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High-efficiency electrotransfection of human primary hematopoietic stem cells ¹

L. H. LI^*, P. MCCARTHY and S. W. HUI^*2

^* Department of Molecular and Cellular Biophysics,
Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263

²Correspondence: Department of Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. E-mail: sekwen.hui{at}roswellpark.org

SPECIFIC AIMS

Themajor obstacle to high-efficiency electrotransfection of hematopoietic stem cells is high cell mortality caused by DNA uptake-induced apoptosis and postpulse colloidal-osmotic swelling. By applying caspase inhibitors (B-D-Fluomethyl Ketone and Z-VAD-FMK) to reduce apoptosis, and by using the postpulse pelleting method to suppress colloidal-osmotic swelling of human primary CD-34⁺ cells, we achieved a transfection efficiency of 25%, which is within the reach of therapeutic applications.

PRINCIPAL FINDINGS

1. Apoptosis is the main cause of cell mortality after electroporation
Human primary CD-34⁺ cells from peripheral blood were used as a model for hematopoietic stem cells. Electrotransfection was accomplished by applying four 400 µs long quasisquare pulses at pulse-field strength up to 2.25 kV/cm, in the presence of 400 µg/ml of plasmids (pEGFP-N1) encoding the enhanced green fluorescence protein. We found that cell viability was not affected 1 h after pulsing but became very low at 24 h after pulsing. By examining the morphological change in cells after electrotransfection, we found that the low viability was due to apoptosis. Most cells, including GFP-expressed cells, showed the shrinkage and fragmentation of nuclei, a characteristic morphology of apoptosis, as demonstrated in Figure 1a, b . These GFP-expressed cells lost most of their GFP at 48 h after pulsing because of late apoptosis, and the percentage of GFP-expressed cells became less than 1% at this time point.

View larger version (60K): [in this window] [in a new window]   Figure 1. The effect of caspase inhibitors on transfection efficiency 24 h after pulsing. Phase and fluorescence micrographs of human primary hematopoietic CD-34+ cells are shown in a, c and b, d, respectively. The cells were electroporated by four 1.7 kV/cm, 400 µs pulses in the presence of plasmids encoding GFP (400 µg/ml) and were incubated in pellet for 15 min after pulse. Following pulsing and incubation, the cells were aliquoted and cultured in the medium either with (c, d) or without caspase inhibitors (a, b).

If apoptosis is the major factor limiting the viability of transfected cells, caspase inhibitors may be applicable in rescuing these cells by reducing apoptosis. To verify this theory, caspase inhibitors (B-D-Fluomethyl Ketone and Z-VAD-FMK) were added in the postpulse culture medium. As expected, a large fraction of cells remained viable after pulsing. It is interesting that most GFP-expressed cells were now nonapoptotic, as shown in Figure 1c, d . The transfection efficiency; that is, the percentage of viable and GFP-expressed cells, reaches as high as 25% under optimal conditions when assayed by microscopy. The percentage of viable cells among GFP-expressed cells was 90% ± 5% and 5% ± 3%, respectively, for cells cultured in media with or without caspase inhibitor. Furthermore, unlike cells cultured in media without caspase inhibitor, the transfection efficiency maintained the same level at 24 h and 48 h after pulsing when caspase inhibitors were used. The experiments were repeated and analyzed also by flow cytometry. At 24 h after pulsing, the percentage of GFP-positive cells (fourth quadrant in Fig. 2 ) was 27% when caspase inhibitors were used. Most GFP-positive cells were viable, as indicated by the fact that these cells were propidium iodide (PI)-negative, as shown in Figure 2 . PI was used to identify nonviable cells. The mortality was 41% (second quadrant). Less than 2% of cells were PI- and GFP-positive (first quadrant). For the same cell sample, the mortality and transfection efficiency were determined as 60% and 14%, respectively, by microscopy. This discrepancy could be because of the higher sensitivity of flow cytometry in detecting GFP fluorescence.

View larger version (34K): [in this window] [in a new window]   Figure 2. Flow cytometry analysis of pulsed human primary hematopoietic CD-34+ cells cultured in the medium with caspase inhibitors. The cells were electroporated by four 1.7 kV/cm, 400 µs pulses in the presence of plasmids encoding GFP (400 µg/ml) and incubated in a pellet for 15 min after pulsing. The cells were stained with PI. Cells defined as and GFP- and PI-positive, PI-positive, GFP- and PI-negative, and GFP-positive are located in quadrants 1 to 4, respectively.

