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Stem Cells, Vol. 19, No. 6, 492-499, November 2001
© 2001 AlphaMed Press

High Efficiency Electroporation of Human Umbilical Cord Blood CD34+ Hematopoietic Precursor Cells

Michael H. Wua, Stephen L. Smitha,b, M. Eileen Dolana

a Section of Hematology-Oncology, Department of Medicine and Cancer Research Center, Committee on Cancer Biology, University of Chicago, Chicago, Illinois USA;
b Present address: Institute for Transfusion Medicine, Clinical Services-Stem Cell Department, Glenview, Ilinois, USA

Key Words. Electroporation • Hematopoietic • Cord blood plasma

M. Eileen Dolan, Ph.D., Section of Hematology-Oncology, University of Chicago, 5841 S. Maryland Ave., Box MC2115, Chicago, Illinois 60637, USA. Telephone: 773-702-4441; Fax 773-702-0963; e-mail: edolan{at}medicine.bsd.uchicago.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human umbilical cord blood provides an alternative source of hematopoietic cells for purposes of transplantation or ex vivo genetic modification. The objective of this study was to evaluate electroporation as a means to introduce foreign genes into human cord blood CD34+ cells and evaluate gene expression in CD34+/CD38dim and committed myeloid progenitors (CD33+, CD11b+). CD34+ cells were cultured in X-VIVO 10 supplemented with thrombopoietin, stem cell factor, and Flt-3 ligand. Electroporation efficiency and cell viability measured by flow cytometry using enhanced green fluorescent protein (EGFP) as a reporter indicated 31% ± 2% EGFP+/CD34+ efficiency and 77% ± 3% viability as determined 48 hours post-electroporation. The addition of allogeneic cord blood plasma increased the efficiency to 44% ± 5% with no effect on viability. Of the total CD34+ cells 48 hours post-electroporation, 20% were CD38dim/EGFP+. CD34+ cells exposed to interleukin-3, GM-CSF and G-CSF for an additional 11 days differentiated into CD33+ and CD11b+ cells, and 9% ± 3% and 8% ± 7% were expressing the reporter gene, respectively. We show that electroporation can be used to introduce foreign genes into early hematopoietic stem cells (CD34+/CD38dim), and that the introduced gene is functionally expressed following expansion into committed myeloid progenitors (CD33+, CD11b+) in response to corresponding cytokines. Further investigation is needed to determine the transgene expression in functional terminal cells derived from the genetically modified CD34+ cells, such as T cells and dendritic cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human umbilical cord blood is an attractive alternative to mobilized peripheral blood and bone marrow as a source of hematopoietic precursor cells (HPCs), and has been used in clinical transplantation during the past several years [1–3]. Cord blood is richer in hematopoietic progenitors than mobilized peripheral blood, is easy and painless to obtain, and is a source of normal human hematopoietic progenitors in accordance with ethical regulations [4–6]. Hematopoietic progenitor cells from cord blood were shown to be sufficient for transplant and to induce durable engraftment in children [7]. In addition, the relative immunological immaturity of neonatal lymphocyte cord blood cells seems to render the graft-versus-host disease less severe in allogeneic, HLA-mismatched transplantation than conventional bone marrow transplantation [8,9].

Ex vivo genetic modification of target cells for transplantation, especially HPCs, is a critical technology in gene therapy. Drug-resistance gene delivery into HPC is an approach that has been studied with a major focus on retroviral or adenoviral transduction. Electroporation is a well-established, nonchemical, nonviral technique for the delivery of exogenous macromolecules into cells [10]. One major advantage for using electroporation is that the potential for exposure to biohazards associated with virus-mediated gene transduction is minimized.

