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Adherent Cells Generated During Long-Term Culture of Human Umbilical Cord Blood CD34⁺ Cells Have Characteristics of Endothelial Cells and Beneficial Effect on Cord Blood Ex Vivo Expansion

^a Department of Pediatrics,
^b Obstetrics/Gynecology,
^c Clinical Pathology,
^d Hematology/Oncology,
^e Medical Research Center, College of Medicine, Ewha Women’s University;
^f Department of Cell Biology and Immunology, Asan Institute for Life Sciences; University of Ulsan, Seoul, Korea;
^g Department of Hematology, Chungnam University, Dae-Jeon, Korea;
^h Graduate School of Biotechnology, Korea University, Seoul, Korea

Key Words. Cord blood CD34 • Ex vivo expansion • Adherent endothelial cell • Apoptosis • Long-term liquid culture

Chu-Myong Seong, M.D., Dept. of Hematology/Oncology, Ewha Wowen’s University, Mock-Dong Hospital, Yangchun-Ku, Mock-6-Dong 911-1, Seoul, Korea 158-056. Telephone: 82-2-650-5015; Fax : 82-2-650-5062; e-mail: cmseong{at}ewha.ac.kr.

	ABSTRACT

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Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study, UCB CD34⁺ cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin, flt3 ligand, and granulocyte-colony stimulating factor. By week 4-5, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor, human vascular cell adhesion molecule-1, human intracellular adhesion molecule-1, human CD31, E-selectin, and human macrophage. Furthermore, when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer, better expansion of total nucleated cells, CD34⁺ cells, and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34⁺ cells, which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells, we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method, one endothelial cell may be found from 314 CD34⁺ cells after 5 weeks of culture. These results suggest that the UCB CD34⁺ cell fraction contains endothelial cell precursors, establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering.

	INTRODUCTION

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Effective hematopoiesis depends on an intimate association of hematopoietic stem cells with the stroma that constitutes the hematopoietic microenvironment [1]. The stroma not only provides a suitable environment for self-renewal, proliferation, and differentiation of hematopoietic stem cells (HSCs), but also other regulatory factors and cytokines [2]. The stroma, as one component of the microenvironment, includes fibroblasts, endothelial cells, macrophages, adipocytes, osteoclasts, osteoblasts, and smooth muscle cells [3]. In recent years, there has been an increasing interest in the stromal cell system, which includes the marrow-derived stromal cells used to support hematopoiesis.

The feasibility of ex vivo expanding umbilical cord blood (UCB) CD34⁺ cells to provide increased cell doses for transplantation is of considerable interest [4]. We have previously shown that during ex vivo expansion of UCB CD34⁺ cells using thrombopoietin (TPO), flt-3 ligand (FL), and/or granulocyte colony-stimulating factor (G-CSF) in stroma-free liquid culture, distinct patterns of apoptosis are associated with modulation of CD44 [5], and phenotypic changes are dependent on the type of cytokines used [6]. Meanwhile, during UCB CD34⁺ cell expansion when using the same triple cytokines, we observed that a fibroblast-like adherent cell layer was found by week 4 or 5. Therefore, we have investigated the ability of UCBs to establish a stromal cell layer in a modified long-term liquid culture using immunohistochemistry and flow cytometry. We have also investigated whether preestablished stromal cells derived from CD34⁺ UCB can provide the beneficial effects on CD34⁺ cell expansion during ex vivo expansion, compared with those without adherent cell layers.

Recently, 7-amino-actinomycin D (7-AAD) has been introduced as a valuable fluorescent dye for assessing apoptosis [7]. The method of 7-AAD enables simultaneous staining of cell-surface antigens, as it does not require permeabilization of the cell membrane [8]. For assessment of apoptotic changes, flow cytometry was applied for quantification of apoptosis on an individual cell level. Finally, we compared the changes of apoptosis during ex vivo expansion with or without a preestablished adherent cell layer, which is derived from UCB CD34⁺ cells.

	MATERIALS AND METHODS

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Cell Source and CD34⁺ Cell Purification
Umbilical cord blood was obtained at the end of full-term deliveries. Mononuclear cells were isolated using a Ficoll-Hypaque (density, 1.077; Pharmacia Biotech, Upsala, Sweden; http://www.pnu.com) density gradient centrifugation. After two cycles of plastic adherence for 60 minutes, the cells were washed and suspended in phosphate-buffered saline (PBS, pH 7.4), which contained 0.1% bovine serum albumin. During these procedures, all adherent cells were removed and only nonadherent cells were recovered. The CD34⁺ cell fraction was isolated with a superparamagnetic microbead selection using monoclonal antibody (QBEND10) and miniMACS columns (Miltenyi Biotec; Bergisch Gladbach, Germany; http://www.miltenyibiotc.com). The efficiency of purification and immunophenotype were verified with flow cytometry and counterstained with a fluorescein isothiocyanate (FITC)-anti-human CD34 (HPCA-2; Becton Dickinson [BD]; Mountain View, California; http://www.bd.com) as previously described [6]. The morphology of isolated CD34⁺ cells was also assessed using the Wright-Giemsa (Sigma; St. Louis, Missouri; http://www.sigmaaldrich.com) stained cytospin preparations.

