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a CRC Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Withington, Manchester, United Kingdom;
b CIEMAT, Department of Molecular and Cellular Biology, Avenida Complutense, Madrid, Spain;
c CRC Section of Tumour and Cell Biology, Paterson Institute;
d AmCell Corp., Sunnyvale, CA, USA
Key Words. CD34+ cells • AC133+ • LTC-IC • NOD/SCID mice • Dendritic cells
Dr. E.A. de Wynter, CRC Section of Haemopoietic Cell and Gene Therapeutics, Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, United Kingdom.
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Abstract |
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Most of the clonogenic cells (67%) were contained in the CD34+AC133+ fraction. Compared with cultures of the CD34+AC133– cells, generation of progenitor cells in long-term culture on bone marrow stroma was consistently 10- to 100-fold higher in cultures initiated with CD34+AC133+ cells and was maintained for the 8-10 weeks of culture. Only the CD34+AC133+ cells were capable of repopulating NOD/SCID mice. Human cells were detectable as early as day 20, with increased levels (67%) apparent 40 days post-transplantation. Five thousand CD34+AC133+ cells engrafted about 20% of the mice, while no engraftment was observed in animals transplanted with up to 1.2 x 105 CD34+AC133– cells. The CD34+AC133+ population was also enriched (sevenfold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133+ cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.
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Introduction |
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Attempts to identify and purify populations of human hemopoietic stem cells have focused on antigens expressed on the cell surface. The most commonly used marker for the isolation, purification, and manipulation of stem and progenitor cells is the CD34 antigen. This molecule is highly expressed on primitive cells but is significantly reduced as cells differentiate and mature. The frequency of CD34+ cells has been estimated to be 1%-4% of all nucleated cells in adult bone marrow and about 1% in cord blood from full-term deliveries [3-5]. CD34 selection has proved to be particularly useful in transplantation studies where selected cells were able to reconstitute hemopoiesis in myeloablated patients, indicating the presence of primitive stem cells in the graft [6]. However, isolated CD34+ cells are heterogeneous, and the stem cell content is a small fraction of the total CD34+ population. It is difficult to assign a specific phenotype to these stem cells, and this has hampered progress in their characterization. However, by assessing coexpression of other markers, including CD38, CD45RA, CD71, Thy-1, c-kit, HLA-DR, and Rh-123, it is possible to distinguish cell populations which are enriched in primitive or progenitor cells [7-12].
In vitro, progenitor cells may be detected using standard colony-forming assays, but the more immature cells require more sophisticated assay techniques. At present, the most primitive human hemopoietic cell which can be assayed in vitro is the LTC-IC. It is characterized by the ability to generate granulocyte-macrophage colony forming cells (GM-CFC) after at least five weeks in culture on a supportive stromal cell layer. In mice, these LTC-IC are also able to reconstitute hemopoiesis. A number of studies have defined the phenotype of these cells. [9, 13, 14]. A more primitive human cell than those identified by in vitro assays has been defined and may be detected by an in vivo functional assay [15]. The method relies on the ability of human hemopoietic cells to repopulate the bone marrow of immune-deficient, non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice after i.v. transplantation.
Recently, a novel antigen expressed on human hemopoietic stem and progenitor cells was reported [16]. This antigen is a unique five-transmembrane (5-TM) molecule, and the monoclonal antibody AC133 raised against the protein recognizes a subset of CD34+ cells in bone marrow, fetal liver, umbilical cord, and peripheral blood. The AC133 selected cells engrafted successfully in a fetal sheep transplantation model in both primary and secondary recipients, indicating the presence of long-term repopulating cells [17].
In this study, we have used the AC133 monoclonal antibody to analyze cells in bone marrow, umbilical cord blood, and apheresis products. Cells clearly expressing AC133 (AC133+) and cells with little or no expression of the antigen (AC133–) were examined with respect to their clonogenic capacity, ability to sustain hemopoiesis on supportive stroma, ability to engraft in the NOD/SCID mouse, and generation of dendritic cells ex vivo. The results indicated that CD34+AC133+ cells from cord blood were highly enriched in long-term culture-initiating cells, NOD/SCID repopulating cells, and dendritic cell progenitors.
