The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions

Endothelial progenitor cells (EPCs) derived from bone marrow are known to be heterogeneous. In this study, we tried to find favorable conditions that induce the differentiation of mononuclear cells (MNCs) from bone marrow into EPCs. The differentiation capacity of MNCs from rat bone marrow was investigated in different conditions, such as different media, different induction times and different culture surfaces. The cell morphology and endothelial biomarkers associated with differentiated MNCs were studied. Our results indicated that MNCs cultured in EGM-2MV (Endothelial cell basal medium-2, plus SingleQuots of growth supplements) developed a bursiform shape, a late EPC-like morphology, while MNCs cultured in complete medium (CM, M199 with 10% FBS, 20 ng/mL VEGF and 10 ng/mL bFGF) showed a spindle shape, an early EPC-like morphology. Cells of both morphologies were able to incorporate DiI-ac-LDL and bind lectin in vitro. MNCs cultured in EGM-2MV exhibited a higher proliferation rate and higher eNOS expression than MNCs cultured in CM. MNCs cultured in EGM-2MV had the ability to form tubes on Matrigel. Flow cytometry results indicated that CD133 expression was highest at day 12 and that the greatest number of cells positive for both FLK-1 and CD133 appeared at day 20 from cells cultured in dishes without fibronectin coating. In addition, the expression levels of CD133, CD31 and FLK-1/CD133 were not significantly different between cells of different shapes. Our experiments suggest that MNCs from bone marrow can be differentiated into late EP-like cells in EGM-2MV, which have the ability to rapidly proliferate. These MNCs can also be differentiated into early EP-like cells in CM. Additionally, fibronectin may not be necessary for the differentiation of EPCs to mature ECs after three generations. Differentiated MNCs from bone marrow in EGM-2MV have the characteristics of EPCs, although the expression levels of EPC markers were lower than previously reported.     

GM-CSF accelerates proliferation of endothelial progenitor cells from murine bone marrow mononuclear cells in vitro

Objective: To test whether the GM-CSF accelerates the proliferation of bone marrow endothelial progenitor cells (BM EPCs). Methods: BM EPCs were induced by endothelial cell conditioned medium (EC-CM). The effect of different concentrations of GM-CSF on the proliferation of BM EPCs was evaluated by the formation of EC-cols, MTT assay, and cell cycle assay. The single progenitor cell growth curves were quantified. Results: The data indicated that GM-CSF accelerated the proliferation of BM EPCs both in colony numbers and colony size. MTT confirmed the effect of GM-CSF on accelerating the proliferation of BM EPCs. The single colony experiments showed that EC-cols expressed different proliferation capacity, suggesting that the EC-cols with different proliferation potentials might have been derived from different levels of immature progenitors. The cell cycle assay showed that the rate of cells entering into S phase was 9.3% in the group treated with GM-CSF and 2.1% in the controls. Furthermore, these cells displayed the specific endothelial cell markers and formed capillary-like structures. Conclusions: GM-CSF accelerates proliferation of BM EPCs. The potential beneficial of GM-CSF in the application of treating vascular ischemic patients is promising.     

Protection of bone marrow progenitor cells by superoxide dismutase

Intravenously administered cuprozinc-superoxide dismutase in X-irradiated mice hastens the recovery of peripheral blood cells. This effect is consistent with protection of the pluripotent stem cells by the enzyme. Amongst the bone marrow cells committed to differentiation along the myeloid pathway, there exists in mice a subpopulation of macrophage progenitor cells that is inactivated by superoxide radicals, generated photochemically or by X-rays. This cell killing effect is inhibited by superoxide dismutase, in part because it acts intracellularly. Human bone marrow also contain a superoxide-sensitive subpopulation of myeloid progenitor cells that is protected by superoxide dismutase but not by catalase. As well, human myeloid progenitor cells contain a subpopulation with enhanced sensitivity to X-rays in vitro. Treatment of these cells with exogenous superoxide dismutase reduces the sensitivity to X-rays by a factor of 2.     

