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Cardiovascular Research 2006 70(1):126-135; doi:10.1016/j.cardiores.2006.01.014
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Copyright © 2006, European Society of Cardiology

Generation of CD133+ cells from CD133– peripheral blood mononuclear cells and their properties

Erik J. Suuronena, Serena Wonga,c, Varun Kapilaa, Geeta Waghraya, Stewart C. Whitmanb,c, Thierry G. Mesanaa and Marc Ruela,c,*

aDivision of Cardiac Surgery, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario, Canada K1Y 4W7
bPathology and Laboratory Medicine, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario, Canada K1Y 4W7
cCellular and Molecular Medicine, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario, Canada K1Y 4W7

* Corresponding author. Division of Cardiac Surgery, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario, Canada K1Y 4W7. Tel.: +1 613 761 4893; fax: +1 613 761 5367. Email address: mruel{at}ottawaheart.ca

Received 25 August 2005; revised 11 January 2006; accepted 13 January 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 References
 
Objective CD133 may be the most specific marker of endothelial progenitor cells (EPCs), which are thought to be largely confined to the bone marrow milieu. This study reports on the phenotypic characterization and functional analysis of human CD133+ cells and their generation from cells in the peripheral circulation.

Methods Adult human CD133+ and CD133– cells were isolated from peripheral blood mononuclear cells, and the generation of CD133+ cells in culture was attempted using different culture combinations. The phenotypic, migratory, adhesive, and angiogenic properties of the native and generated populations were investigated.

Results In adherent and in suspension culture systems, CD133+ cells also expressing CD34 and VEGFR-2 were successfully derived from a previously CD133– population. The migratory potential of CD133+ cells was enhanced by the presence of the CD133– cells. Also, the CD133+ cells derived from the CD133– cells demonstrated improved adhesion to extracellular matrix and endothelial monolayer substrates, and their contribution to in vitro angiogenesis was enhanced compared to freshly isolated CD133+ cells.

Conclusions These results demonstrate a source of blood CD133+ cells other than direct mobilization from the bone marrow. Cellular interaction was observed between fractions, with CD133+ cells showing better in vitro function in the presence of CD133– cells. These findings provide a novel source for CD133+ cells and a rationale for the investigation of angiogenic cell recruitment or delivery strategies involving more than one cell type at ischemic sites.

KEYWORDS Angiogenesis; Cell differentiation; Stem cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 References
 
The regeneration or formation of blood vessels in the adult heart may occur simultaneously by both vasculogenesis and angiogenesis [1]. Endothelial progenitor cells participate in both processes and are important for vascular repair and maintenance [2]. Specifically, endothelial progenitors have been shown to contribute to adult blood vessel formation, to be involved in neovascularization after ischemic events, and to participate in vascular homeostasis [3,4]. For example, patients at risk for coronary artery disease have decreased numbers of circulating endothelial progenitors, whose activity may also be impaired [5]. Impaired progenitor activity is also associated with age-related endothelial dysfunction [6]. Recently, we showed that procedural modifications can enhance the functional preservation of endothelial progenitor cells during coronary artery bypass surgery [7].

The majority of endothelial progenitor cells originate from the bone marrow, where they exist in a quiescent state. These cells, upon specific stimulation, are mobilized into the systemic circulation. The endothelial progenitor cell has been characterized by the expression of CD34, CD133 and vascular endothelial growth factor receptor-2 (VEGFR-2) [8,9]. Multipotent adult progenitor cells expressing CD133 and VEGFR-2, but not CD34, are possible origins of the circulating EPC [10]. CD34 is not expressed exclusively on progenitor cells, but also on mature endothelial cells and CD133 is absent on mature endothelial and monocytic cells [9]. It is therefore believed that CD133 is a more specific marker of stem cells than CD34 [11].

Other cells within the peripheral blood contribute to vascular formation and may be involved in regulating the function of endothelial progenitor cells. Subsets of human peripheral blood monocytes have been identified with the ability to differentiate into, amongst others, endothelial cells [12,13]. In addition, bone marrow monocytic lineage cells can adhere to regions of injured endothelium and accelerate re-endothelialization by endothelial progenitors [14,15]. Recently, it was demonstrated that interactions with the cellular microenvironment influence the surface receptor expression and function of CD34+ cells in the peripheral circulation [16]. The relationship between the cell types within the peripheral blood and how they relate to angiogenic potential of CD133+ cells remains to be investigated, and constitutes a focus of the present study.

