Anja Neumann

Rebecca Jahn

Curt Diehm

Elke Driller

Franz Hessel

Gerald Lux

Oliver Ommen

Holger Pfaff

Uwe Siebert

Dr David Pittrow
email: david.pittrow@mailbox.tu-dresden.de

Juergen Wasem

Prof. Dr Med David Pittrow

Dr Herrn Prof

Dr Wilhelm Kirch
email: wilhelm.kirch@mailbox.tu-dresden.de

Outcomes of medical management of peripheral arterial disease in general practice: follow-up results of the PACE-PAD Study

Keywords: Guideline orientation, peripheral arterial disease, outcomes, intermittent claudication, observational study

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In addition, we added 2 additional publications to the reference list, which were published very recently and fit well in the context of this study (Diehm 2010[START_REF] Meves | Peripheral arterial disease as an independent predictor for excess stroke morbidity and mortality in primary-care patients: 5-year results of the getABI study[END_REF].

In Tables 4a and 4b the sequence of n and % was changed (now: % (n)), to have a consistent format across all tables.

Background

Substantial advances have been made in the understanding of peripheral arterial disease (PAD) and its implications [START_REF] Belch | Critical Issues in Peripheral Arterial Disease Detection and Management: A Call to Action[END_REF]. A series of studies have shown that PAD is highly prevalent in the primary care setting and the general population. For example, the getABI in 6,880 patients aged at least 65 years found PAD in 21.0% of patients [asymptomatic PAD as evidenced by a low ankle-brachial index [ABI] <0.9 in 12.3%, and symptomatic PAD in 8.7%] (Diehm et al 2009). The German Heinz Nixdorf Recall Study (4814 unselected patients aged 45-75 years) reported PAD in 8.2% in men and 5.5.% in women (asymptomatic PAD: 6.4% in men and 5.1% in women) [START_REF] Kroger | Prevalence of peripheral arterial disease -results of the Heinz Nixdorf recall study[END_REF].

Further, the increasing awareness of the association of PAD with generalised atherosclerosis in other vascular territories has secured its role in primary and secondary prevention of cardiovascular disease. PAD patients carry a high risk of premature death, and of cardiovascular and cerebrovascular events [START_REF] Meves | Peripheral arterial disease as an independent predictor for excess stroke morbidity and mortality in primary-care patients: 5-year results of the getABI study[END_REF]. According to a recent metaanalysis of Heald et al., this risk is doubled, even if all other conventional cardiovascular risk factors are accounted for [START_REF] Heald | Risk of mortality and cardiovascular disease associated with the ankle-brachial index: Systematic review[END_REF]. This is of particular importance, as PAD signals a risk higher than -for example -smoking or diabetes mellitus, and can therefore guide physicians in selecting patients at particularly high risk. Against the background of the high disease burden of PAD associated with increased risk of vascular events, an aggressive approach to treat cardiovascular risk factors is recommended in all major guidelines, e.g. those of the TransAtlantic Inter-Society Consensus (TASC II) [START_REF] Norgren | Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II)[END_REF], the American Heart Association (AHA)/ American College of Cardiology (ACC) [START_REF] Hirsch | ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation[END_REF] The primary care setting is of particular importance for the management of PAD patients.

The family physician needs to be efficient in identifying high risk patients, as he serves as a gatekeeper for the selection of patients for further assessment or treatment by specialists [START_REF] Grumbach | Resolving the gatekeeper conundrum: what patients value in primary care and referrals to specialists[END_REF], and has a pivotal role in the long-term management of patients [START_REF] Kröger | Management of patients with peripheral arterial disease in primary care: a cross-sectional study in Germany[END_REF].

Methods

Design and study flow

PACE-PAD is a multicentre, cluster-randomised, observational, non-interventional prospective longitudinal study, which followed patients with confirmed PAD in stage Fontaine I-IV at 3 visits over 18 months (Figure 1). The study was conducted according to the principles of "Good Epidemiological Practice (GEP)" (Arbeitsgruppe Epidemiologische Methoden der Deutschen Arbeitsgemeinschaft Epidemiologie (DAE)). Protection of patient and centre data was ensured. According to a statement of the legal department of the University Duisburg-Essen, for this non-interventional study a formal approval was not necessary.