Role of interleukin-1 in 4-hydroperoxycyclophosphamide toxicity to bone marrow progenitor cells: A review

We have demonstrated that in vitro preincubation with IL-1 or TNFa for 20 hours can protect human hematopoietic progenitors from lethal doses of 4-HC. On the other hand, preincubation with IL-6 or IL-3, in a similar fashion, did not provide any protection but in fact demonstrated a slight increase in 4-HC toxicity in the same experiments. The observation that IL-1 was still protective even when a purified cell population depleted of accessory cells was used is suggestive of a direct effect of IL-1. Our data also suggest that early progenitor cells including the replatable B;-CFC are the main target of that protection. We believe that using this in vitro assay system will enable us to investigate the possible mechanisms responsible for the protection of these primitive progenitors. From a clinical perspective, future studies should attempt to clarify whether protection by IL-1 is selective for normal hematopoietic cells versus malignant cells and whether these protected primitive progenitors represent the pluripotent stem cells responsible for engraftment of transplanted bone marrow by using an animal model system.     

Identification of cultured progenitor cells from human marrow stroma

The marrow stromal cells (MSC) are essential for regulation of bone remodeling and hematopoiesis. It is of prime importance to isolate MSC and to expand the proliferating cells ex vivo. In this study, we analyzed cultured MSC for various cellular parameters, including cell morphology, cell cycle, and expression of cell surface antigens by flow cytometry. MSC were divided based on cell size to small (S-cells) and large (L-cells) and were visualized by light and electron microscope. The S-cells were proliferating cells correlated with G0/G1 phase of cell cycle, and expressed cFOS. The expression of surface markers CD-34, -44, -51, -61, -62E, -62P, -62L was quantified using flow cytometry. CD-44 was ubiquitously expressed by S and L cells, CD-51 and -61 were expressed by 30%-38% of S-cells. CD-34 and -62 expressed 20% positive of the analyzed cells that were of the proliferating progenitors (S-cells). This study enables the identification of subpopulations from MSC with special attention paid to the proliferating cells from ex vivo cultures of marrow stroma.     

Enrichment and cryopreservation of bone marrow progenitor cells for autologous reinfusion

From 20 patients with solid tumors or acute nonlymphocytic leukemia in remission, hemopoietic progenitor cells were taken and stored in liquid nitrogen, for use in autologous bone marrow transplantation. Bone marrow aspiration resulted in a volume of 920(+/- 170) ml containing 16.8(+/-6.0) x 10(9) nucleated bone marrow cells and 7.2(+/-4.4) x 10(6) myeloid progenitor cells (CFUc). With use of the Haemonetics blood cell separator a progenitor cell-enriched fraction is obtained. This fraction is depleted of 90(+/-6)% of the erythrocytes and 59(+/-15)% of the neutrophils contained in the original. The original aspirate volume is reduced to one-fifth (21 +/- 3%) while containing 88(+/-38)% of the original CFUc's and 52(+/-11)% of the nucleated bone marrow cells. This technique of bone marrow enrichment has the advantage of a minimum of open-air contact, being independent of extensive laboratory facilities and manpower. The enriched fraction is frozen in autologous plasma and a final concentration of 10% (v/v) DMSO, using a program-controlled freezer (L'Air Liquide). Materials are stored at liquid nitrogen temperature in bags (Gambro) and test vials. Total CFUc recovery in test vials after thawing was 81(+/-32)%.     

Sildenafil attenuates hypoxic pulmonary remodelling by inhibiting bone marrow progenitor cells