Stroma-Free Liquid Culture
In 10 independent experiments from different cord blood donors, the purified CD34⁺ cell fractions were suspended in Iscove’s-modified Dulbecco’s medium ([IMDM] GIBCO; Grand Island, New York; http://www.lifetech.com) at a density of 5.0 x 10⁵ cells/ml and then supplemented with 10% fetal bovine serum ([FBS] BioWhittaker; Walkersville, Maryland), recombinant humanized (rh) TPO (10 U/ml), rhFL (50 ng/ml), and rhG-CSF (100 U/ml) as previously described [5]. Custom-made coverslips were placed at the bottom of each 24-well culture plate and incubated at 37°C in 5% CO₂. Twice a week, the cells were fed with the removal of one-half of the culture volume, which was replaced with fresh medium and growth factors. The cells were cultured for 5-6 weeks. During ex vivo expansion, an inverted light-microscopic examination was done. When a firmly adherent monolayer was fully formed, the adherent cell layers from the culture were harvested for morphological, immunophenotypic, and functional studies.

Immunohistochemistry
The adherent cell layers grown on customized coverslips were washed with PBS and then fixed in acetone or 4% paraformaldehyde for 15 minutes. Immunostaining was performed using a three-step streptoavidin-biotin complex immunoperoxidase technique. Briefly, endogenous peroxidase activity was blocked with 3% H₂O₂ in water for 5 minutes and washed in PBS three times for 5 minutes. Primary monoclonal antibodies at different dilutions were incubated with a fixed cell layer for 2 hours at room temperature and then the cells were washed in PBS for 10 minutes. Afterwards, the secondary biotinylated antibodies (goat anti-mouse IgG or goat anti-rabbit IgG [DAKO; Carpenteria, CA; http://www.dako.dk]) were applied for 30 minutes. The control was treated with a secondary antibody. Endogenous biotin activity was blocked by incubation of slides with avidin-biotin complex for 30 minutes and the staining was visualized with a 3-amino-9-ethylcarbazole (in N,N-dimethylformamide) substrate. The cells were then counterstained with hematoxylin solution. Next, slides were mounted in aqueous-based mounting media (DAKO). Primary antibodies were as follows: von Willebrand factor ([vWF] DAKO), human vascular cell adhesion molecule-1 ([VCAM-1] DAKO), human intracellular adhesion molecule-1 ([ICAM-1] Novocastra; Burlingame, CA; http://www.novocastra.com ), human CD31 ([PECAM-1] DAKO), E-selectin (endothelial leukocyte adhesion molecule-1; DAKO), and human macrophage.

Cocultures Using Preestablished Adherent Cell Layers
Preestablished adherent cell monolayers from UCB were irradiated with 1,500 cGy. This was done in order to eliminate endogenous hematopoietic foci before the inoculation of the cells. In 10 independent experiments from different cord blood donors, freshly isolated UCB CD34⁺ cells in 24-well plates (BD Falcon) were cultured with and without the irradiated adherent cell layer. All experiments were duplicated. This was done at 5.0 x 10⁴ cells/ml in IMDM supplemented with 10% FBS and cytokines (rhTPO [10 U/ml], rhFL [50 ng/ml], and rhG-CSF [100 U/ml]). These cells were cultured by the above methods for up to 5 weeks. At initiation and weekly intervals thereafter, the harvested cells were tested for a total cell number, CD34, colony-forming units granulocyte-macrophage (CFU-GM), and CFU-granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) with and without the association of an adherent cell layer, generated during the long-term in vitro culture of HCB CD34⁺ cells.

Fluorescence-Activated Cell Sorting (FACS) Analysis and Measurement of Apoptosis
Approximately 1 x 10⁵ EDTA-detached cells in adherent cell layers were stained at 4°C for 30 minutes with FITC-anti-human CD34 (anti-HPCA-2; BD) and phycoerythrin (PE)-anti-human CD14 (TUK4; Chemicon; Temecula, CA; http://www.chemicon.com). During coculture on the adherent cell layer, the harvested cells were stained with 20 µg/ml 7-AAD (Sigma) at 4°C for 30 minutes [7, 8]. The negative control consisted of incubation with isotype-matched irrelevant antibodies (mouse IgG1-FITC [BD]; mouse IgG2a-PE [Chemicon]). Samples were analyzed on a FACSCalibur flow cytometer (BD). Ten thousand events were acquired for each analysis and data were analyzed using CellQuest (BD) or Attractors (BD) software. Results were expressed as mean ± standard deviation (SD) of percentage of positive cells from four separate samples.