-here insert Figur 1-

Study course

Baseline. Patients were eligible for inclusion, if they had suspected symptomatic PAD, i.e., newly diagnosed intermittent claudication (IC) or IC-like complaints with PAD suspected by the treating physician. Further, patients with suspected asymptomatic PAD were eligible for inclusion, if they were aged 55 years or above and had (1) previous myocardial infarction and/or (2) previous ischemic stroke and/or (3) manifest type 1 or type 2 diabetes mellitus and/or (4) current smoking (for more than 10 years each). If PAD had been diagnosed prior to baseline, patients were not eligible for further evaluations (exclusion criterion).

Follow-up/longitudinal study. If the suspicion of PAD was confirmed at the baseline visit, patients were followed up for 18 months. Three follow-up visits (F/U 1 at 6 month, F/U 2 at 12 month, and F/U 3 at 18 month) were scheduled to collect the following information: incidence of cardiovascular events (stable or unstable angina pectoris, myocardial infarction or coronary revascularisation with or without stenting), cerebrovascular events (stroke or transient ischemic attacks [TIA], prolonged ischemic neurological deficit [PRIND], revascularisation of the carotids) or peripheral revascularisation or amputation, respectively.

The longitudinal study endpoints were myocardial infarction, stroke or minor/major amputation due to PAD. Further, it was assessed whether patients received guideline-oriented management during follow-up, which was assessed using 6 quality indicators for each patient, using a standardised questionnaire (Table 1). The quality indicators were defined and assessed by an interdisciplinary board (composed of clinical experts in the fields of angiography, cardiology and neurology as well as a methodical specialist regarding evidenced based medicine). They comprised prescription of exercise training in the Fontaine stages I and II, recommendation of smoking cessation, management of arterial hypertension, diabetes mellitus and hypercholesterolemia as well as the use of antiplatelet therapy. Guideline-oriented treatment was assumed for example, if a patient with diabetes mellitus received dietary measures or at least 1 antidiabetic drug.

-here insert Table 1-

Statistics

The original sample size was calculated based on the assumption that the cumulative incidence of vascular events after 18 months was 6.8% in PAD patients with guidelineoriented therapy versus 9.8% in PAD patients not receiving such therapy. A sample of 3,483 symptomatic patients (of whom at least 85% were assumed to have diagnosed PAD) and of 20,485 patients with risk factors (of whom at least 10% were assumed to have diagnosed PAD) was required to obtain a power of 80% at a significance level of 5%. It was planned, to test primarily the hypothesis that the cumulative incidence of cardiac, cerebrovascular and peripheral vascular events during the follow-up period would be lower in PAD patients with guideline-oriented management compared to PAD patients without such management.

Using cross tables, frequency distributions and descriptive statistics, the distributions of variables between the two patient strata were compared. In all analyses, a 2-sided-or the chi-square p-value <0.05 (to evaluate differences between proportions for two or more than two groups) was considered to denote statistical significance. All analyses were performed with SPSS Version 15 for Windows (SPSS Inc, Chicago, Ill, USA).

Results

Characteristics. Patients were included by 2,768 physicians. The flowchart in Figure 1 displays the disposition of patients in the baseline part of the study, and during the 3 followup examinations. Of the 6,129 PAD confirmed patients who were originally included at baseline, 22.5% were in Fontaine Stage I, 64.7% in Stage II, 7.8% in Stage III and 3.5% in Stage IV (1.5% not specified). In Follow-up 1, 4,645 PAD patients (75.8%), in Follow-up 2 4,201 patients (68.5%) and in Follow-up 3 3,910 Patients (63.8%) were documented.

Overall there were 5,099 patients with at least one documented follow-up visit which could be included in the analyses.

-here insert Figure 2-Guideline orientation. Only 28.4% of all PAD patients met all applicable (i.e., up to 6) quality indicators for guideline-oriented treatment. Nonetheless, the majority of PAD patients were to a large extent managed in line with guidelines. Figure 2 shows individual quality indicators and the adherence rates over the whole study period of 18 months. With exception of exercise training intervention (41.8%), guideline adherence rates with regard to the various risk factors were high (82.0% to 92.5%). The great majority of the current smokers received the general advice to stop smoking (90.7%). Management of hypertension, diabetes mellitus, hypercholesterolemia and antiplatelet therapy in line with the respective guidelines, was reported in no less than 88% of the PAD patients each (see details in Figure 3).