The recruitment of bone marrow (BM)‐derived progenitor cells to the lung is related to pulmonary remodelling and the pathogenesis of pulmonary hypertension (PH). Although sildenafil is a known target in PH treatment, the underlying molecular mechanism is still elusive. To test the hypothesis that the therapeutic effect of sildenafil is linked to the reduced recruitment of BM‐derived progenitor cells, we induced pulmonary remodelling in rats by two‐week exposure to chronic hypoxia (CH, 10% oxygen), a trigger of BM‐derived progenitor cells. Rats were treated with either placebo (saline) or sildenafil (1.4 mg/kg/day ip) during CH. Control rats were kept in room air (21% oxygen) with no treatment. As expected, sildenafil attenuated the CH‐induced increase in right ventricular systolic pressure and right ventricular hypertrophy. However, sildenafil suppressed the CH‐induced increase in c‐kit⁺ cells in the adventitia of pulmonary arteries. Moreover, sildenafil reduced the number of c‐kit⁺ cells that colocalize with tyrosine kinase receptor 2 (VEGF‐R2) and CD68 (a marker for macrophages), indicating a positive effect on moderating hypoxia‐induced smooth muscle cell proliferation and inflammation without affecting the pulmonary levels of hypoxia‐inducible factor (HIF)‐1α. Furthermore, sildenafil depressed the number of CXCR4⁺ cells. Collectively, these findings indicate that the improvement in pulmonary haemodynamic by sildenafil is linked to decreased recruitment of BM‐derived c‐kit⁺ cells in the pulmonary tissue. The attenuation of the recruitment of BM‐derived c‐kit⁺ cells by sildenafil may provide novel therapeutic insights into the control of pulmonary remodelling.     

Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow

There is great interest in the therapeutic potential of non-hematopoietic stem cells obtained from bone marrow called mesenchymal stem cells (MSCs). Rare myogenic progenitor cells in MSC cultures have been shown to convert into skeletal muscle cells in vitro and also in vivo after transplantation of bone marrow into mice. To be clinically useful, however, isolation and expansion of myogenic progenitor cells is important to improve the efficacy of cell transplantation in generating normal skeletal muscle cells. We introduced into MSCs obtained from mouse bone marrow, a plasmid vector in which an antibiotic (Zeocin) resistance gene is driven by MyoD and Myf5 enhancer elements, which are selectively active in skeletal muscle progenitor cells. Myogenic precursor cells were then isolated by antibiotic selection, expanded in culture, and shown to differentiate appropriately into multinucleate myotubes in vitro. Our results show that using a genetic selection strategy, an enriched population of myogenic progenitor cells, which will be useful for cell transplantation therapies, can be isolated from MSCs.     

Purification and growth of endothelial progenitor cells from murine bone marrow mononuclear cells

This study reports the culture and purification of murine bone marrow endothelial progenitor cells (EPCs) using endothelial cell-conditioned medium (EC-CM). Endothelial-like cells appeared at day 5 in culture of bone marrow mononuclear cells in the presence of EC-CM in the culture system, and these cells incorporated acetylated low-density lipoproteins (Ac-LDL) and reacted with endothelial-specific Ulex Europaeus Lectin. Continued incubation of these cells at low density with EC-CM for longer than 10 days resulted in the formation of endothelial cell colonies which gave rise to colonies of endothelial progeny and can be passed for many generations in the EC-CM culture system. Cells derived from these colonies expressed endothelial cell markers such as vWF and CD31, incorporated Dil-Ac-LDL, stained positive for Ulex Europaeus Lectin, formed capillary-like structures on Matrigel, and demonstrated a high proliferative capacity in culture. These bone marrow-derived adherent cells were identified as EPCs. The purification and the formation of EPC colonies by using EC-CM were associated with the cytokines secreted in the EC-CM. VEGF, bFGF, and GM-CSF in the EC-CM stimulated the proliferation and growth of EPCs, whereas AcSDKP (tetrapeptide NAc-Ser-Asp-Lys-Pro) in EC-CM suppressed the growth of mesenchymal stem cells (MSC) and fibroblasts. This approach is efficient for isolation/purification and outgrowth of bone marrow EPCs in vitro, a very important cell source in angiogenic therapies and regenerative medicine.     

肝细胞生长因子对骨髓内皮祖细胞的动员作用

目的: 分析肝细胞生长因子(HGF)能否动员骨髓内皮祖细胞,以及动员的内皮祖细胞能否参与创伤修复时的血管新生和内皮修复.方法: 将腺病毒HGF载体(adenovirus vector encoding HGF gene, Ad-HGF)经尾静脉注射到Balb/c小鼠体内,用ELISA方法检测血浆HGF水平的变化;用流式细胞术检测外周血CD34 细胞含量变化;对外周血单个核细胞进行分离、培养,并对生长的细胞克隆进行内皮细胞表面标志Tie-2、vW因子的免疫组化检测.建立雌性小鼠CCl4肝损伤模型,静脉移植HGF处理后雄性小鼠外周血单个核细胞到其体内,4 W后利用原位杂交技术检测新生肝组织中是否存在雄性细胞.结果: 注射Ad-HGF能明显提高小鼠血浆的HGF水平,并使外周血中以CD34、Tie-2和vW因子等为标志的内皮祖细胞的数量显著增多.这些细胞参与肝损伤修复时的血管新生.结论: HGF对骨髓内皮祖细胞具有明显的动员作用.     