Clonogenic Assays
Clonogenic assays were performed as follows: 1 x 10³ CD34⁺ UCB cells were cultured at two plates per point in complete methylcellulose medium (HCC-4434; Methocult; Stem Cell Technologies, Vancouver, BC, Canada; http://www.stemcell.com). It was supplemented with 50 ng/ml stem cell factor (SCF), 10 ng/ml interleukin-3, GM-CSF, and 1 U/ml recombinant human erythropoietin at 37°C in a humidified atmosphere at 5% CO₂. After 12 to 14 days of incubation, colonies were enumerated with the aid of an inverted microscope and counted by morphologic criteria.

Limiting Dilution Assay of UCB CD34⁺ Cells
Isolated UCB CD34⁺ cells using monoclonal antibody (QBEND10) and mini-MACS columns were seeded in 24-well plates at four dilutions, with 20 replicate wells for each. It was assumed in this model that there was homogenous suspension of endothelial cells in each dilution, and a single endothelial cell in a well resulted in a positive adherent cell. Wells that did not contain endothelial cells after 5 weeks of culture were counted and the number of endothelial cells in any other inoculum were determined by using Poisson distribution. A minimum of 1 x 10² cells/ml and a maximum of 1 x 10⁴ CD34⁺ cells/ml were plated. The dilutions were chosen to be maximally informative at weeks 4 to 5 of the assay, yielding 10%-37% negative cells for fibroblastic adherent cell layers. Twice a week, the cells were fed with the removal of one-half of the culture volume, which was replaced with fresh medium and growth factors. After forming the fibroblastic adherent cell layer, we used immunohistochemistry using vWF antibody to confirm endothelial cells. Estimates of endothelial cell proportions were analyzed by standard limiting dilution assay techniques. Wells that did not contain endothelial cells after 5 weeks of culture were counted and the number of endothelial cells in any other inoculum were determined by using Poisson distribution.

Statistical Analysis
The results are presented as the mean ± SD of the data obtained from three or more experiments performed in duplicates. Statistical significance was determined using the Student’s t-test.

	RESULTS

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Purity and Morphology of Purified CD34⁺ Cells
The percentage of CD34⁺ and CD34⁺/CD38^- cells in the cell fraction containing purified cells was 93.6% ± 4.8% and 31.1% ± 11.0%, respectively (n = 10) (Fig. 1). The morphology of the purified cells using Wright-Geimsa staining predominantly showed rather small blasts, but mature stromal cells were not seen (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 1. Representative scattergram for purified cells. A) Purified cells and mononuclear cells after elimination of adherent cells were stained with FITC-anti-CD34 (HPCA-2) antibody. B) Purified cells stained with isotype-matched irrelevant antibodies. C) Purified cells stained with FITC-anti-CD34 and PE-anti-CD38 (Leu 17) antibodies. Abbreviations: UL = upper left; UR = upper right; LL = lower left; LR = lower right.

View larger version (889K): [in this window] [in a new window]   Figure 2. Immunohistochemical staining of fibroblast-like adherent monolayer cells using a three-step streptoavidin-biotin complex immunoperoxidase technique. Controls were treated with secondary antibody alone (A). The positive results were revealed as the following primary antibodies: (B) vWF, (C) VCAM-1, (D) ICAM-1, (E) CD31, (F) E-selectin.

View larger version (13K): [in this window] [in a new window]   Figure 3. Representative FACScan scattergrams for the phenotype of CD34 and CD14 on fibroblast-like adherent cell-derived UCB CD34+ cells.

View larger version (11K): [in this window] [in a new window]   Figure 4. The frequency of endothelial cells in UCB CD34+ cells by limiting dilution analysis. The number of inoculated CB CD34+ cells was plotted on the X axis and the logarithm of the proportion of negative culture endothelial cells was plotted on the Y axis.

	DISCUSSION

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These endothelial cells may be generated from CD34⁺ cells using TPO, FL, and G-CSF because we used highly purified (93.6% ± 4.8%) CD34⁺ cells for culture (Fig. 1), which were consistently shown in the previous two reports by our lab [5, 6], although the possibility that residual endothelial cell fractions from unfractionated UCB-formed endothelial cells cannot be ruled out. Despite the fact that large numbers of endothelial cells subsequently developed in the culture wells, mature endothelial cells were not observed on the cytospins (data not shown). Meanwhile, adherent fibroblast-like cells were negative for macrophage antigens. Estimates of endothelial cell proportions were analyzed by standard limiting dilution assay techniques. The number of tested cells (linear scale) producing 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. Based on this method, one endothelial cell was found from 314 CD34⁺ cells after 5 weeks of culture. In addition, similar approaches of quantification of precursor cells for stromal cells in the CD34⁺ UCB should be done on more purified subsets of CD34⁺ cells (i.e., CD34⁺/CD38^-/Thy1-⁺/Lin-, Rh123^dim).