-here insert Figure 3-Characterisation. Table 2 provides an overview of demographic and clinical patient characteristics in the total PAD follow-up cohort, and grouped by guideline-orientation status. There were (limited) differences between groups, particularly with regards to mean patient age and diabetes status. Mean patient age was slightly lower in the group treated according to guidelines compared to the group which was not treated in such a way (66.9 vs. 68.5 years). Diabetes was more prevalent in the latter group. In contrast the rate of patients with previous ischemic events (e. g. myocardial infarctions) and atherothrombotic manifestations (e.g. angina pectoris) were substantially more frequent in the treatmentorientation group.

-here insert table 2-An analysis by Fontaine status showed that guideline orientation was higher in the early PAD disease stages: while in stage I 37.1% of patients were treated according to guidelines, in stage III only 9.8% and in stage IV 18.0% were managed accordingly (Table 3).

-here insert table 3-Outcomes after 18 months. During the 18 month follow-up period in the total PAD cohort, 224 cerebrovascular/coronary deaths, 457 all-cause deaths, 140 ischemic strokes, 116 myocardial infarctions, 100 minor and 68 major amputations were reported (Fig. 4a). Figure 4b displays the overall event rates (first event only) at the different follow-up visits.

Overall, there were 973 events reported during the 18 month follow-up period, 468 at Follow-up 1, 280 at Follow-up 2 and 225 at Follow-up 3.

-here insert figure 4a--here insert figure 4b-Table 4a provides an overview of all vascular event rates grouped by guideline-orientation status, Table 4b of first event only. There was a statistically significant difference between the two subgroups with more overall, cerebrovascular, and peripheral events in the group not treated according to guidelines. However, a lower number of vascular deaths (coronary, cerebrovascular) and all-cause deaths were reported in this group (Table 4a).

-here insert table 4a-With the exception of follow-up 3, where in the group not treated according to guidelines more first events occurred, there was no statistically difference concerning the first event (Table 4b).

-here insert table 4b-

Discussion

While the role of PAD as marker of high risk for cardiovascular disease has been fostered, data regarding to actual everyday care of patients with PAD are limited. According to the present study, firstly, guideline-oriented therapy which accounts for all major quality indicators is the exception rather than the rule. Secondly, unexpected results were noted in terms of long-term outcomes with no major differentiation between the subgroups which were differentiated by orientation vs. non-orientation to guidelines.

In the recent years, a number of studies have investigated treatment rates and intensity in PAD patients. More than a decade ago, one of the most authoritative studies about the treatment situation of PAD patients, PARTNERS, reported relatively low treatment rates in 1,865 PAD patients (54% on antiplatelets; 56% on lipid lowering drugs; 88% on antihypertensives, 85% on antidiabetics). The "German epidemiological trial on Ankle Brachial Index, getABI", is an ongoing non-interventional study in 6,880 unselected individuals aged 65 years or older to assess risk factor profile, treatment and prognosis of PAD patients in primary care. In the patients with PAD alone assessed at the baseline examination in 2001, antiplatelets were prescribed in 53% (versus 65% if coronary or cerebrovascular disease was present), lipid-lowering agents in 40.0%, antihypertensives in 93.3%, and antidiabetics in 74.4% [START_REF] Pittrow | The German Trial on Ankle Brachial Index (getABI): high prevalence and evidence for antiplatelet undertreatment of peripheral arterial disease in primary care [abstract[END_REF]. Further, in the pooled analysis of three large French observational studies (ECLAT 1, PRISMA, APRES), patients with PAD alone received antiplatelets in 78.9%, lipid-lowering agents in 40.4%, antihypertensives in 25.8%, and antidiabetics in 17.0% [START_REF] Bongard | Comparison of cardiovascular risk factors and drug use in 14,544 French patients with a history of myocardial infarction, ischaemic stroke and/or peripheral arterial disease[END_REF]. The large contemporary REACH registry reported in patients with manifest PAD and the respective concomitant disease or condition, drug treatment rates of 92% for hypertension, 86% for diabetes, 70% for hyperlipidemia, and 82% for antiplatelet use [START_REF] Bhatt | International Prevalence, Recognition, and Treatment of Cardiovascular Risk Factors in Outpatients With Atherothrombosis[END_REF]. While at first glance these rates appear satisfactory, in that registry only a minority of patients were at target goals for blood pressure, glucose, cholesterol, body weight and non-use of tobacco [START_REF] Bhatt | International Prevalence, Recognition, and Treatment of Cardiovascular Risk Factors in Outpatients With Atherothrombosis[END_REF].

Notably, in all reported studies, treatment intensity was dependent on comorbidity: if in addition to PAD, coronary heart disease or cerebrovascular disease was concomitantly present, treatment rates had consistently improved.