A previous report demonstrated that non-adherent CD133+CD34– cells could be generated from a population of cultured adherent CD133+ cells and repopulate SCID mice [17]. We sought to examine the characteristics and relationship between the CD133+ and CD133– fractions of the mononuclear cells from the peripheral blood. Here we show that CD133+ cells can be generated from the CD133– population of peripheral blood mononuclear cells, both in suspension and in adherent culture systems. The CD133+ cells derived from the CD133– cells demonstrated improved adhesive and angiogenic properties compared to freshly isolated CD133+ cells. In addition, interaction with the CD133– cells affected the function of the CD133+ population.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 References
 
2.1 Cell isolation and culture
The study was approved by the Human Research Ethics Board of the University of Ottawa Heart Institute and conformed to the Declaration of Helsinki. Informed consent was obtained from all participants. Total peripheral blood mononuclear cells (PBMCs) were isolated from healthy adults, as described previously [7]. CD133+ cells were separated from PBMCs using CD133-bound microbeads and a magnetically activated cell sorter (autoMACS; Miltenyi Biotec, Bergisch-Gladback, Germany) following the manufacturer's protocol. Purity of the sorted CD133+ cells was 97.1±0.9% and effectively 100.0±0.0% for the CD133– cells. To maintain the natural cellular environment, initial culture work was performed based on ratios of CD133+/ – cells equivalent to that of the original PBMC isolate as follows:



Formula

Fractions were then cultured on fibronectin-coated 12-well culture plates in the following combinations: a) 3 million CD133– cells; b) X million CD133+ cells; c) 3 million CD 133– cells+X million CD133+ cells; and d) control cultures of 3 million non-separated PBMCs. Cultures were supplemented with endothelial basal medium (EBM-2; Clonetics, Guelph, Canada) with EGM-2-MV-SingleQuots (Clonetics) as described [7]. Cultures were maintained for 2 weeks with two different protocols: protocol one investigated the adherent and non-adherent cells, and protocol two investigated the adherent population only. Cells in protocol one had fresh media added without aspiration, whereas cells in protocol two had fresh media added every 2 days after aspiration of the old media. After 2 weeks, all cells were lifted and separated by autoMACS using the CD133 microbeads and cell numbers in each fraction were determined by a cell counter (Beckman Coulter, Mississauga, Canada). Due to variability in the initial number of CD133+ cells between donors, data are expressed relative to the initial number of CD133+ isolated for each donor. Since it was pre-determined that control and recombined fractions were not significantly different in cell number or CD133+/ – ratio (data not shown), only autoMACS-separated cells were used in functional analyses.

2.2 Proliferation in suspension
A ratio of CD133+ and CD133– cells equivalent to the original PBMC isolate were used for the following cultures in 10 ml of EBM medium in polypropylene tubes: a) unsorted control; b) CD133+ cells; c) CD133– cells; and d) CD133+ and CD133– cells. After 48 h incubation at 37 °C in a water bath, placed on a shaker to prevent cell adhesion or clotting, cells were centrifuged and separated by autoMACS using the CD133 marker. Cell numbers and viability were then determined as above.

2.3 Migration assay
Assays were performed using VEGF (50 ng/ml; Sigma) as a migratory stimulus for 24 h as described previously in Ref. [7]. Using freshly isolated cells, the experimental groups were: a) CD133+ cells; b) CD133– cells; c) combined CD133+ and CD133– cells (in a ratio equivalent to the original separation ratio); and d) CD133+ cells with the supernatant from 24-h cultured CD133– cells. These groups (except for d) were also tested using CD133+ cells derived from the CD133– fraction after 2 weeks of culture (hereafter referred to as 2-week derived CD133+ cells). CD133+ and CD133– cells were labeled with the fluorescent dye CellTracker Orange and CFDA (both from Molecular Probes, Eugene, OR, USA), respectively. For quantification, migrated cells were counted in six random microscopic fields in a blinded fashion.