Recently, we reported the electroporation of human peripheral blood CD34+ cells with efficiencies >=20% in transient (48 hours), colony-forming unit (CFU) (2 weeks) and long-term culture-initiating cell (LTC-IC) (7 weeks), and provided evidence that the introduced reporter gene is integrated into the genome of LTC-IC [11]. We extend these studies to evaluate electroporation as a means to introduce genes into human umbilical cord blood HPCs. We demonstrate that electroporation can be used to deliver foreign genes into the more primitive subpopulations of CD34+ hematopoietic cells (CD34+/CD38dim). We also show that the introduced gene is functionally expressed following expansion into committed myeloid progenitors (CD33, CD11b) in response to corresponding cytokines in liquid culture.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The ECM 600 electroporator was from Genetronics, Inc. (San Diego, CA). The FACScan flow cytometer, CellQuest software, conjugated monoclonal antibodies including mouse IgG1/fluorescein isothiocyanate (FITC), HPCA-2 anti-CD34-phycoerythrin (PE)/PerCP, anti-CD38-PE/FITC, anti-CD33-PE, anti-CD11b-PE, and anti-CD14-PE were from Becton Dickinson Immunocytometry Systems (San Jose, CA; http://www.bd.com). The anti-CD41a-PE was from Beckman Coulter, Inc. (Fullerton, CA; http://www.coulter.com). The Miltenyi CD34 isolation kit and separation columns were from Miltenyi Biotec (Auburn, CA; http://www.miltenyibiotec.com). Hetastarch (Hespan) was from Baxter Healthcare Corp. (Deerfield, IL; http://www.baxter.com). Human immunoglobulin solution (Gammagard) and human serum albumin (HSA) were from Hyland Division of Baxter Healthcare Corp. (Glendale, CA). The plasmid DNA, pEGFP-N1, was purchased from Clontech Laboratories, Inc. (Palo Alto, CA; http://www.clontech.com). Dulbecco's phosphate-buffered saline ([DPBS], Ca2+ and Mg2+-free) and X-VIVO 10 culture medium were from BioWhitaker, Inc. (Walkersville, MD; http://www.biowhittaker.com). Stem cell factor (SCF) was from R&D Systems (Minneapolis, MN; http://www.rndsystems.com) and Flt-3L was kindly provided by Immunex Corp. (Seattle, WA; http://www.immunex.com). Thrombopoietin (TPO) was from Genzyme Corp. (Cambridge, MA) or R&D Systems. Methylcult H5100 was from Stem Cell Technologies (Vancouver, Canada; http://www.stemcell.com). All other medium and biochemicals were from Sigma Chemical Co. (St. Louis, MO; http://www.sigma-aldrich.com). Statistical analyses were performed using StatView 5.01 from SAS Institute Inc. (Cary, NC).

CD34+ Cell Selection
Individual fresh human umbilical cord blood was used as the source for CD34+ cell selection using the Miltenyi CD34 isolation kit on the Midimax separation columns. Use of human cord blood was in accordance with institutional and federal guidelines for the use of human tissues. Pooled cord blood samples were selected on the CellPro clinical scale device (Nexell; Irvine, CA) according to the manufacturer's instructions. The selected CD34+ cells were cultured as previously described [12].

Culture Conditions
For the preparation of plasma, 1 ml of 6% hetastarch (Hespan) was added to 4 ml of cord blood in the original cord blood collection bag and incubated on a plasma extractor for 1 hour at room temperature. The top plasma layer was then collected and centrifuged at 1,800 x g for 20 minutes to remove platelets. The supernatant (plasma stock) was stored at 4°C for up to 1 month. For CD34+ cells cultured with 5% plasma, 1% plasma volume by volume (v/v) was also included in the electroporation buffer. Extended cultures of electroporated CD34+ HPCs were designed to expand committed precursor cells. Seventy-two hours post-electroporation, a small fraction of the cells (approx. 0.5-1 x 106 cells) was pelleted and resuspended in Methylcult H5100 supplemented with interleukin-3 (IL-3) (50 ng/ml), G-CSF (100 ng/ml), and GM-CSF (100 ng/ml). After 11 days in culture, the cells were collected and evaluated by flow cytometry for the expression of EGFP, CD33, CD11b, CD14, and CD41 antigens.

Electroporation Optimization
Immediately before electroporation, cells were pelleted by centrifugation for 10 minutes at 250 x g at room temperature and resuspended at 2 x 105 viable cells/ml in electroporation buffer (X-VIVO 10 supplemented with 1% HSA). The plasmids pEGFP-N1 (2 µg/µl), pcDNA3 (2 µg/µl, control), or no DNA in sterile DPBS were added to 4 mm gap sterile disposable electroporation cuvettes, followed by addition of 500 µl cell suspension (1-2 x 105 cells). The cell-DNA mixtures were electroporated and added to 2 ml X–VIVO 10 supplemented with TPO/SCF/Flt-3L with or without 5% plasma in 24-well plates and incubated at 37°C, 5% CO2 for 48 hours before evaluation by flow cytometry. Electroporation parameters were 220 V (550 V/cm), 1,600 µF (38 milliseconds [ms]), 30 µg DNA, 2 x 105 cells in 500 µl of X-VIVO 10 supplemented with 1% HSA at room temperature. CFU and LTC-IC assays were performed as described previously [12].