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Materials and Methods |
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Apheresis samples were obtained from peripheral blood of patients with lymphoma, breast carcinoma, teratoma, and Ewing's sarcoma after treatment with G-CSF and appropriate chemotherapy.
Growth Factors
Purified recombinant human G-CSF and recombinant stem cell factor (SCF) were obtained from Amgen, Inc. (Thousand Oaks, CA). Recombinant interleukin-3 (IL-3) and IL-6 were obtained from Sandoz (Basel, Switzerland). Recombinant erythropoietin (Epo) was obtained from Boehringer (Mannheim, Germany). Flt-3 ligand (FL), transforming growth factor-beta (TGF-ß), and tumor necrosis factor-alpha (TNF-
) were purchased from R & D Systems (Abingdon, Oxon, UK). GM-CSF was obtained from Glaxo (Geneva, Switzerland).
CD34 and AC133 Analysis
Fifty µl aliquots of cord blood, apheresis, or bone marrow were dispensed into three tubes. CD45-FITC and CD34-PE monoclonal antibodies were added to one tube, CD45-FITC and AC133-PE to a second tube, and CD45-FITC and an isotype control-PE to the control tube. After incubation for 20-30 min, red cells were lysed by addition of lysing solution (Ortho Diagnostics; Raritan, NJ). Samples were then centrifuged, washed, and the cell pellet resuspended in phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) for flow cytometric analysis. A minimum of 75,000 events was analyzed for each test sample to ensure a sufficient number of positive events, and the percentage of AC133 and CD34 was determined using the ISHAGE protocol [18].
Mononuclear Cell Preparation
Cord blood samples were diluted 1:1 with PBS and the mononuclear cells separated by layering the samples over an equal volume of Ficoll-Hypaque 1.077 g/ml (Life Technologies; Paisley, Scotland). After centrifugation for 25 min at 400 x g, the cells at the interface were removed and washed once with PBE (PBS/0.5% bovine serum albumin/5mM EDTA).
Isolation of CD34+ and AC133+ Cells
CD34+ cells were isolated by incubating 1-2 x 108 mononuclear cells in 300 µl of PBE with 100 µl of CD34 (QBEND 10)-conjugated magnetic beads (Multisort beads, Miltenyi Biotec; Bergisch Gladbach, Germany), 100 µl of human IgG (8 mg/ml) followed by incubation for 30 min at 4°-8°C. After incubation, the cells were washed with ice-cold PBE and processed through a MACS magnetic separation column (Miltenyi Biotec). Cells labeled with microbeads were passed through a column placed in a magnetic field and the target cells retained. After washing the column thoroughly with ice-cold PBE, the target cells were recovered by removing the magnetic field and flushing the column with 1 ml of PBE.
Fluorescence-Activated Cell Sorting (FACS)
To obtain the CD34+AC133+ and CD34+AC133– populations by FACS sorting, CD34+ cells were first isolated as described above then labeled with a CD34-FITC-conjugated antibody (HPCA-2-FITC, Becton Dickinson) and an AC133-PE-conjugated antibody (AmCell Inc; Thousand Oaks, CA) for 15-20 min at room temperature. The cells were then washed with PBE and analyzed. CD34+AC133+ and CD34+AC133– fractions were sorted on a FACS Vantage flow cytometer (Becton Dickinson; San Jose, CA) equipped with a 488 nm argon laser. Isotype matched controls conjugated to FITC and PE were used to set the sorting gates.