2.4 Cytokine antibody array
Raybio® human cytokine antibody array V (RayBiotech, Norcross, GA, USA) was used to assay over 75 proteins in the supernatants of CD133– cell cultures. Specifically, differences in the soluble factors were assayed by comparing the growth factor and cytokine levels within culture media and the supernatant of cultured cells (Kodak 1D imager; Kodak, Rochester, NJ, USA). Protein levels were quantified against internal controls in the array and against other samples as fold increases.

2.5 Adhesion assay
Twenty-four-well culture plates were coated with type I collagen (100 µg/ml; Becton-Dickinson, Oakville, Canada) or fibronectin (100 µg/ml; Sigma) for 1 h at 37 °C. Wells were then blocked with 1% bovine serum albumin (BSA) for 1 h and 2 x 104 cells, labeled as above, and the following were added to the wells: a) CD133+ cells; b) CD133– cells; c) combined CD133+ and CD133– cells (1 x 104 each); and d) 2-week derived CD133+ cells. The cells were allowed to attach for 1 h at 37 °C and the medium and non-adherent cells were then aspirated. The adherent population was fixed with 4% paraformaldehyde (PFA) for 20 min and stored in 0.1 M PBS. The numbers of cells were quantified from counts in 6 random microscopic fields.

For adhesion to endothelial cells, a monolayer of human umbilical vein endothelial cells (HUVECs) was established 48 h prior to the assay by plating 2 x 104 cells in 24-well plates. Cultures were supplemented with M199 medium (Invitrogen, Burlington, Canada) containing 10% FBS (Invitrogen), 90 mg/l heparin (Sigma), 2 mM L-glutamine (Invitrogen), 50 µg/ml endothelial cell growth supplement (Sigma) and 5 mg/ml gentamycin (Invitrogen) termed HUVEC medium. HUVECs were treated with TNF-{alpha} (1 ng/ml; Sigma) for 12 h prior to the addition of the CD133 fractioned cells. Then, 2 x 104 labeled cells were added to the wells in the following groups: a) CD133+ cells; b) CD133– cells; c) combined CD133+ and CD133– cells (1 x 104 each); and d) 2-week derived CD133+ cells. The cells were incubated for 3 h at 37 °C. The medium and non-adherent cells were then aspirated and the adherent population was fixed with 4% PFA for 20 min and stored in 0.1 M PBS. The numbers of cells were quantified from counts in 6 random microscopic fields.

2.6 In vitro angiogenesis assay
ECMatrix mix (Chemicon, Temecula, CA) was prepared as per the manufacturer's protocol and 80 µl of the mix was added into wells of a 96-well plate and incubated at 37 °C for 1 h. As reported previously [18], the progenitor cells were seeded on the matrix with an endothelial cell population. All matrices were seeded with 1 x 104 HUVECs and 1 x 104 cells of interest (labeled as above) were added to the wells in the following groups: a) CD133+ cells; b) combined CD133+ and CD133– cells (0.5 x 104 each); and c) 2-week derived CD133+ cells. Cultures were supplemented with EBM medium+10% FBS and incubated at 37 °C for 24 h. The number of cells contributing to in vitro tubule formation was then determined by counting the number of labeled cells in capillary-like structures in 6 random microscopic fields.

2.7 Flow cytometry
To examine cell phenotype, cells were examined by flow cytometry as described previously [7]. Cells were labeled for 20 min with mouse antihuman antibodies against different combinations of the following antigens: CD31, CD34, CD133, VEGFR-2, and vascular endothelial (VE) cadherin. Antibodies were from Beckman Coulter (Mississauga, ON, Canada) except CD133 (Miltenyi Biotec) and VEGFR-2 (R&D Systems, Minneapolis, MN, USA). Cells were analyzed by Cytomics FC500 (Beckman Coulter). In controls, cells were incubated with mouse immunoglobulin G conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC).

2.8 Statistical analysis
Values are expressed as mean±standard error of the mean. Statistical analyses were performed in Intercooled Stata 8 (Stata, College Station, TX, USA). Comparisons of continuous data between groups were performed with a two-tailed Student's t test, using Bonferroni corrections as appropriate. Probability values of P<0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
References
 
Culture of CD133+ and CD133– cells
3.1.1 Percentage CD133+ cells in culture relative to the initial number isolated
For the combined adherent and non-adherent cells (protocol 1), all cultures showed a loss of CD133+ cells after 2 weeks, except for the CD133– group (Fig 1A). CD133+ cells cultured alone resulted in the loss of all but a very small fraction of CD133+ cells (3±1% remaining). The PBMC control and mixed CD133+/ – groups showed losses of CD133+ cells to 21±9% and 43±11% of the initial CD133+ numbers, respectively. Seeding CD133– cells resulted in the generation of CD133+ cells equivalent to 68±23% of the initial CD133+ numbers in the original PBMC isolate.