It should be noted that current treatment recommendations for PAD patients are based on various strength of evidence. The recommendation for smoking cessation (counselling, nicotine replacement, bupropion) is based on evidence level I B (consistent evidence, based on a single randomised or on non-randomised studies), for physical exercise on IA (several randomised studies or metaanalyses available). In contrast, the evidence is considered to be weaker for lipid lowering (level II B), and diabetes treatment (II C). While the evidence for the use of antiplatelet drugs is strong (I A), it should be noted that the underlying data are based on symptomatic patients. While antiplatelet therapy has been recommended in diabetic patients and/or PAD patients (American Diabetes Association 2003, American Diabetes Association 2007), the recent results of the POPADAD study in patients with diabetes and concomitant asymptomatic PAD did not provide evidence to support the use of ASS or antioxidants in primary prevention of cardiovascular events and mortality. [START_REF] Belch | The prevention of progression of arterial disease and diabetes (POPADAD) trial: factorial randomised placebo controlled trial of aspirin and antioxidants in patients with diabetes and asymptomatic peripheral arterial disease[END_REF] Similarly, in the large Japanese JPAD study, primary prevention in diabetic patients with low dose acetylic salicylic therapy did not reduce vascular event rates (Ogawa et al 2008). Very recently, Fowkes et al reported the results of the AAA study: Among participants without clinical cardiovascular disease, identified with a low ABI based on screening a general population, the administration of acetylic salicylic acid compared with placebo did not result in a significant reduction in vascular events [START_REF] Fowkes | Aspirin for Prevention of Cardiovascular Events in a General Population Screened for a Low Ankle Brachial Index: A Randomized Controlled Trial[END_REF].

Other reasons beyond the varying strength of evidence may account for the reluctance of physicians to adhere to the PAD guidelines. Basically, many physicians still underestimate the importance of PAD in relation to CHD or CVD [START_REF] Belch | Critical Issues in Peripheral Arterial Disease Detection and Management: A Call to Action[END_REF]. Second, the expectation in the management of younger patients that life style change or other nonpharmacological treatment may be sufficient [START_REF] Pickering | Lifestyle modification and blood pressure control: is the glass half full or half empty?[END_REF], and in elderly the fear of doing harm by applying too-intensive treatment for example with regards to antihypertensive therapy [START_REF] Berlowitz | Inadequate management of blood pressure in a hypertensive population[END_REF][START_REF] Chobanian | Control of hypertension -an important national priority[END_REF]) accounts for undertreatment. Further, the high number of guidelines in various fields, and their length and complexity makes it difficult for the physician to keep abreast with the plethora of information. Clinical inertia on the side of physicians might be the result. [START_REF] Phillips | Clinical inertia[END_REF] With respect to outcomes at 1.5 years, in our study the total incidence of deaths and vascular events was 23.5%, an unexpectedly high rate, which hints at the inclusion of patients with advanced disease (or multiple risk factors). For comparison, in PAD patients the 1-year incidences of death plus major vascular events were 8.3% in getABI (Lange et al 2005) and 5.4% in REACH [START_REF] Steg | Outcomes of medical management of peripheral arterial disease in general practice: follow-up results of the PACE-PAD Study Anja Neumann (1), Rebecca Jahn (1)[END_REF].

For our investigation, we chose a real-life, non-interventional study design in order to obtain data on actual practice patterns. This ensured that various types of PAD patients with a broad variety of concomitant diseases or medications could be investigated. However, when interpreting the results of the present study, several limitations have to be taken into account. First, the study was observational and not randomised, which means that unknown biases cannot be ruled out. [START_REF] Benson | A Comparison of Observational Studies and Randomized, Controlled Trials[END_REF][START_REF] Sinatra | Efficacy and safety of single and repeated administration of 1 gram intravenous acetaminophen injection(paracetamol) for pain management after major orthopedic surgery[END_REF] While physicians were requested to document eligible patients consecutively, there was no additional log-file of all patients in the practice to exclude possible selection processes. Further, no data are available about those patients who could not be followed up. The rate of lost-to-follow-up patients was substantial (about a quarter), and it can be hypothesized that these patients are less well treated than more compliant patients. No information was collected on reasons why guideline-oriented treatment was not pursued (example: contraindication to exercise training in patients with musculoskeletal disease or due to acute cold). The missing difference between outcomes in the guideline-oriented and non-guideline-oriented groups may be due to various factors, which comprise low treatment intensity and therefore unsatisfactory clinical effect, underestimation of the implications of PAD [START_REF] Hirsch | Gaps in public knowledge of peripheral arterial disease: the first national PAD public awareness survey[END_REF] with resulting patient non-compliance or non-adherence with therapy. Further, a lack of effect in certain subgroups, e.g. missing effect of antithrombotic therapy in the asymptomatic patients as noted in the AAA study [START_REF] Fowkes | Aspirin for Prevention of Cardiovascular Events in a General Population Screened for a Low Ankle Brachial Index: A Randomized Controlled Trial[END_REF], or in the high Fontaine stages, might account for the non-differentiation. Finally, methodological issues cannot be fully excluded, such as misclassification of events.

