key: cord-0844661-ey7yv8n4
authors: Tada, Masako; Tada, Takashi
title: Nuclear Reprogramming of Somatic Nucleus Hybridized With Embryonic Stem Cells by Electrofusion
date: 2006
journal: Embryonic Stem Cell Protocols
DOI: 10.1385/1-59745-037-5:411
sha: 43fe2228e6146e0971d09d3e2e6aac6c38c27fd9
doc_id: 844661
cord_uid: ey7yv8n4

Cell fusion is a powerful tool for understanding the molecular mechanisms of epigenetic reprogramming. In hybrid cells of somatic cells and pluripotential stem cells, including embryonic stem (ES) and embryonic germ cells, somatic nuclei acquire pluripotential competence. ES and embryonic germ cells retain intrinsic trans activity to induce epigenetic reprogramming. For generating hybrid cells, we have used the technique of electrofusion. Electrofusion is a highly effective, reproducible, and biomedically safe in vitro system. For successful cell fusion, two sequential steps of electric pulse stimulation are required for the alignment (pearl chain formation) of two different types of cells between electrodes in response to alternating current stimulation and for the fusion of cytoplasmic membranes by direct current stimulation. Optimal conditions for electrofusion with a pulse generator are introduced for ES and somatic cell fusion. Topics in the field of stem cell research include the successful production of cloned animals via the epigenetic reprogramming of somatic cells and contribution of spontaneous cell fusion to generating intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of epigenetic reprogramming and regenerative medicine.

Cell fusion is a phenomenon combining genetic and epigenetic information between two different types of cells, resulting in the creation of a new cellular phenotype and function. Fused cells can grow as polynuclear cells, called heterokaryons. In other cases, one combined nucleus is formed through cell division after cytoplasmic membrane fusion. The phenotype of hybrid cells varies depending on the combination of parental cells for fusion. Specialized gene products from one of the parental cells are maintained; others are often extinguished via a tissue-specific repressive mechanism, a phenomenon called extinction (1). Thus, it has been believed that epigenetic information fusion with polyethylene glycol (11). The electrofusion parameters, however, were optimized for making hybridomas for the stable production of antibodies.

In this chapter, we introduce an electrofusion procedure to create hybrid cells from pluripotential stem cells and adult lymphocytes. The overall procedure is summarized (see Fig. 1 ). The electrofusion procedure is subdivided into several parts: (1) cell culture of ES and fused cells; (2) pretreatment of somatic cells; (3) setup of the Electro Cell Manipulator (ECM) 2001 (BTX), AC (alternating current)/DC (direct current) pulse generator with a Microslide chamber; (4) operation of the electrofusion generator; (5) selection of postfusion cells; and (6) isolation and cloning of fused cells. In the process of electrofusion, AC pulses induce the alignment and compression of the cells, and DC pulses transiently make revertible pores in cytoplasmic membranes to initiate the process of fusion among cells. The alignment voltage, pulse length, and electroporation voltage and the number of DC pulses should be precisely controlled.

After cell fusion, ES hybrid cells with somatic cells should be isolated with selection medium. Thus, genetically marked ES cells or somatic cells are needed for cell fusion experiments. For example, normal ES cells were hybridized with thymocytes derived from ROSA26 transgenic mice, which carry the ubiquitously expressed neo/lacZ transgene (12). Consequently, ES cells hybridized with the thymocytes can survive and grow only in medium supplemented with a protein synthesis inhibitor, G418. The ES hybrid cells and their derivatives are visualized as cells positive for X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) staining, which allows one to observe their contribution in chimeric embryos and tissues (3,5,8).

In another selection system, male ES cells deficient in the Hprt gene on the X chromosome are used for isolating ES hybrid cells with wild-type somatic cells. The somatic cell-derived Hprt gene rescues the HPRT activity in the hybrid cells. In DNA synthesis, the purine nucleotide can be synthesized by the de novo pathway and recycled by the salvage pathway. When electrofusion-treated cells are cultured in medium with HAT (hypoxantine, aminopterin, and thymidine), the de novo pathway is interfered with, and only the salvage pathway functions. As a consequence, the Hprt-deficient ES cells die; ES hybrid cells are able to survive and grow (5,6).

To succeed in obtaining ES hybrid cells, we recommend that (1) genetic markers highly expressed in the undifferentiated state are used for isolating hybrid clones, (2) the origin of somatic cells is carefully chosen for creating undifferentiated hybrid cells, and (3) 

Inactivated neo r PEFs are routinely used as feeder cells for culture of ES and hybrid cells and for selection of hybrid cell colonies with G418. Use 4 × 10 5 feeder cells/30-mm culture dish, 1 × 10 6 feeder cells/60-mm culture dish, and 2.5 × 10 6 feeder cells/100-mm culture dish. 1 . Coat 60-mm culture dishes with 0.1% gelatin for at least 30 min at 37°C. 2. Incubate neo r PEFs with 10 μg/mL MMC for 2 h at 37°C in the CO 2 incubator. 3. Prepare the frozen stock of MMC-treated feeder cells at a cell concentration of 5 × 10 6 cells/mL for each cryotube. 4. Store in liquid nitrogen.

1. One day before cell fusion, coat 30-mm culture wells (6-well culture plate) with 0.1% gelatin for at least 30 min at 37°C. 2. Prepare inactivated PEFs (4 × 10 5 cells/30-mm well) in 3 mL ES medium.

One of the most important factors for cell fusion experiments is that the culture conditions be optimized for maintaining the pluripotential competence and full set of chromosomes (80 chromosomes) derived from mouse ES cells and somatic cells through numerous cell divisions (see Note 2).

culture medium once or twice a day. 2. Ascertain that complete sets of chromosomes are maintained in the ES cells before cell fusion.