Figure 1
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  		Fig. 1 CD133 expression for PBMC control, CD133+, CD133– and CD133+/ – groups after culture in different conditions. (A) The number of CD133+ cells in cultures of combined adherent+non-adherent cells on fibronectin, expressed as a percentage relative to the initial number of CD133+ cells in the original PBMC sample. *P<0.001 versus other culture conditions at 2 weeks. (B) The proportion of cells that express CD133 in different culture conditions of adherent±non-adherent cells on fibronectin. *P<0.05 versus other cell types within culture condition. (C) The number of CD133+ cells after culture for 48 h in suspension. *P<0.01 versus other groups. (D) The proportion of cells that express CD133 after suspension culture. *P<0.05 versus other groups; {blacksquare}P<0.05 versus PBMC group.

 
3.1.2 Proportion of CD133+ cells in culture
The proportion of cells taken from 2-week cultures that expressed CD133 was also determined (Fig 1B). For the combined adherent and non-adherent population (protocol 1), the proportion of cells that were CD133+ was equivalent for three groups (4.9±1.8%, 12.3±3.2% and 8.6±1.4% for PBMC control, CD133– and CD133+/ – groups, respectively) and were significantly greater than percentages of CD133+ cells initially seeded. However, the group of CD133+ cells cultured alone had a loss in the proportion of CD133+ cells (31.5±8.9%) compared to initial seeding (100%), but this proportion was still greater than that of the other groups. In looking at the adherent cells alone (protocol 2), the proportion of cells that were CD133+ again increased in the PBMC control, CD133– and CD133+/ – groups (12.6±3.7%, 33.6±5.0% and 31.6±2.6%, respectively) after 2 weeks of culture. In comparing the combined adherent and non-adherent population versus the adherent population alone, the constant removal of the non-adherent population of cells resulted in a greater proportion of cells expressing CD133 in the CD133+, CD133– and CD133+/ – groups, but not in the PBMC controls (Fig. 1B). Therefore, in the absence of non-adherent cells, the proportion of cells expressing CD133 increased. In both protocols, the culture of CD133+ cells alone resulted in the generation of CD133– cells, as expected [17].

3.2 Generation of CD133+ cells in suspension
After 48 h incubation in suspension, the number of CD133+ cells in each culture group was examined (Fig. 1C). The CD133– cell population generated CD133+ cells (to 36±15% of the initial CD133+ numbers) equivalent in number to that of the CD133+/ – mixed (42±18%) and unsorted control (36±18%) populations (P=1.0 and 0.8, respectively). Very few CD133+ cells (0.4±0.3% of the initial CD133+ numbers) remained when CD133+ cells were cultured alone. The proportion of the cells in suspension culture that were CD133+ was also determined (Fig. 1D). The proportion of cells in the CD133+ culture group that remained positive for the expression of CD133 was 17.9±9.0%, indicating that approximately 82% of the cells in this population lost CD133 expression. The proportion of CD133+ cells in the CD133– and CD133+/ – groups were equivalent (2.0±0.6% and 3.6±1.7%, respectively). These values were all greater than that observed for the control unsorted group (0.7±0.2%).

3.3 Cell phenotype analysis by flow cytometry
Flow cytometry was performed to characterize the phenotype of the 2-week derived CD133+ cells. The 2-week derived cells were gated on the expression of CD133+ (Fig. 2A) and flow analysis demonstrated that 88.5±3.8% were also positive for CD34 and VEGFR-2 expression (Fig. 2B). Flow analysis was also performed to examine VEGFR-2 expression of the CD133– cells to better define the population that gave rise to the derived CD133+ cells. At the time of isolation, 4.16±2.03% of the CD133– cells expressed VEGFR-2 (Fig. 2C and D) and after 2 weeks of culture, expression increased to 28.51±5.19% (Fig. 2E and F). Within the 2-week CD133– population, a shift was observed along the CD133 axis resulting in 2 peaks. Most of the CD133– cells (95%) located within the population shift towards CD133 positivity also expressed VEGFR-2 (Fig. 2G and H).