Clonogenic Assays
Clonogenic assays for colony-forming cells (CFC) were performed as previously described [11]. Briefly, 1,500 sorted cells were added to a 1 ml mixture containing 30% (v/v) fetal calf serum, 10% (v/v) 5637 conditioned medium (from the EJ bladder carcinoma cell line), 1% (v/v) bovine serum albumin, 2 units Epo, and 1.35% (v/v) methylcellulose. Triplicate cultures were plated and incubated at 37°C in a humidified atmosphere of 5% CO2 and 5% O2 for 14 days. Colonies were then identified and scored as GM-CFC or BFU-E according to standard criteria [19].
In a serum-free clonogenic assay, 1,000 cells were added to a mixture containing 350 µl X-VIVO 10 medium (BioWhittaker; Wokingham, UK), 100 µl 10% deionized BSA, and human recombinant growth factors at final concentrations of 100 ng/ml SCF, 5 x 104 U/ml G-CSF, 10 ng/ml IL-3, 200 U/ml IL-6, and 2 U/ml Epo. Cultures were plated in triplicate, incubated at 37°C for 14 days, then identified and scored as outlined above.
Long-Term Bone Marrow Cultures
Adherent stromal layers were established as previously described [19]. Cultures were initiated with bone marrow cells at a concentration of 2 x 106/ml in Iscove's modified Dulbecco's medium at 350 mOsm/kg containing 10% (v/v) fetal calf serum, 10% (v/v) horse serum, and 5 x 10–7 M hydrocortisone in 25 cm2 tissue flasks. The flasks were gassed with CO2 in air and incubated at 33°C. All cultures were maintained by weekly demi-depletion of the supernatant and replacement with fresh medium. The adherent layers were confluent after three to four weeks, and each culture was then irradiated with 15 Gy and used as stromal support to test hemopoietic function of the FACS sorted cell populations.
Transplantation of Purified Cells into NOD/SCID Mice
Six- to eight-week-old NOD/LtSz-scid/scid (NOD/SCID) mice were used as recipients of the human hemopoietic cells. Mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in microisolaters in a pathogen-free environment. Mice were given total body irradiation of 2.5-3.0 Gy (x-rays; 300kV, 10mA; 1.03 Gy/min) and transplanted with varying numbers of cells from the CD34+AC133+ and CD34+AC133– populations. The cells were injected i.v. within four h of irradiation. In three other separate experiments mice aged eight to nine weeks were irradiated with a sublethal dose of 3.5 Gy (0.83 Gy/h) from a Cobalt 
-irradiator immediately prior to use, as previously described [20]. 5 x 103 cells from the CD34+AC133+ or the CD34+AC133– subpopulations were injected into each mouse via the tail vein and the mice were studied eight to nine weeks after transplantation.
Reconstitution of NOD/SCID Mice with Human Hemopoietic Cells
The presence of human hemopoietic cells in the mouse bone marrow was examined periodically using flow cytometry to detect CD45-labeled cells. Bone marrow samples were obtained under anesthesia from individual reconstituted mice 20 days and 40 days after transplantation, as previously described [21]. Bone marrow was aspirated from one femur by puncture through the knee joint with a 22-gauge needle. Aliquots of 1-5 x 105 bone marrow cells were labeled with CD45-FITC-conjugated antibody (Pharmingen; San Diego, CA) for 25 min at 4°C. Additional aliquots were double-stained with CD45-FITC and CD34-PE (HPCA-2). Bone marrow cells from transplanted NOD/SCID mice were labeled with isotype-conjugated antibodies. In addition, bone marrow cells from untransplanted NOD/SCID mice stained with CD45-FITC and CD34-PE antibodies served as controls for nonspecific staining of human and mouse cells. After labeling, red blood cells were lysed, washed in PBS/BSA, and resuspended in the same buffer containing 2 mg/ml propidium iodide (PI). For each FACS analysis, a total of 15,000 cells (PI–) was collected.