Determination of Transfection Efficiency and Viability by Flow Cytometry
Forty-eight hours after electroporation, CD34+ cells were washed in DPBS with 0.5% bovine serum albumin, and incubated with 20 µl of anti-CD34-PE monoclonal antibody for 15-30 minutes at ambient temperature in the dark. The cells were then washed and resuspended in 0.5 ml of DPBS with 0.5% bovine serum albumin. Propidium iodide was added at 10 µg/ml to each tube immediately prior to flow acquisition. Percent viable cells were determined by gating on propidium iodide negative cells on a dot plot displaying side scatter (SSC) on the X-axis and FL-3 (propidium iodide) on the Y-axis, and the gated viable CD34+ cells were then evaluated on a dot plot displaying FL-1 (EGFP) on the X-axis and FL-2 (CD34-PE) on the Y-axis. For analyses of CD34+/CD38dim (48 hours post-electroporation), CD33+, CD11b+, CD14+, and CD41+ (11 days in extended culture), the cells were stained with appropriate antibodies and acquisition events collected in the presence of propidium iodide.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Optimization of Electroporation Parameters
We were interested in the variability among cord blood samples in terms of electroporation efficiency and viability. Due to the low percentage of CD34+ cells (approximately 0.5% of nucleated cells), we chose to use fresh, large volume (80-100 ml) cord blood so that an individual cord blood sample could provide us with sufficient CD34+ cells for electroporation and flow cytometry of various parameters. Approximately 1-2 x 106 CD34+ cells were selected from individual fresh human umbilical cord blood, cultured for 48 hours in X-VIVO 10 containing TPO/SCF/Flt-3L prior to electroporation, and then cultured for an additional 48 hours prior to evaluation by flow cytometry. We observed that for peripheral blood CD34+ cells, using these same recombinant human growth factors resulted in 28% of the CD34+ cells entering into S-phase and 21% CD34+ cells expressing EGFP 48 hours post-electroporation, and that a 24-48 hour incubation period is necessary for efficient electroporation [11,12]. Figure 1Go illustrates the percentage of EGFP+ and percentage of viable CD34+ cells isolated from cord blood using increasing voltages (Fig. 1AGo) and increasing pulse lengths (Fig. 1BGo). As shown in Figure 1AGo, CD34+ cell viability dropped almost linearly as the voltage increased from 200V to 300V. The percentage of EGFP+ cells was highest between 220V and 260V. Considering both efficiency and viability, we chose 220V as optimal for subsequent experiments. For the optimization of pulse length, CD34+ cells were electroporated with 30 µg DNA/500 µl at a voltage of 220V and selected pulse lengths between 23 ms and 67 ms (Fig. 1BGo). The percentage of EGFP+ cells remained the same from 37 ms to 54 ms. Taking into account viability, we chose 37 ms for further studies. Using a voltage of 220V (550V/cm) and pulse length of 37 ms achieved a 31% ± 2% EGFP+/CD34+ efficiency at 48 hours post-electroporation and 77% ± 3% viability. The optimal conditions for electroporation of CD34+ cells from cord blood and peripheral blood [11] are similar. The mean percentages of EGFP+-CFU and EGFP+-LTC-IC from four independent experiments each using the optimal conditions are 41% ± 13% and 25% ± 4%, respectively.



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  		Figure 1. Electroporation optimization of cord blood CD34+cells. CD34+ cells were cultured for 48 hours in TPO/SCF/Flt-3L, electroporated in a 4 mm gap cuvette, at 2 x 105 cells/500µl, 30 µg DNA/500 µl, and evaluated 48 hours post-electroporation for EGFP expression (filled circle) and cell viability (open circle). (A) Increasing voltages ranging from 180V to 300V were applied to the cells. (B) Increasing capacitance was used to give resultant pulse length as shown on the X-axis. Shown are the means from three individual human cord blood samples with standard error bars (± SE).