Conclusions

The present PAD cohort was a high-risk sample with an unexpectedly high rate of deaths and vascular events. The proportion of patients treated comprehensively according to the guidelines was relatively low, which calls for an optimisation. There was a lack of differentiation between the guideline-oriented on non-guideline oriented therapy in terms of outcomes, which may be due to patient-related or other factors, and calls for further research.

Figure 1. Flow chart with disposition of patients

  and the newly released joint recommendations of the German Society of Angiology (DGA) and other societies.(Deutsche Gesellschaft für Angiologie und Gesellschaft für Gefäßmedizin 2009)

Figure 2 .Figure 3 .

 23 Figure 2. Guideline orientation, by individual quality indicators

Table 2 . Fontaine stages in the total cohort, and by overall guideline orientation status

 2

		All PAD patients	PAD treated	PAD not treated	Comparison between
				according to	according to	PAD subgroups
				guidelines	guidelines	(p value)
			% (N)	% (n)	% (n)
	All patients	100 (5099)	28.4 (1450)	71.6 (3649)
	Fontaine stages
	I (asymptomatic)	100 (1145)	30.3 (347)	69.7 (798)	0.115
	IIa (mild claudication)	100 (1765)	31.6 (557)	68.4 (1208)	<0.05
	IIb (moderate to severe	100 (1534)	29.1 (446)	70.9 (1088)	0.521
	claudication)
	III (ischemic rest pain)	100 (399)	9.8 (39)	90.2 (360)	<0.05
	IV (ulceration or gangrene)	100 (178)	18.0 (32)	82.0 (146)	<0.05
	Missing/multiple stages	100	(78)	37.2 (29)	62.8 (49)	<0.05

Table 3 . Patient characteristics overall and by guideline-orientation

 3

	Parameter	All PAD patients	PAD treated	PAD not treated	Comparison
			according to	according to	between subgroups
			guidelines	guidelines	(p value)
		(N=5099)	(n=1450)	(n= 3649)
	Age (years), mean ± SD	68.0+-9.0	66.9 ± 8	68.5± 9	<0.05
	< 65 years, %	36.2	41.2	34.2	<0,05
	≥ 65 years, %	63.8	58.8	65.8	<0,05
	Males: females, %	68.5:31.5	72.8:27.2	66.8:33.2	<0.05
	Body Mass Index (kg/m 2 )	27.7	27.8	27.7	0.33
	Systolic and diastolic BP, mean	140.4/82.1	140.0/82.3	140.5/81.9	0.34; 0.15
	(mmHg)
	Complaints (intermittent	85.7	85.6	85.7	0.89
	claudication), %
	Vascular risk factors
	None/1/2/3/4, n	320/2.427/1.815/48	90/736/560/151/	230/1.691/1.255/336/	0.90
		7/50	15	35
	Current Smoking,	53.2 (2711)	52.0 (807)	53.7 (1904)	0.27
	> 10 years	94.6 (2565)	93.9 (758)	94.9 (1807)	0.17
	Diabetes mellitus	54.9 (2801)	52.0 (807)	56.2 (1994)	<0.05
	> 10 years	74.6 (2091)	76.9 (621)	73.7 (1470)	<0.05
	Arterial hypertension	76.0 (3877)	77.3 (1199)	75.5 (2678)	0.19
	> 10 years	70.3 (2727)	69.8 (837)	70.6 (1890)	0.33
	Hypercholesterolemia	69.4 (3537)	70.6 (1096)	68.8 (2441)	0.21

Values indicate % (n), if not specified otherwise. AP, angina pectoris; TIA, transient ischemic attack; PRIND, prolonged reversible ischemic neurological deficit