Figure 2
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  		Fig. 2 Phenotypic flow cytometry characterization of CD133+ and CD133– cells. Representative analysis of: (A) CD133 expression on CD133– cells after 2 weeks of culture demonstrating generation of CD133+ cells. (B) Two-week derived CD133+ cells co-express VEGFR-2 and CD34 (~89%). (C, D) CD133– cells at isolation (t=0) show little VEGFR-2 expression (~4%). (E, F) CD133– cells after 2 weeks of culture express more VEGFR-2 (~29%). (G, H) CD133– cells within the small shift along the CD133 fluorescence intensity axis greatly co-express VEGFR-2 and CD34. All data was analyzed from 150,000 counted events.

 
3.4 Cell migration, cytokines and growth factors
For freshly isolated CD133+ and CD133– cells plated separately at day 0, the number of migrating CD133+ cells per high power field (5.2±0.7; Fig. 3A) was significantly less than the number of migrating CD133– cells (65±7.6; Fig. 3B). The number of migrating CD133+ cells was significantly increased when plated in combination with the CD133– fraction or its supernatant (26.9±1.6 and 29.6±2.6, respectively; Fig. 3C and D). This was also observed with 2-week derived CD133+ cells (Fig. 3D) when comparing these cells tested alone (3.9±0.3) versus those cultured with the CD133– fraction (18.2±2.8).


Figure 3
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  		Fig. 3 Migration of CD133+ cells. Representative migration assay images of (A) CD133+ cells plated alone, (B) CD133– cells alone, and (C) the co-culture of CD133+ and CD133– cells. Scale bar=75 µm. (D) The number per high-powered field (HPF) of migrating CD133+ cells, freshly isolated (0 days) or derived after 2 weeks of culture (2-week cells) when cultured alone or with the CD133– cells or its supernatant. *P<0.01 versus in combination and supernatant cultures. Representative cytokine array for (E) culture medium and (F) CD133– cell culture supernatant after 24 h incubation.

 
The supernatant of the cultured CD133– cells contained elevated levels of several cytokines and growth factors compared to the control medium (Fig. 3E and F). Among these were included (fold increase in parentheses, P<0.05): GRO (2.6); IL-1 β (2.1); IL-6 (28.0); IL-8 (16.3); IL-10 (7.9); MCP-1 (10.8); MIP-1β (2.6); and RANTES (3.9).

3.5 Cell adhesive properties
The adhesive properties of CD133+ cells were tested on the ECM proteins fibronectin and collagen, and also on a monolayer of HUVECs. Freshly isolated CD133+ cells adhered to fibronectin (Fig. 4A), and the number of attached cells was not affected by co-culture with the CD133– fraction (Fig. 4B). The 2-week derived CD133+ cells also demonstrated adhesion to fibronectin (Fig. 4C). For collagen, freshly isolated CD133+ cells adhered equally when cultured alone (Fig. 4D) or in combination with the CD133– fraction (Fig. 4E). The 2-week derived CD133+ cells demonstrated the greatest adhesion to collagen (Fig. 4F). Upon culture on a monolayer of HUVECs, adhesion of the freshly isolated CD133+ cells cultured alone (Fig. 4G), or with the CD133– cells (Fig. 4H) was observed. Adhesion to the monolayer was greatest for the 2-week derived CD133+ cells (Fig. 4I).


Figure 4
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  		Fig. 4 Adhesion potential of CD133+ cells. Examples of adhesion of CD133+ cells (red) and CD133– cells (green) to (A–C) fibronectin (FN), (D–F) collagen (COLL) and (G–I) a monolayer of HUVECs after 1 h (fibronectin and collagen) or 3 h (HUVECs) of culture. (A, D and G). Freshly isolated CD133+ cells; (B, E and H) CD133+ cells (t=0) with CD133– cells; and (C, F and I) 2-week derived CD133+ cells. Scale bar=75 µm. (J) Number of adherent CD133+ cells per HPF on different substrates with different CD133+ populations and culture conditions. *P<0.05 versus all substrates and cell groups; {blacksquare}P<0.01 versus all other cell groups within substrate.