 
Effect of Allogeneic Umbilical Cord Blood Plasma on Electroporation Efficiency and CD34+ HPC Expansion
To determine the effect of plasma on electroporation efficiency, CD34+ cells were cultured for 48 hours in TPO/SCF/Flt-3L supplemented with 5% allogeneic plasma, electroporated in electroporation buffer supplemented with 1% plasma (v/v), and then cultured for 48 hours in TPO/SCF/Flt-3L with 5% allogeneic plasma. As shown in Figure 2Go, the presence of plasma during the 96-hour culture period resulted in an increase in the percentage of EGFP+/CD34+ cells from 32% to 42% while maintaining a high mean fluorescence intensity (CD34 brightness). No plasma was used in culturing and electroporation for the control (-Plasma). Figure 3Go illustrates the mean (± standard error [SE]) of three cord blood samples evaluated in the absence and presence of plasma for efficiency of electroporation and CD34+ viability. The percentage of EGFP+/CD34+ cells increased from 31% ± 2% to 44% ± 5% (p < 0.01) with no significant effect on viability. The use of plasma is shown to enhance ex vivo cell expansion, including those expressing EGFP with an increase of 2.5-fold in the total number of EGFP+/CD34+ cells (p < 0.01) (Fig. 3BGo).



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  		Figure 2. Dot plot analysis of cord blood CD34+ cells in the presence and absence of plasma. (A) Non-electroporated CD34+ cells (negative control). CD34+ cells 48 hours post-electroporation in the (B) absence or (C) presence of 5% plasma for 48 hours prior to, 1% plasma during, and 5% plasma for an additional 48 hours. Shown are the fluorescence intensity on the X-axis and CD34-PE on the Y-axis.

 


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  		Figure 3. Effect of allogeneic plasma on electroporation efficiency and cell expansion. Selected cord blood CD34+ cells were cultured in the absence (-Plasma) and presence (+Plasma) throughout the 96-hour culture period as described in Figure 2Go. Each culture contained 1.0 x 105 viable cells immediately prior to electroporation. (A) Percentages of EGFP+/CD34+ and viable cells in the culture measured by flow cytometry 48 hours post-electroporation. (B) Total number of CD34+/EGFP+ and viable cells 48 hours post-electroporation. Each bar represents the mean (± SE) from three individual human cord blood samples. ** indicates statistical significance at Student's t-probability value p < 0.01.

 
Effect of Cord Blood Plasma on CD38 Antigen Expression of CD34+ Cells
We were interested in the effect of plasma on the primitive state of the CD34+ cells. Pooled cryopreserved umbilical cord blood CD34+ cells were cultured in the presence and absence of 5% plasma in TPO/SCF/Flt-3L and were analyzed for the early subset marker CD38 (Fig. 4Go). Immediately after selection (day 0), 5.7% of the CD34+ cells were CD38dim (Fig. 4BGo). After 4 days in culture in the presence of 5% plasma and TPO/SCF/Flt-3L, 10.7% of non-electroporated CD34+ cells were CD38dim (Fig. 4CGo). For CD34+ cells electroporated with pEGFP-N1 in the presence of plasma (Fig 4DGo), 47.8% were EGFP+/CD34+. As shown in Figure 4EGo, 32.5% of the CD34+ cells are EGFP+/CD38bright and 20.8% are EGFP+/CD38dim 48 hours post-electroporation in the presence of plasma. It is noteworthy that the percentage of CD38dim cells not expressing EGFP (8.1%) is similar to non-electroporated cells at day 0 (Fig. 4BGo) and day 4 (Fig. 4CGo).



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  		Figure 4. Dot plot analysis of umbilical cord blood CD34+ subsets. CD34+ cells were cultured for 48 hours with plasma prior to, during and after electroporation, and evaluated by flow cytometry. Cells at day 0 (immediately after selection) were analyzed with (A) mouse IgG1-PE/FITC and (B) CD34-PE and CD38-FITC. Forty-eight hours after electroporation, CD34+ cells non-electroporated (C) or electroporated with pEGFP-N1 (D and E) were analyzed for expression of (C) CD34/CD38, (D) CD34/EGFP, and (E) CD34/ CD38/EGFP. Cells in A-C and E were gated on CD34-Cy5 for analysis. To exclude all debris and dead cells for subset analysis, cells were gated on low side scatter (FSC) and intermediate forward scatter cells displayed on an SSC and FSC dot plot, commonly referred to as the lymphocyte region.