Culture of Dendritic Cells
Dendritic cells were cultured in serum-free medium according to the method of Strobl et al. [22, 23]. CD34+AC133+ and CD34+AC133– cells from cord blood were cultured in 24-well plates (Falcon; Oxford, UK) at 1-5 x 103 cells/well in 1 ml of serum-free medium (X-VIVO 10) supplemented with L-glutamine (2.5 mmol/l), SCF 20 ng/ml, TGF-ß 0.5 ng/ml, TNF- 10 ng/ml, GM-CSF 100 ng/ml and FL 100 ng/ml. Cultures were incubated at 37°C in a humidified atmosphere in the presence of 5% CO2 for 10-11 days. At the end of the culture period, the cells were harvested, analyzed for the presence of CD1a+ and CD83+ cells, and function was assessed in a mixed lymphocyte reaction.
Preparation of T cells for Allogeneic Mixed Lymphocyte Reaction (MLR)
Fresh heparinized blood samples were obtained from normal healthy volunteers. Mononuclear cells from 10-ml aliquots were obtained as previously described and washed three times in PBS. The peripheral blood mononuclear cells were then placed in RPMI 1640 medium (GIBCO, Life Technologies; Paisley, Scotland) supplemented with either 10% human antibody (AB) serum or 10% autologous plasma at a concentration of 2.5-5 x 106 cells/ml, and incubated in a volume of 3 ml per well on six-well plates (Falcon). The cells were incubated at 37°C for two h to allow monocytes to adhere to the base of the well. At the end of the incubation period, nonadherent cells were then placed on a sterile nylon wool column pre-equilibrated in RPMI 1640/10% AB serum and incubated for one h to allow B cells to adhere to the column. The cells were then flushed through the column with fresh medium and the nonadherent cells harvested. These were predominantly T cells with a mean T-cell content of 88% CD3+ as determined by flow cytometry.
MLR
The dendritic cells from the cultures described above were collected, washed in RPMI 1640/10% AB serum to remove the cytokines and irradiated with 30 Gy using a -ray-emitting Cesium source. Irradiated cells at 102, 103, and 104 were dispensed into 96-well plates and 5-10 x 104 T cells added to each well. After six days of incubation at 37°C in 5% CO2, each well was pulsed with 74 kBq of [3H-] thymidine for four h, and the cells from each well harvested onto Packard unifilter plates using the Packard 196 harvester. Plates were allowed to dry overnight and the thymidine incorporation into the harvested cells counted. The [3H-] thymidine incorporation into the responding T cells is taken as a measure of proliferation in response to the antigen-presenting cells. Tests were performed in triplicate, and results were expressed as mean counts per minute (cpm) ± standard deviation (SD).
Statistics
For the analysis of AC133 expression on CD34+ cells, the significance of the medians was assessed with the Kruskal-Wallis test. Paired comparisons were made with the Mann-Whitney test.
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Results |
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shows a summary of results from three different sample sources. AC133 could be detected in all samples assessed, and the percentage of cells labeled with AC133 was always lower than that of CD34. It has been shown that only CD34+ cells from fetal liver, fetal bone marrow, adult bone marrow, cord blood, and peripheral blood express AC133 [17], and we did not observe any cells which stained for AC133 but not for CD34. Therefore, the proportion of AC133+ cells in the CD34+ population was determined. On average, the CD34+ cells mobilized to peripheral blood contained the highest proportion of AC133+ cells at 75.3%, compared to only 36.3% in normal bone marrow and 51% in cord blood. Although the difference between cord blood and bone marrow was not significant, when the apheresis samples were compared with either bone marrow or cord blood, a highly significant difference (p < 0.0001) was demonstrated.
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. The majority of cells in the lymphocyte gate were CD34+ cells, as indicated in Figures 1A and 1B. There was a range of fluorescence intensity of AC133-expressing cells, with all of these cells positive for the CD34 antigen (Fig. 1D). Less than 10% of cells in the lymphocyte gate were unlabeled.
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shows that clonogenic cells were present in both CD34+AC133+ and CD34+AC133– populations, although the majority of GM-CFC, BFU-E, and CFC-Mix were contained in the CD34+AC133+ population.