Differential analysis of animal bone marrow by flow cytometry.

A simple procedure was developed for rapid analysis of animal bone marrow by flow cytometry using the lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]. The batch process allows differentiation of bone marrow cells into lymphoid, erythroid, and myeloid populations and enables classification of erythroid and myeloid cells into proliferating and maturing subpopulations. From these data, myeloid:erythroid (M:E) ratios and maturation indices for erythroid and myeloid cells (EMI and MMI, respectively) can be derived. This procedure provides the opportunity to analyze bone marrow quantitatively and offers distinct advantages to current manual methods in terms of simplicity, throughput, and reproducibility. The method has been tested successfully using marrow from Wistar rats, B6C3F1 mice, beagle dogs, and cynomolgus monkeys. This technique facilitates the evaluation of bone marrow samples taken from preclinical safety studies or from animal colonies of large size.     

Differentiation of adult rat bone marrow stem cells into epithelial progenitor cells in culture

We have previously obtained monoclonal bone marrow stem cells from adult rats (rMSCs) and induced them into phenotypic neurons. In the present study, we aimed to induce rMSCs into epithelial cells by culturing them onto compartmentalized permeable supports, which have been used for growing a variety of polarized epithelia in culture. Hematoxylin staining showed that after 4 days grown on permeable supports, rMSCs formed an epithelial-like monolayer. Immunofluorescence of the permeably-supported monolayers, but not the rMSCs grown in culture flasks, showed positive signals for epithelial markers, cytokeratin 5 & 8. RT-PCR results also showed the mRNA expression of epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) as well as tight junction protein ZO-1 in the rMSC-derived monolayers grown on permeable supports but absent from those grown in culture flasks. However, western blot only detected protein expression of ZO-1 but not ENaC nor CFTR. The short-circuit current measurements showed that the rMSC-derived monolayers grown on permeable supports exhibited a trans-monolayer resistance of 30-50 Omega cm(2); however, the monolayers did not respond to activators or blockers of CFTR or ENaC. The results suggest that compartmentalized or polarized culture conditions provide a suitable environment for rMSCs to differentiate into epithelial progenitor cells with tight junction formation; however, this condition is not sufficient for functional expression of epithelial ion channels associated with well-differentiated epithelia.     

The bone marrow plasma cell labeling index by flow cytometry

The bone marrow plasma cell labeling index is the most important prognostic indicator for patients with multiple myeloma. Traditionally, this test has been performed as a two color immunofluorescent microscope technique which is time consuming and requires a degree of subjectivity in its interpretation. We have assessed various adaptations of this method to flow cytometry. A bromodeoxyuridine method has been compared with a propidium iodide DNA method to detect cells in S phase and CD38-FITC has been compared with CD38-FITC + CD138-FITC and CD38-biotin + streptavidin FITC to identify plasma cells. The mean channel fluorescent intensity of the plasma cell peaks for each of these markers was 12. 7, 17.4 and 35.3 respectively demonstrating the superiority of CD38-biotin + streptavidin FITC. Analysis after propidium iodide staining provided a good correlation with the slide technique (r = 0. 71; P < 0.0001) but the bromodeoxyuridine method did not correlate with the slide method (r = 0.09; P = NS). The labeling index values obtained from either of the flow methods were greater than the microscopic method. Thus a labeling index of >4% will replace the traditional >1% threshold for identifying patients with a significantly increased labeling index. The advantages of the new method are that it takes less time to perform, is more objective and provides additional data on ploidy and cell cycle status.     

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hematopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen I, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis.     