 
Expression of EGFP Gene in Committed Progenitors Derived from Electroporated Cord Blood CD34+ HPCs
To demonstrate the expression of the introduced genes in committed progenitors derived from the electroporated cord blood CD34+ cells, we exposed the electroporated CD34+ cells to IL-3-, GM-CSF-, and G-CSF-supplemented medium for a further 11 days and analyzed for the expression of CD33/CD11b (early myeloid), CD14 (monocytes), and CD41a (megakaryocyte). As shown in Figure 5Go, 74% ± 1% and 63% ± 4% of the electroporated cord blood CD34+ cells differentiated into CD33+ and CD11b+ cells, respectively. Of these, 9% ± 3% and 8% ± 7% were expressing the reporter gene. There were 50% ± 1% CD14+ and 3% ± 1% CD41+ cells in the culture, but no EGFP+ cells were observed in this more differentiated subpopulation of cells. Although the percent of EGFP+ cells decreased over time, there was a 130-fold increase in the total number of EGFP+/CD33+ cells obtained during the 11 days. No CD34+ cells were detected in this culture upon staining with anti-CD34-PE (data not shown). A representative fluorescent microphotograph of these cells is shown in Figure 6AGo using a normal light. Figure 6BGo represents the same cells under FITC filter. It is clear that the level of EGFP expression in these committed cells varies and some cells are brighter than others. This is consistent with dot plot analysis of the sample (data not shown).



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  		Figure 5. Percentage of committed myeloid progenitor cells 11 days post-electroporation in culture with IL-3, GM-CSF, and G-CSF. Percentages of viable (filled bar) and EGFP+ (open bar) of CD33+, CD11b+, CD14+, and CD41a+ were evaluated. Each bar represents the mean (±SE) from three individual human cord blood samples.

 


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  		Figure 6. Microphotograph of committed progenitors derived from human cord blood CD34+ cells 11 days post-electroporation in X-VIVO 10 supplemented with IL-3, GM-CSF, and G-CSF. Shown are cells under normal light (A) and FITC filter (B). Magnification: 100x.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report herein the optimization of electroporation parameters for selected CD34+ HPCs from human umbilical cord blood. Our optimized electroporation conditions resulted in 31% ± 2% CD34+ HPCs expressing the EGFP reporter gene 48 hours post-electroporation in serum-free medium supplemented with TPO, SCF, and Flt-3L with 77% ± 3% viability. The electroporation efficiency for cord blood CD34+ cells is 10% higher (p < 0.01) than that determined for peripheral blood CD34+ cells using the same culture and electroporation conditions [11]. CFU and LTC-IC studies revealed 41% ± 13% and 25% ± 4% EGFP+ cells. The presence of pooled allogeneic plasma during the culture period further increased electroporation efficiency to 44% ± 5% with no detectable effect on cell viability. Electroporation can be used to introduce foreign genes into HPCs that do not express the differentiation marker CD38 (CD34+/CD38dim), which are believed to be more pluripotent than CD34+/CD38bright [13–15]. Of the electroporated cord blood CD34+ cells differentiated into CD33+ and CD11b+ cells, 9% ± 3% and 8% ± 7% were expressing the reporter gene following 11 days in culture, respectively.

Ex vivo studies have revealed several characteristics that could potentially provide a comparable amount of hematopoietic cells for transplant and gene therapy. In short- and long-term colony-forming assays, the number of HPCs from an average term cord have been shown to be similar to, or higher than from bone marrow [7,16,17]. The ex vivo expansion capacity of CD34+/HLA-DR+ cells from cord blood is greater than that of cells derived from bone marrow. Greater frequencies of cells with an immature phenotype (CD38dim, CD45RA–, HLA-DR–, or Thy1+) in CD34+ cord blood cells have been shown to have higher ex vivo expansion potential compared to those from bone marrow [15–20]. In studies on multilineage hematopoiesis of human stem cells in xenogeneic animals such as the severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice, it was shown that cord blood has a higher frequency of SCID-repopulating cells than bone marrow and mobilized peripheral blood [21]. In the presence of SCF, cord blood cells exit G0/G1 phase more rapidly [22,23] and replated cord blood CFU-granulocyte, erythroid, macrophage, megakaryocyte (GEMM) give rise to CFU-GEMM, BFU-E, CFU-GM, whereas replated bone marrow CFU-GEMM gives rise mainly to the more committed CFU-GM in secondary cultures [24].