In our study, the established endothelial cell layers from UCB CD34⁺ cells supported to maintain and amplify the ex vivo expansion of CD34⁺ cells and CFU-GM (p < 0.05) and CFU-GEMM (p < 0.05, data not shown) during the 5 weeks of cultures compared with those without preestablished endothelial cell layers (Table 1). By Philpott et al., the 7-AAD^dim cells have been demonstrated to be apoptotic by morphological observation, DNA gel electrophoresis, and terminal deoxynucleotidyl transferase-mediated deoxynucleotidyl (dUTP) nick end labeling [8]. On a scatter versus 7-AAD fluorescence, the three populations can be discriminated not only from one another but also from cell debris or clumps [5, 8]. The apoptosis of expanded hematopoietic progenitor cells were downregulated in preestablished endothelial cell cocultures groups (Table 2). Traycoff et al. reported that the decline of primitive hematopoietic progenitor cell (HPC) activity during ex vivo expansion of human CD34⁺ cells was proliferation associated and might be a result of apoptosis [12]. Previously, we found some of the apoptotic fractions on at least myeloid differentiation of HCB CD34⁺ cells during ex vivo expansion were positive to CD64 and CD32 [6]. These results suggested that downregulation of apoptosis by endothelial cells that excrete endogenous cytokines during ex vivo expansion could be one of the mechanisms of more effective ex vivo expansion of HPCs. Human umbilical endothelial cells produce many cytokines: macrophage-colony stimulating factor, Kit ligand, fibroblast growth factor, transforming growth factor-ß, IL-1, tumor necrosis factor, IL-4, IL-6, GM-CSF, and G-CSF [13, 14]. However, which cytokines have key roles on apoptosis needs to be elucidated.

There may be some potential clinical applications of the stromal precursor cells for cellular therapies. Lazarus et al. and Liu et al. suggested that reconstitution of bone marrow stromal cells is enhanced by infusion of stromal progenitors [15, 16]. This observation has useful applications to cancer patients who have a severely damaged bone marrow microenvironment by cytotoxic drugs and radiation therapy after high-dose cytotoxic drugs [17]. Brandt et al. reported that the proliferation of HSCs in the presence of cytokines, without stromal cell support, may result in impairment of engraftment capacity, which may be overcome by coculturing with porcine endothelial cell lines [18]. However, further investigations are warranted to investigate if infusion of an ex vivo-generated adherent cell layer enhances the engraftment in patients transplanted with UCB CD34⁺ progenitors. Transplantation of the cultured donor adherent cells together with the ex vivo expanded CD34⁺ CB cells might not only accelerate hematologic recovery but also prevent acute graft-versus-host disease in the allogeneic setting. Moreover, the comparison of the effects produced by adherent cells with respect to other coculture systems (i.e., other stromal cell lines or primary bone marrow stromas) should be done in the future. In conclusion, our study found the establishment of the adherent endothelial cell layer, which does not express the macrophage-type antigens from UCB CD34⁺ cells during ex vivo expansion with TPO, FL, and G-CSF. These newly formed adherent endothelial cell layers seem to provide a better environment for UCB CD34⁺ cells during ex vivo expansion through downregulation of apoptosis. These observations may provide insight for the understanding of UCB transplantation and future cellular therapy or graft engineering.

	ACKNOWLEDGMENT

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This work was supported by grant (No: 2000-1-21100-001-3) from the Basic Research Program of the Korea Science and Engineering and M.O.S.T., Frontier Project (No: SC-02-C2), and the Intramural Research Grant of Ewha Women’s University (E.S. Yoo). We are grateful for the generous gift of G-CSF from Kirin Brewery Co., Tokyo, Japan.

	REFERENCES

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15. Lazarus HM, Haynesworth SE, Gerson SL et al. Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 1995;16:557–564.[Medline]

16. Liu X, Rapp N, Cheng, L. Human mesenchymal stem cells enhance ex vivo expansion of human megakaryocyte, erythroid and myeloid progenitors from purified cord blood CD34⁺ cells. Proceedings from American Society of Hematology Meeting 1998;725a.

17. Carlo-Stella C, Tabilio A, Regazzi E et al. Effect of chemotherapy for acute myelogenous leukemia on hematopoietic and fibroblast marrow progenitors. Bone Marrow Transplant 1997;20:465–471.[CrossRef][Medline]

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