2. Apoptosis caused by electroporation or cytokine depletion
Because human primary hematopoietic CD-34⁺ cells undergo spontaneous apoptosis when cultured ex vivo due to cytokine depletion, we compared the effects of added cytokines (a mixture of SCF and GM-CSF) and caspase inhibitors to clarify the relative importance of spontaneous and electrotransfection-induced apoptosis. We found that the viability of the control cells was enhanced to about the same level either by adding cytokines alone (from 16+1% to 55+7%), caspase inhibitors alone (to 58+5%), or both cytokines and caspase inhibitors (to 64+2%). This result suggested that the fraction of apoptotic cells that could be rescued in nonpulsed control sample was caused by the depletion of cytokines. However, the viability of pulsed cells could not be rescued by cytokines alone (4+2% viability for pulsed cells with or without cytokines), but could be rescued only by caspase inhibitors (to 22+1% or 29+7% with or without cytokines, respectively). This finding indicated that electrotransfection triggered an apoptotic signal that differed from that by the depletion of cytokines.

3. Inhibition of colloidal-osmotic swelling by postpulse centrifugation
Forming a cell pellet after pulsing will reduce the postpulse colloid-osmotic swelling. The pelleting method was found effective in increasing the transfection efficiency of many hematopoietic cell lines, such as NK-L cells, NFS-70 pro-B cells, and L1210 subclone 3–3 cells. However, without applying caspase inhibitors, the numbers of viable transfected cells were too few to compare the effect with or without pelleting. Apparently, the attempt to improve the transfection efficiency by restraining the colloid-osmotic effect alone is futile if recovered cells would enter apoptosis. When caspase inhibitors were used, the transfection efficiency was up to 3 times greater when pulsed cells were incubated in a pellet than that when cells were incubated in suspension. This finding demonstrated that postpulse colloidal-osmotic effect was a significant contributor to cell mortality; the effect was masked by apoptosis when no caspase inhibitors were used.

After successfully reducing the mortality rate, we proceeded to determine the optimal pulse conditions. All cells were incubated in pellet after pulsing. The optimal field found was 1.7 kV/cm – 2 kV/cm, which resulted in 20% transfection efficiency when assayed by microscopy.

CONCLUSIONS AND SIGNIFICANCE

Because of the expected impact on therapeutics and tissue replacement, much effort has been made to improve the transfection efficiency of human primary hematopoietic stem cells. Electrotransfection is an attractive approach because it is a physical method and is applicable to all cells types, including nonendocytic primary hematopoietic cells. Also, it is free from biocontamination and immune reaction concerns. Electroporation has been successfully applied to transfect nonphagocytic and nonproliferating cells such as peripheral leukocytes and stem cells, which are usually refractory to chemical transfection vectors. However, the electrotransfection efficiency is limited by the ‘toxicity’ of this method. The major achievement of this work is the identification and overcoming factors limiting the electrotransfection efficiency. It is known that, in most cases, the electroporation process does not electrocute cells directly when electrical pulses are within the range of recommended protocols. Instead, the electroporation process creates minipores in cell membranes and allows small ions, but not macromolecules, to pass through the membranes. This selective passage of small ions causes colloidal-osmotic swelling, which kills cells. Recently, we also found that DNA-uptake induced by electrotransfection could lead to large-scale apoptosis. Many hematopoietic cells enter readily into the apoptotic pathway because of this effect.

Because either cytokines or caspase inhibitors were found to rescue spontaneous apoptosis of control cells to the same extent and without additional effect, whereas caspase inhibitors alone could rescue apoptosis of pulsed cell, we believe that two independent mechanisms of apoptosis involve the activation of caspase(s). The first mechanism, accounting for 22% of cells, is by electrotransfection. The second one, accounting for 43% of cells, is by the depletion of cytokines (growth factors). About 16% survive without cytokines, within which 4% of cells remain viable even with pulse. The remaining 41% are nonviable cells in our experiment. Assuming that the surviving and rescuable populations from both apoptotic mechanisms overlap to the most extent, we describe a likely outcome diagram as depicted in Figure 3 .

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic representation of different apoptosis-susceptible populations of human primary hematopoietic CD-34+ cells. There is 10% overlap between the cytokine-dependent cells segment (rescuable by cytokines or caspase inhibitors, 33%+10%) and the electrotransfection-sensitive segment (rescuable by caspase inhibitors only, 12%+10%). Unaffected viable cells segment (4%), and nonrescuable cells segment (41%) are also presented.

The mechanism(s) of electrotransfection-induced apoptosis of human primary CD-34⁺ cells was not identified by this study. It is not a result of the electroporation process, because electroporation in the absence of DNA causes no additional apoptosis as compared with unpulsed cells. The apoptosis process is likely to be triggered by the uptake of exogenous DNA. Similar exogenous DNA-induced apoptosis in human hematopoietic cells associated with electroporation have been reported. In any case, the 20% of transfection efficiency of the primary human hematopoietic stem cells by electroporation is a significant advancement towards ex vivo gene delivery for therapeutic purposes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0447fje ; to cite this article, use FASEB J. (January 19, 2001) 10.1096/fj.00-0447fje

This Article Full Text (PDF) All Versions of this Article: 15/3/586 00-0447fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by LI, L. H. Articles by HUI, S. W. Search for Related Content PubMed PubMed Citation Articles by LI, L. H. Articles by HUI, S. W.