Electroporation has been one of the technologies used for gene delivery into human hematopoietic cells and differentiated lineages, from the early works of Toneguzzo and Keating [25–27] to the more recent investigations by us [11] and others [28,29]. Our recent demonstration that electroporated peripheral blood CD34+ cells resulted in a stably integrated reporter gene in LTC-IC [11] indicates that electroporation may prove useful for stable transfection of stem cells with genes of therapeutic interest. Since the culture and electroporation conditions are similar for CD34+ cells from peripheral blood and cord blood, it may be plausible to expect that the electroporated cord blood CD34+ cells are stable in the expression of the transgene.

Although relatively lower levels of CD33+ cells were expressing EGFP+ (9%), the number of EGFP+/CD34+ cells were expanded 130-fold ex vivo into EGFP+/CD33+ myeloid progenitors in 11 days. However, the more committed CD14+ monocytes and CD41a megakaryocytes did not express the reporter gene. Further investigation is necessary to elucidate the relationship between foreign gene expression and stem cell differentiation. It is too early to predict from these data whether transfected CD34+ cells sustain gene expression in functional terminal lymphocytes, such as T cells, B cells, and the antigen-presenting dendritic cells.

We demonstrate a 10% increase in CD34+ cells expressing EGFP in the presence of plasma compared to the absence of plasma. In addition to improvement of electroporation efficiency, there may be other advantages of using cord blood plasma as a component in the CD34+ cell culture. It has been shown that allogeneic cord blood plasma enhances the primitive cord blood CFU-GEMM expansion, whereas peripheral blood plasma and fetal bovine serum favor the more mature subsets of cells [30,31]. Human cord blood plasma was also known to enhance the ex vivo expansion of human cord blood CD34+/HLA-DR+ cells and promote their exit from G0/G1 phase more rapidly [22,23]. The expansion capacity and the functionality of the progeny myeloid cells in serum-free bone marrow and mobilized bone marrow cultures could be restored by autologous plasma in the expansion culture [32]. One report showed that autologous cord blood plasma enhances retrovirus-mediated transduction of committed progenitors, but not LTC-IC [32].

The CD34+/CD38dim immunophenotype defines highly primitive populations in both bone marrow and cord blood, and the percentage of CD34+ cells in cycle is directly correlated with increasing CD38 expression [33]. Our data showed that 96 hours in TPO/SCF/Flt-3L increased the percentage of CD38dim in the cord blood CD34+ population from 5.7% to 10.7%. Interestingly, 20.8% of the CD34+ cells were EGFP+/CD38dim. It has been demonstrated that CD34+/CD38dim cord blood cells have a higher cloning efficiency, proliferate more rapidly in response to cytokine stimulation, and generate approximately sevenfold more progeny than do their counterparts in bone marrow [33]. A recent study [15] revealed that the functional differences between CD34+/CD38dim and CD34+/CD38bright cells were less pronounced in fetal liver and umbilical cord blood than in their bone marrow counterparts by showing that interferon-{gamma} was only a selective inhibitor of primitive CD34+/CD38dim from bone marrow cells, yet it inhibited both CD34+/CD38dim and CD34+/CD38bright cells from fetal liver and cord blood cells to the same extent. Although the cycling of CD34+ cells appears to promote expression of the differentiation antigen CD38, it remains to be determined whether this is indicative of pluripotency loss for human cord blood CD34+ HPCs.

The ability to stimulate cycling of CD34+ stem cells while maintaining their pluripotency is essential for gene transduction with implication in therapeutic trials. We conclude from our studies that the addition of allogeneic plasma increases the percent of CD34+ cells expressing EGFP+ following introduction of pEGFP-N1 by electroporation and that the introduced gene is expressed in certain committed lineages upon differentiation. The true clinical values of this technology, however, rely on the expression of foreign genes in functional terminal cells derived from the electroporated HPCs.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
M. Eileen Dolan is supported by U.S. Public Health Service grants CA57725, CA81485, and a breast cancer planning grant awarded to The University of Chicago Cancer Center CA66132.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received May 23, 2001; accepted for publication July 13, 2001.




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