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illustrates a representative experiment showing that throughout the culture period GM-CFC production was always markedly higher with the CD34+AC133+ cells than with the CD34+AC133–. All cultures initiated with the CD34+AC133+ cells produced GM-CFC up to weeks 8-10, when the cultures were terminated. GM-CFC production from CD34+AC133+ cells was 100-fold greater over the first few weeks of culture, rising to 1,000-fold at week 6 compared with CD34+AC133– cells (Fig. 2A). The number of nonadherent cells produced in the same period was also consistently higher with the CD34+AC133+ population (Fig. 2B) and reflected the difference seen in GM-CFC production. In two experiments, when the adherent layers were examined for the presence of progenitors at week 8, the cultures initiated with CD34+AC133+ cells still had significant numbers, indicating that the cultures were still active when terminated. In contrast, few progenitors were detected in the CD34+AC133– group (Fig. 3).
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). After 40 days, the percentage of CD45 cells had increased to almost 70% in the mice injected with the higher numbers of CD34+AC133+. At the same time point, little or no engraftment was detectable in mice injected with CD34+AC133– cells over the dose range 104 - 1.2 x 105. In three additional experiments where the mice received a limiting dose of 5 x 103 CD34+AC133+ cells per animal, 20% (3 of 14) of the animals engrafted at eight weeks post-transplantation, while none of the animals transplanted with 5,000 CD34+AC133– cells did. Individual animals were scored as positive only when both human CD45 and CD34 cells were present as determined by flow cytometry (Table 4). The proportion of CD45-labeled cells ranged from 0.5% to 8.7% and the proportion of CD34-labeled cells from 0.3% to 2.9% in the animals transplanted with CD34+AC133+ cells. In the animals injected with CD34+AC133– cells, the CD45 fraction ranged from 0% to 0.2%, and the CD34 was never greater than background (<0.2%).
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). The fold expansion over input cell numbers was 26-fold for the CD34+AC133+ and 8-fold for the CD34+ AC133– cells (data not shown).
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). Stimulation was detectable at low input cell numbers from the CD34+AC133+ fraction and increased dramatically. In contrast, in cells from the CD34+AC133– population, activity was significantly reduced.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Also, analyses were performed on mononuclear cells obtained by Ficoll gradient and not unfractionated samples as in this study. In general, we can confirm that the majority of the CFC (67%) are contained in the CD34+AC133+ population, with the remainder in the CD34+AC133– fraction. AC133 is expressed on a proportion of CFC, including GM-CFC, BFU-E, and CFC-Mix. All three types of clonogenic cells were present in both the CD34+AC133+ and CD34+AC133– populations. We could not distinguish an enriched population of erythroid precursors (BFU-E) in the CD34+AC133– population using our standard clonogenic assays. However, in a serum-free assay with recombinant growth factors, we did observe that the CD34+AC133– population was enriched for erythroid colonies as reported by Yin et al. [17]. These authors also noted that granulomonocytic committed progenitor cells showed decreased staining with AC133, although the mean fluorescence intensity (MFI) for this subset was higher than the MFI for the lymphoid and erythroid committed progenitors. When we selected the most intensely stained AC133 cells and examined them in clonogenic assays, the colonies generated were exclusively GM-CFC (data not shown). Although AC133 will be seen as a valuable indicator of primitive cells (see below), it is most highly expressed on the committed progenitors.
Long-term cultures allow generation of progenitor cells in vitro, and we used this system to investigate the behavior of the two AC133 populations. The number of GM-CFC recovered after week 5 or later reflects the presence of LTC-IC. Hemopoiesis, as measured by the level of progenitor cell production, was clearly enhanced in the CD34+AC133+ cells compared with CD34+AC133–, indicating that the former cells are greatly enriched in LTC-IC. At week 8, the cultures were still active, and progenitor cells were still present in large numbers in both the supernatant and the adherent layers. These results confirm that AC133 is expressed in a population of cells which is particularly enriched in LTC-IC, the most primitive human cells which can be assayed in vitro. As we noted previously, there is significant variation in the proportion of AC133+ cells in normal and apheresis samples, so it would be of interest to determine whether this is reflected in the absolute numbers of LTC-IC contained in the different samples. In addition, as the proportion of LTC-IC is higher in mobilized peripheral blood than in cord blood, [25, 26] the AC133 antigen may be a valuable marker for LTC-IC selection. Experiments testing these possibilities are the subject of current work as well as further purification of primitive cells by subfractionation of the AC133-labeled population.