Set the optimized electrical parameters to fuse ES cells and thymocytes (see Fig. 2A,B) . 

The automatic operation switch is used to initiate AC followed by DC. AC is utilized to induce a nonhomogeneous electric field, resulting in cell alignment and pearl chain formation (see Fig. 2E ,F) (see Note 5). DC is utilized to produce reversible temporary pores in the cytoplasmic membranes. When juxtaposed pores in the physically associated cells reseal, cells have a chance to be hybridized by cytoplasmic membrane fusion. AC application after the DC pulse induces the compression of cells, which helps the process of fusion between the cell membranes. 1 . Press the automatic operation switch of the ECM 2001. 2. Add 40 μL DMEM to the fusion mixture between electrodes to induce recovery of the membrane reformation immediately after electroporation. 3. Leave the cell mixture for 10 min at room temperature. 4. Transfer the cell mixture directly to a 30-mm culture dish containing inactivated PEFs with 3 mL ES medium with LIF. 5. Repeat the cell fusion procedure sequentially using several Microslides. Usually, the cell suspension recovered from three Microslides (80 μL × 3) is plated into one 30-mm culture dish. As a control, plate the untreated cell mixture and culture it under the same conditions. 6. Incubate the cells at 37°C in the CO 2 incubator for 24 h. 7. Change the medium to ES medium with the proper supplement to select ES hybrid cells 24 h after cell fusion. 8. Change the selection medium once a day.

As a result of the 7-d treatment, nonfused ES cells and hybrid cells derived from ES cells are killed, and thymocytes are nonadherent. Thus, the hybrid cells of ES cells and somatic cells survive and form colonies. Several colonies of hybrid cells per 10 4 host ES cells appear using the previously mentioned procedures for electrofusion.

D5796), add 50 mL fetal bovine serum (FBS); 5 mL 200 mM glutamine (Sigma, cat. no. G7513); and 5 mL 10,000 IU/mL penicillin and 10 mg/mL streptomycin (100X penicillin-streptomycin

ES medium: prepare the culture medium for ES cells in a 500-mL DMEM/nutrient mixture F12 HAM (F12) bottle and store it at 4°C. To 500 mL DMEM/F12 (Sigma, cat

Sterilize all dissection instruments (scissors and forceps) by immersion in 70% ethanol, followed by flaming

Sacrifice a 6-to 8-wk-old adult mouse humanely and dissect out the thymus in a clean room if a clean bench is not available

Wash the tissues with sterilized PBS twice in 60-mm Petri dishes

Place the half lobe of thymus in the barrel of a sterile 2.5-mL syringe with a sterile 18-gage needle

Expel and draw up the thymus gently through the needle via the tip of the needle several times in a 50-mL conical tube with 2 mL DMEM to dissociate the thymus into a single-cell suspension

Transfer the supernatant excluding cell clumps to a 15-mL conical tube and add 10 mL DMEM

Spin down the thymocytes in 15-mL conical tubes at 300g for 5 min

Coat 60-mm culture dishes with 0.1% gelatin for at least 30 min at 37°C. 2. Trypsinize the ES cells and remove excess trypsin quickly

Add 3 mL ES medium to inactivate the trypsin and dissociate the cells into a single-cell suspension by gentle pipetting

Plate the cells on a fresh gelatin-coated 60-mm culture dish

Incubate the ES cells in the CO 2 incubator for 30 min to separate feeder cells from ES cells

Collect unattached ES cells and harvest them by centrifugation at 300g for 5 min

Resuspend the cell pellet in 10 mL DMEM and transfer the cell suspension into a 15-mL conical tube

Spin down the ES cells and the thymocytes in 15-mL conical tubes at 300g for 5 min, separately

Wash the cells with 10 mL DMEM and spin them down at 300g for 5 min and repeat to remove FBS completely

Add 5-10 mL DMEM and adjust the density of ES cells and thymocytes to 1 × 10 6 cells/mL

Pellet a 1:5 mixture of ES cells and thymocytes (1 mL ES cell suspension and 5 mL thymocyte suspension). Keep the remaining cells for control experiments

Spin down and resuspend the cell pellet in 0.3 M mannitol to the appropriate density of 6 × 10 6 cells/mL (see Note 3). Use them for electrofusion immediately

Setup of Electro Cell Manipulator ECM

Setup of AC/DC Pulse Generator, ECM

the Microslides by immersion in 70% ethanol followed by flaming. 2. Set a Microslide in a 100-mm plastic dish chamber made from a bacterial dish

Apply 40 μL cell mixture into the 1-mm electrode gap on the Microslide at room temperature (see Fig. 2C)

Place the chamber on an inverted microscope to allow observation of cell alignment (see Note 4)

Perform the automatic operation of electrofusion of the mixture of normal ES cells and thymocytes collected from the 6-to 8-wk-old ROSA26 transgenic mice carrying the ubiquitously expressed neo/lacZ transgene

Culture the electrofusion-treated cells in ES medium for 24 h

Change the medium to the ES medium supplemented with G418. ES hybrid cell colonies can be detected at 7-10 d

Prepare a 24-well culture plate containing 1 × 10 5 inactivated neo r PEFs per 10-mm well and 0.8 mL ES medium with G418 supplement for selection

Pick up the colonies with a micropipet and transfer each colony into the 10-mm well of the 24-well culture plate on inactivated PEFs

Subculture the colonies every 2 or 3 d and gradually expand the number of cells in 30-and 60-mm culture dishes with ES medium on inactivated PEFs (see Fig. 2G,H) (see Note 6)

Extinction by indirect means

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