CellGro®/ CellGenix™, 20802-0500)
  4. Fetal Bovine Serum (FBS) heat inactivated (Gibco, 10100-147)
  5. Recombinant Human SCF (PeproTech, AF-300-07)
  6. Recombinant Human Flt3-Ligand (PeproTech, AF-300-19)
  7. Recombinant Human IL-15(PeproTech, 200-15)
  8. Recombinant human IGF-1 (PeproTech, AF-100-11)
  9. Fixation/Permeabilization Solution Kit (BD Pharmingen, 554714): including both the Fixation and Permeabilization Solution (BD Pharmingen, 554722) and the - Perm/Wash™ buffer (BD Pharmingen, 554723)

Equipment

  1. Cell culture disposables: 96, 24 and 6 well plates, 50 ml and15 ml centrifuge tubes, 1.7 ml microcentrifuge tubes, pipettes, pipette tips
  2. miniMACS separator (Miltenyi Biotec)
  3. MACS columns (Miltenyi Biotec, 130-042-201)
  4. Flow cytometer (FACS Calibur, BD Pharmingen)
  5. FACS tubes round bottom
  6. CO2 incubator (Forma, 3111)
  7. Centrifuge suitable for 15 ml and 50 ml tubes (Hettich)
  8. Microcentrifuge suitable for 1.7 ml tubes (Sigma, 1-15K)

Procedure

  1. Collect fresh cord blood (typically 60–80 ml samples) from the umbilical cords of the placentas of normal, full-term, non-stressed newborns of consenting mothers.
  2. Cord blood samples were diluted 1:2 in PBS. Cord blood mononuclear cells (CBMC) were isolated by Ficoll-Hypaque centrifugation at 400 g for 30 minutes using standard procedures and washed extensively in PBS.
    • ▲ CRITICAL STEP Ensure that the cells were processed within 4 h of collection.
  3. Isolate CD34+ cells from CBMCs with the Miltenyi human CD34 Microbead Kit according to the manufacturer’s instructions (Miltenyi Biotec).
    • ▲ CRITICAL STEP Cell purity analysis by FACS, purity should be routinely higher than 90%.
  4. Determine the number of the CD34+ cells by gently mixing 10 μl of the above isolated cells with 80 μl of PBS and 10 μl of Trypan Blue. Use a Neubauer chamber to count the number of viable (not stained) CD34+ cells.
  5. The isolated CD34+ cells were cultured in warm SCGM supplemented with 100 U/ml Penicillin/Streptomycin and 10% FBS in the presence of recombinant human SCF (30 ng/ml), Flt3-L (50 ng/ml), IL-15 (50 ng/ml) and IGF-1 (100 ng/ml).
    • ▲ CRITICAL STEP Depending on the cell number of the starting population (5×10e4 cells or 1×10e5 cells per culture), cultures were grown in 0.1 or 1.0 ml medium in 96- or 24-well culture plates, respectively.
  6. The cultures were incubated at 37°C in a humidified atmosphere with 5% CO2 for 4 weeks.
    • ▲ CRITICAL STEP Every 3–4 days, half of the medium volume was replaced with fresh medium containing the same concentration of freshly added cytokines. Cell density was thereby adjusted to 1×10e6 cells/ ml in 6-well culture plates.
  7. Total number of viable cells differentiated from CD34+ cells by various cytokine combinations, as counted for up to 4 weeks.
  8. At day 28, collect cells, the percentage of CD3- CD56+ cells was measured by FACS analysis.
  9. NK cell function was evaluated through flow cytometry monitoring of perforin and CD107a expression.
    • (1) For perforin assay: Intracellular staining for perforin was performed following surface staining for CD56+ CD3neg cells (to identify NK cells). Stained cells were fixed and permeabilized with the Fixation/Permeabilization Solution Kit (BD Pharmingen), as detailed in manufacturers’ instructions:
      • a. Thoroughly resuspend cells with 250 μl of BD Cytofix/Cytoperm solution in the 1.5 ml Eppendorf tube and incubate for 20 min. at 4°C.
      • b. Wash cells two times in 1 ml BD Perm/Wash buffer.
      • c. Thoroughly resuspend fixed/permeabilized cells in 50 μl of BD Perm/Wash buffer containing a predetermined optimal concentration of a PE-conjugated anti-perforin Ab or appropriate negative control. Incubate at 4°C for 30 minutes in the dark.
      • d. Wash cells 2 times with BD Perm/Wash buffer and resuspend in staining buffer and then analyzed by flow cytometry.
    • (2) For CD107a assay, the cytokine-differentiated NK cells were isolated, then 2×10e5 NK cells and 2×10e5 cells of the erythroleukemia cell line K562 (American Type Culture Collection) were mixed by gentle pipetting, spun down for 1 min at 200 g, and incubated for 2 h at 37°C in 200 µl of SCGM media. NK cells were then stained with an Alexa-647-labeled anti-CD56-Ab, PE-Cy5-labeled anti-CD3-Ab, and degranulation was detected with a PE-labeled anti-CD107a-Ab (all Abs from BD Biosciences).