Cells expressing AC133 engraft in primary and secondary recipients in a fetal sheep xenogeneic transplantation model, demonstrating the long-term repopulating potential of the AC133 cells [17]. By using the knee joint sampling technique of Drize et al. [21], engraftment in individual mice can be monitored over a period of time. Our data compared the ability of both CD34+AC133+ and CD34+AC133– cells to engraft in individual NOD/SCID mice. Our results showed that CD34+AC133+ cells do engraft and home to the bone marrow. Human cells were detectable as early as day 20 and continued to increase to day 40 with no engraftment observed in mice transplanted with up to 1.5 x 105 CD34+AC133– cells per mouse.
A recent study has shown that SCID-repopulating cells are contained exclusively in the CD34+38- fraction of cord blood. As few as 100 CD34+ 38– cells engrafted in a portion of the mice at low levels (0.1%), although a sensitive Southern blot detection system was required to detect the human DNA component in the mouse bone marrow [27]. There was, however, a dose-response relationship, and mice transplanted with 5,000 cells had levels of engraftment of about 10%. In the present study, using 5,000 CD34+AC133+ cells and CD45/CD34 detection by flow cytometry, we were able to identify human cells in mouse bone marrow. When 5,000 cells were injected per mouse in 14 animals, three mice engrafted. This implies that at least three repopulating cells (SRC) in 70,000 cells engrafted, or 1:23,000. About 20% of transplanted cells get to the bone marrow [28] which reduces this figure to 1:4,700. Previous reports indicate that the frequency of SRC in cord blood is 1 in 9.3 x 105 cells [29, 30]. The SRC therefore appear to have been enriched about 200-fold in the CD34+AC133+ population.
Several reports have shown that dendritic cells (DC) and monocytes develop from a common CD34+ progenitor cell in the bone marrow and that CD34+ cells can be manipulated to generate large numbers of functionally mature DC in the presence of the appropriate cytokines [31-33]. It was clear that the cells capable of giving rise to functional dendritic cells reside in the CD34+AC133+ population. Cells were defined as dendritic based on several criteria: typical dendritic morphology, expression of surface membrane CD1a and CD83, and the capacity to induce proliferation of allogeneic T cells in the primary mixed lymphocyte response. As the CD34+AC133+ cells represent about 50% of the starting CD34+ population in cord blood, it is possible to eliminate the "noise" produced by unwanted cells by initiating cultures with AC133+ cells. Furthermore, these cells represent a better defined starting point to examine the ontogeny of dendritic cells.
The AC133 antibody may provide a means of enriching for primitive hemopoietic stem cells with obvious implications for gene therapy. The observations reported here should be helpful in defining the phenotype of primitive cord blood cells, with the additional advantage that the cells can be obtained by a simple, one-step, positive selection procedure for AC133 alone.
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Acknowledgments |
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This work was supported by the Cancer Research Campaign.
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Y. J. Summers, C. M. Heyworth, E. A. de Wynter, C. A. Hart, J. Chang, and N. G. Testa AC133+ G0 Cells from Cord Blood Show a High Incidence of Long-Term Culture-Initiating Cells and a Capacity for More Than 100 Million-Fold Amplification of Colony-Forming Cells In Vitro Stem Cells, September 1, 2004; 22(5): 704 - 715. [Abstract] [Full Text] [PDF]
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) and CD34+AC133– (
) cells seeded per flask on irradiated marrow stroma. B) Number of non-adherent cells generated from the same culture.