Phenotypic analysis of a large number of normal human bone marrow sample by flow cytometry

Bone marrow aspirates from 48 healthy donors (34 adults, 14 children) were analyzed by flow cytometry (FACS Analyzer) after purification of low-density bone marrow cells (Ld BMC) on a density gradient (d = 1,077) and labelling with 23 anti-hematopoietic cell monoclonal antibodies. Based on physical properties, these Ld BMC could be divided into four different populations called E, My, Mo and L, which comprised 14% +/- 9%, 31% +/- 16%, 10% +/- 5% and 45% +/- 14% of these cells, respectively. The phenotypic analysis of these different populations enabled the identification in E, of erythrocytes (Glycophorin A+, Rhesus D+, but negative for early erythroid differentiation markers such as the transferrin receptor (Tf. R) and the FA6-152 antigen); in My of cells of the myeloid lineage (VIM2+, HLA DR-); in Mo of cells of the monocytic lineage (VIM2+, CD14+) plus some myeloblasts (VIM2+, CD14-, HLADR+) and finally in L of a heterogeneous population including: 1. T lymphocytes labelled to the same extent by CD2, CD3, CD5 and CD6 (28% +/- 10%), B lymphocytes assessed by CD19 and CD20 (12% +/- 8%), Pre-B cells (CD10+ = 8% +/- 7%), less than 5% of "natural killer" cells (CD16+ or Leu7+) and finally, less than 6% of myelomonocytes (CD14+ and/or VIM2+). 2. The erythroid lineage (rhesus D+ = 42% +/- 20%, Tf.R+ and FA6-152+ = 32% +/- 12%). 3. Undifferentiated cells or progenitor cells (CD34+ = 7% +/- 5%). 4. Cells unlabelled by any antibodies (approximately 6%). We observed no difference between bone marrow samples from adults or children, with respect to physical properties, and with all but four immunological markers. A significantly higher proportion of B cells (CD19+ and CD10+) (P less than 0.001) and undifferentiated cells (CD34+ and HLADR+) (P less than 0.02) was observed in children. These data, obtained from a large number of bone marrow samples, could be used to quantify the imbalance of some bone marrow disorders.     

Human mesenchymal progenitor cells derived from alveolar bone and human bone marrow stromal cells: a comparative study

The aim of the present study was to evaluate the potential of intraoral harvested alveolar bone as an alternative source of multipotent mesenchymal stromal cells for future applications in oral and maxillofacial tissue engineering. Explant cultures were established from 20 alveolar bone samples harvested from the oblique line immediately before wisdom tooth removal. Morphology and proliferation characteristics of the in vitro expanded cells, referred to as human alveolar bone-derived cells (hABDCs), were studied using phase-contrast microscopy. Immunocytochemical analysis of their surface marker expression was conducted using monoclonal antibodies defining mesenchymal stromal cells. To evaluate their multilineage differentiation potential, hABDCs were induced to differentiate along the osteogenic, adipogenic, and chondrogenic lineage and compared to bone marrow mesenchymal stromal cells (hBMSCs) on mRNA and protein levels applying RT-PCR and cytochemical staining methods. hABDCs showed typical morphological characteristics comparable to those of hBMSCs such as being mononuclear, fibroblast-like, spindle-shaped, and plastic adherent. Immunophenotypically, cells were positive for CD105, CD90, and CD73 while negative for CD45, CD34, CD14, CD79α, and HLA-DR surface molecules, indicating an antigen expression pattern considered typical for multipotent mesenchymal stromal cells. As evidenced by RT-PCR and cytochemistry, hABDCs showed multilineage differentiation and similar chondrogenic and osteogenic differentiation potentials when compared to hBMSCs. Our findings demonstrate that human alveolar bone contains mesenchymal progenitor cells that can be isolated and expanded in vitro and are capable of trilineage differentiation, providing a reservoir of multipotent mesenchymal cells from an easily accessible tissue source.     

Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells

Bone marrow stroma provides the microenvironment for hematopoiesis and is also the source of mesenchymal progenitors (mesenchymal or marrow stromal cells [MSC]) that may serve as long-lasting precursors for bone, cartilage, lung, and muscle. While several studies have indicated the differentiation potential of MSC, few studies have been performed on the cells themselves. In an attempt to further expand our knowledge on these cells, we have performed studies on their cell cycle, immuno- and adhesive-phenotype, ex vivo expansion, and differentiation properties. MSC cultures have been initiated from human bone marrow low-density mononuclear cells and maintained in the absence of differentiation stimuli and hematopoietic cells. The homogenous layer of adherent cells thus formed exhibits a typical fibroblastlike morphology, a population doubling time of 33 h, a large expansive potential, and cell cycle characteristics including a subset (20%) of quiescent cells. The antigenic phenotype of MSC is not unique, borrowing features of mesenchymal, endothelial, and epithelial cells. Together, MSC express several adhesion-related antigens, like the integrin subunits α4, α5, β1, integrins αvβ3 and αvβ5, ICAM-1, and CD44H. MSC produce and functionally adhere to extracellular matrix molecules. When incubated under proper stimuli, MSC differentiate into osteoblasts or adipocytes. Taken together, these results demonstrate that adherent marrow-derived cells cultured in the absence of hematopoietic cells and differentiation stimulus give rise to a population of cells with phenotypical and functional features of mesenchymal progenitors. The existence of a subset of quiescent cells in MSC cultures seems to be extremely significant, since their number and properties should be enough to sustain a steady supply of cells that upon proliferation and commitment may serve as precursors for a number of nonhematopoietic tissues. J. Cell. Physiol. 181:67–73, 1999. © 1999 Wiley-Liss, Inc.     

In vitro differentiation characteristics of cultured human mononuclear cells-implications for endothelial progenitor cell biology

Endothelial progenitor cells (EPCs) have been implicated in the pathogenesis and treatment of cardiovascular disease. By use of quantitative uptake of DiLDL and lectin staining, EPCs have been characterized reliably. However, the exact nature and function of this cell population still remains poorly defined. In an attempt to further clarify the cell surface characteristics of EPCs, mononuclear cells (MNCs) were isolated from human blood and cell surface expression patterns were defined by FACS analysis before and after differentiation for 1-10 days in cell culture. "Classical" double staining for DiLDL and Ulex europaeus increases to 89.2 /- 0.05 after 10 days in culture. Looking at EPC-specific markers by FACS analysis, 0.18 +/- 0.11% of freshly isolated MNCs express CD34, 0.13 +/- 0.08% CD133, 0.59 +/-0.51% VEGFr2, 0.01 +/- 0.02% CD34/VEGFr2, 0.09 +/- 0.05% CD34/CD133, 0.58 +/- 0.13% CD34/CD31, and 0.02 +/- 0.01% CD34/CD146, respectively. Induction of the endothelial phenotype is evidenced by positive staining for VEGFr2, CD146, and CD31, and occurs in co-expression with stem cell markers in less than 2 +/- 0.52% of cultured cells. Expression of CD34 increases to 0.38 +/- 0.10% after 10 days, whereas the CD133(+) cell population shows an initial peak at 24h (0.29 +/- 0.18%) before decreasing to 0.15 +/- 0.02% at day 10. EPCs co-expressing CD34/CD133 increase to 0.19 +/- 0.09% after 10 days, and EPCs double-positive for CD34/VEGFr2 increase to 1.45 +/- 1.03%. Looking at leukocyte, lymphocyte, and monocyte lineage markers, 56.27 +/- 0.15% of freshly isolated MNCs express CD45, 7.13 +/- 0.02% CD14, and 38.65 +/- 0.01% CD3. Over the 10-day culture period, expression of CD45 decreases to 28.48 +/- 0.18%, CD3 to 23.11 +/- 0.02%, and CD14 to 0.09 +/- 0.02%. Cells co-expressing CD3/CD45 decrease from 38.88 +/- 0.33% to 24.86 +/- 2.49% after 10 days in culture. These findings extend present knowledge by showing that human MNCs differentiate at a very low rate to EPCs, while a majority of the cultured cell population remain committed to the leukocyte or lymphocyte lineage. Careful surface marker analysis might be necessary when using in vitro EPC differentiation systems.