Anticipated Results

Following this protocol, purified CD34+ HSCs from human umbilical cord blood (UCB) were maintained with Flt3-L and SCF, IL-15 and IGF-1 for up to 4 weeks. To monitor cell expansion in response to cytokine stimulation, cells were counted weekly. Our data showed that proliferation was increased substantially in CD34+ cell cultures containing SCF, Flt3-L, IL-15 and IGF-1 (Fig. 1). Moreover, a significant increase was observed in the percentages and absolute cell numbers of CD3- CD56+ NK cells when cells were cultured in the above multiple cytokines with IGF-1 (Fig. 2).

NK cell function was evaluated through flow cytometry monitoring of perforin and CD107a expression. We found that SCF, Flt3-L and IL-15 with IGF-1 significantly increased perforin expression and CD107a release in human CD34+-derived CD56+ NK cells compared with that of CD56+ effectors derived from the same CD34+ population in the presence of SCF, Flt3-L and IL-15 alone (Fig.3).

References

  1. Smyth, M.J., Hayakawa, Y., Takeda, K. & Yagita, H. New aspects of natural-killer-cell surveillance and therapy of cancer. Nat. Rev. Cancer.2, 850–861(2002).
  2. Lodoen, M.B. & Lanier, L.L. Natural killer cells as an initial defense against pathogens. Curr. Opin. Immunol. 18, 391–398 (2006).
  3. Miller, J.S., Alley, K.A., McGlave, P. Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34+7+ NK progenitor. Blood. 83, 2594-2601(1994).
  4. Lee, S.H. et al. RasGRP1 is required for human NK cell function. J Immunol. 183, 7931-7938(2009).
  5. Mrozek, E., Anderson, P., Caligiuri, M.A. Role of interleukin-15 in the development of human CD56+ natural killer cells from CD34+ hematopoietic progenitor cells. Blood. 87, 2632-2640(1996).
  6. O’Connor, J.C. et al. Regulation of IGF-I function by proinflammatory cytokines: at the interface of immunology and endocrinology. Cell Immunol.252, 91-110 (2008).

Acknowledgements

This work was supported by the Ministry of Science & Technology of China (973 Basic Science Project #2013CB944902, #2009CB522403, #2012CB519004) and the National Natural Science Foundation of China (#31021061, #30730084).

Figures

Figure 1: Expansion of CD34+ cells in multiple cytokines-treated cultures

Fig 1

(a)Total number of viable cells differentiated from CD34+ cells by various cytokine combinations, as counted for up to 4 weeks. (b) Fold expansion of UCB CD34+ cells after 4 weeks of culture with various cytokine combinations.

Figure 2: The generation of NK cells cultured with combined cytokines

Fig 2

(a) Representative flow cytometry analysis of the relative ratio of NK cells cultured with either IL-15 or the combination of IL-15 and IGF-1 at day 28. (b) The absolute number of CD56+CD3− NK cells analyzed in (a). *P < 0.05 (Student’s t-test).

Figure 3: Analysis of the cytotoxic potential of cytokine-differentiated NK cells

Fig 3

Flow cytometry for perforin and CD107a expression in human UCB/CD34+-derived NK cells cultured for 28 days. Data are representative of three independent experiments. The graphs show the average relative frequency of perforin+ and CD107a+ NK cells as above. *P < 0.05 (Student’s t-test).

Associated Publications

IGF-1 promotes the development and cytotoxic activity of human NK cells. Fang Ni, Rui Sun, Binqing Fu, Fuyan Wang, Chuang Guo, Zhigang Tian, and Haiming Wei. Nature Communications 4 () 12/02/2013 doi:10.1038/ncomms2484

Author information

Fang Ni, Binqing Fu, Fuyan Wang, Chuang Guo & Haiming Wei, Wei's Lab(USTC)

Rui Sun & Zhigang Tian, Institute of Immunology,School of Life Sciences, University of Science and Technology of China, 443 Huang-Shan Road, Hefei, Anhui Province 230027, China

Correspondence to: Zhigang Tian ([email protected]) Haiming Wei ([email protected])

Source: Protocol Exchange (2013) doi:10.1038/protex.2013.025. Originally published online 18 February 2013.

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