 
The 2-week derived CD133+ cells demonstrated significantly greater adhesion to fibronectin, collagen, and the cell monolayer than the freshly isolated CD133+ cells cultured alone or with CD133– cells (Fig. 4J). Only a minimal number of the 2-week derived CD133+ cells expressed the known endothelial adhesion proteins CD31 (0.79±0.07%) and VE-cadherin (0.46±0.17%) as determined by flow cytometry. For the 2-week derived CD133+ cells, significantly greater binding efficiency was observed to collagen than to fibronectin or the endothelial monolayer. Also, their co-culture with CD133– cells had no effect on adhesion (data not shown). For freshly isolated CD133+ cells, there was no difference in adhesion to fibronectin or collagen (Fig. 4J).

3.6 CD133+ cells and angiogenic potential
In in vitro angiogenesis assays, CD133+ cells did not result in the formation of capillary-like structures when cultured alone (data not shown). Co-culture with HUVECs was necessary for a contribution from CD133+ cells (Fig. 5A). CD133– cells also participated in capillary-like structure formation when plated with the HUVECs and CD133+ cells (Fig. 5B). The 2-week CD133+ cells derived from the CD133– fraction also contributed to the formation of capillary-like structures (Fig. 5C). Contribution to angiogenesis was significantly greater from the 2-week derived CD133+ cells than from freshly isolated CD133+ cells cultured alone or with the CD133– cells (Fig. 5D). The presence of CD133– cells had no effect on the contribution of CD133+ cells to capillarization.


Figure 5
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  		Fig. 5 Contribution of CD133+ cells to in vitro angiogenesis. Representative images of the participation of CD133+ cells (red) and CD133– cells (green) to the formation of capillary structures using HUVECs seeded along with (A) freshly isolated CD133+ cells alone, (B) CD133+ cells and CD133– cells together and (C) with 2-week derived CD133+ cells. Scale bar=75 µm. (D) Number of CD133+ cells within tubules per HPF with different CD133+ populations and culture conditions. *P<0.001 versus other conditions.

 
4. Discussion
It is generally accepted that increased numbers of endothelial progenitor cells (CD34+CD133+VEGFR2+ cells) in the peripheral circulation may be mobilized from the bone marrow in response to environmental cues such as ischemia. CD133 is regarded as an important marker to identify various stem/progenitor cell populations, including endothelial progenitor cells [19]. In the present study, we demonstrated that CD133+ cells can be derived from the CD133– population of peripheral blood mononuclear cells. The majority (~89%) of these cells were also positive for CD34 and VEGFR-2 expression, therefore characterizing them as endothelial progenitor cells [8,9].

The concept that CD133+ cells can be generated from cells within the peripheral blood in culture has been demonstrated previously by Kuci et al. [17]. In their study, CD133+ cells were positively selected from fresh PBMCs and cultured, and adherent cells generated CD133+CD34– non-adherent cells. On the other hand, our results demonstrate the ability to generate CD133+ cells from CD133– cells rather than from the CD133+ fraction. In addition, in our study, this phenomenon was not dependent on adhesion, as CD133+ cells were generated in both adherent and suspension culture systems. This demonstrates the ability of a subset of peripheral blood CD133– cells to respond to environmental cues to generate a population of CD133+ cells without direct mobilization from the bone marrow.

The CD133– cell source of these derived CD133+ cells remains elusive. It is possible that these progenitors were generated from myeloid precursors or monocytes that can act as pluripotent stem cells [12], express endothelial markers and participate in angiogenesis [13,20]. Alternatively, the generation of CD133+ cells from the CD133– fraction could be a result of de-differentiation. This process has recently been described in lineage-committed mammalian cells such as myoblasts and pancreatic cells [21,22]. Possible sources for the de-differentiation of CD133– cells within the bloodstream include mature late endothelial progenitor cells [23,24] that have not yet become mature endothelial cells or the adult human hemangioblasts described recently, which express CD133 and CD34, and possess the dual capacity to differentiate into cells of hematopoietic or endothelial lineage [25]. Potentially, upon loss of CD133 expression, these cells may be the source of CD133– cells that possess the ability to re-generate CD133+ cells.

We investigated the expression of VEGFR-2 in CD133– cells to better characterize the population that was generating the CD133+ cells. The majority of cells within the peak shifting towards CD133 positivity were CD133–VEGFR-2+. This supports the likelihood that CD133– cells expressing VEGFR-2 were the source for the generated CD133+ cells. Further investigation of the derived CD133+ cells is needed to identify the source of their derivation.

This study demonstrated an effect of the CD133– cells on the functional properties of the CD133+ cells. It has been shown that endothelial progenitor cells have enhanced angiomyogenic capacity when pre-conditioned with VEGF165, brain-derived nerve growth factor and other endothelial signals [26,27]. It has also been demonstrated that upon incubation with mononuclear cells, the migration capacity of isolated peripheral blood CD34+ cells increases [16]. Similarly, in the present study, migration of CD133+ cells was significantly increased upon co-culture with the CD133– fraction. This was observed with both CD133+ cells at t=0, and those derived from the CD133– cells after 2 weeks in culture.

The supernatant of CD133– cell cultures equally promoted migration of the CD133+ cells. A RayBio Human cytokine/growth factor antibody array identified several elevated factors in the supernatant, including inflammatory mediators with demonstrated roles in migration of mononuclear cells: IL-1β, Il-6, IL-8, IL-10, GRO, MCP-1, MIP-1β, and RANTES [28,29]. Inflammation precedes angiogenesis, and several of the elevated cytokines from the CD133– fraction (IL-8, GRO, MCP-1 and RANTES) have been implicated in regulating angiogenesis [30,31]. Studies have previously demonstrated interaction of progenitor cells and other sub-populations from the peripheral blood through cytokines, including the secretion of IL-8 and MCP-1 from CD14+ cells [15,32]. In fact, the mixed transplantation of different sub-populations of peripheral blood mononuclear cells results in synergistic neovascularization, in part through cytokines [32]. Therefore, the CD133– cells provide a pre-conditioning environment that can promote the angiogenic potential of CD133+ cells.

Compared to freshly isolated cells, the 2-week derived CD133+ cells demonstrated increased participation in angiogenesis in vitro and adhesion to three substrates: fibronectin, collagen and an endothelial monolayer. The greatest level of adhesion was seen on the collagen substrate. The mechanism for this improved adhesion is unknown, but is not due to endothelial adhesion molecules CD31 or VE-cadherin. Both CD31 and VE-cadherin showed little expression in derived CD133+ cells as determined by flow cytometry. The CD133+CD34+VEGFR-2+VE-cadherin–CD31– phenotype of these derived cells is the same as the CD133+ cells isolated from the human bone marrow by Quirici et al. [24], suggesting a more primitive phenotype.

The 2-week derived CD133+ cells used for both the adhesion and angiogenesis assays were exposed to the cultured CD133– cells from which they were derived. The interaction with this population of cells produced functionally active CD133+ cells, similar to that reported by Hristov et al. between endothelial cells and endothelial progenitor cells that resulted in improved functional capacity of the progenitors [27]. Such a relationship between the progenitor cells and their neighbouring cells in the peripheral circulation may help to explain the successful revascularization observed after transplantation of different subsets of PBMCs referred to as endothelial progenitor cells by various groups (for review, see Ref. [33]).

Overall, this study demonstrates a source of CD133+CD34+VEGFR-2+ blood cells, other than mobilization from the bone marrow, for neovascularization in ischemic tissue. These cells, derived from the CD133– cells in the peripheral blood, demonstrated improved adhesive and angiogenic properties compared to freshly isolated CD133+ cells. The function of the CD133+ cells depends, at least in part, on interaction with CD133– cells within the circulation. These findings justify the development of angiogenic therapies, such as cell-based tissue engineered delivery strategies, involving the recruitment of more than one cell type to the site of injury.


   Acknowledgements
 
This work was supported by grant MOP-77536 from the Canadian Institutes of Health Research (to Drs. Ruel and Suuronen), and by award 7346 from the Canadian Foundation for Innovation (to Dr. Ruel), and by a Heart and Stroke Foundation of Canada/AstraZeneca Canada Inc. Fellowship (to Dr. Suuronen).


   Notes
 
Time for primary review 19 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 References
 

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