While in other countries the situation of PAD patients in primary care has been repeatedly addressed[START_REF] Hirsch | The Minnesota Regional Peripheral Arterial Disease Screening Program: toward a definition of community standards of care[END_REF][START_REF] Khan | Characteristics and treatments of patients with peripheral arterial disease referred to UK vascular clinics: results of a prospective registry[END_REF], corresponding data from the German healthcare system are limited. Against this background, the Patient Care Evaluation -Peripheral Arterial Disease (PACE-PAD) Study was initiated. We have previously described the baseline results of the study, with focus on the current diagnostics and therapy (i.e., management) of patients with newly diagnosed, suspected or confirmed PAD, with particular focus on the interaction between general physician and specialist care.[START_REF] Neumann | Presentation and medical management of peripheral arterial disease in general practice: rationale, aims, design and baseline results of the PACE-PAD Study[END_REF] In the present article on the 18-month follow-up, we report (1) the differences of patient characteristics and other factors between patients treated according to guidelines and those who were not and (2) the outcomes of guideline-oriented therapy on the incidence of cardiovascular, cerebrovascular or peripheral vascular events in patients with newly diagnosed PAD.

Acknowledgements

The study was supported by an unrestricted educational grant from Sanofi-Aventis Pharma.

We thank Anna Neumann for her strong commitment in the project office. Further, we appreciate the help of the participating GPs for collecting the data for the study and their practice staff for their assistance.

Competing interests

The study was supported with an unrestricted grant from Sanofi-Aventis, Berlin, Germany. The authors declare that they have no competing interests.

Authors' contributions

AN, JW, FH and RJ participated in study conception and design, acquisition of data, analysis and interpretation of data, funding acquisition, and drafting and critical revision of the paper for important intellectual content. HP, ED, OO, DP and CD advised on the study design and focussed especially on patients' compliance and coping with disease. US did the sample size calculations and GL the statistical calculations. All authors accept responsibility for the scientific content of the paper.


Ph.D Irena Koutna
email: qkoutna@fi.muni.cz

MSc Martina Peterkova

MSc Pavel Simara

Stanislav Stejskal MSc Lenka Tesarova

MSc Michal Kozubek

Proliferation and differentiation potential of CD133+ and CD34+ populations from the bone marrow and mobilized peripheral blood

Keywords: CD34, CD133, HSCs transplantation, VEGFR

Objective: CD34 is the most frequently used marker for the selection of cells for bone marrow (BM) transplantation. The use of CD133 as an alternative marker is an open research topic.

The goal of this study was to evaluate the proliferation and differentiation potential for hematopoiesis (short and long term) of CD133+ and CD34+ populations from bone marrow and mobilized peripheral blood.

Patients and Methods: Eight cell populations were compared: CD34+ and CD133+ cells from both the BM (CML Ph-, CML Ph+ and healthy volunteers) and mobilized peripheral blood cells. Multicolor flow cytometry and cultivation experiments were used to measure expression and differentiation of the individual populations.

Results: It was observed that the CD133+ BM population showed higher cell expansion.

Another finding is that during a six-day cultivation with CFSE, more cells remained in division D0 (non-dividing cells). There was a higher percentage of CD38-cells observed on the CD133+ BM population. It was also observed that the studied populations contained very similar but not the same pools of progenitors: erythroid, lymphoid, and myeloid. This was confirmed by CFU-GM and CFU-E experiments.

The VEGFR antigen was used to monitor subpopulations of endothelial sinusoidal progenitors. The CD133+ BM population contained significantly more VEGFR+ cells.

Conclusion:

Our findings suggest that the CD133+ population from the BM shows better proliferation activity and a higher distribution of primitive progenitors than any other studied population.

Introduction

In recent years, the CD34+ antigen has been the most frequently used marker for the identification of hematopoietic stem cells (HSCs). The main source of those cells for bone marrow (BM) transplantations is peripheral blood stem cells (PBSCs) obtained using socalled mobilization of hematopoietic stem cells from healthy donors (in cases of allogeneic transplantation) or patients in remission (in cases of autologous transplantation). BM transplantation is becoming an increasingly less significant method [START_REF] Gratwohl | Current trends in hematopoietic stem cell transplantation in Europe[END_REF]. CD34+ cells represent a heterogeneous population consisting mainly of lineage-committed progenitors and only a small hematopoietic cell population [2]. The percentage of cells capable of rapid reconstitution of sinusoidal endothelial cells (which is mandatory for the regeneration of socalled vascular niches) is thus low [START_REF] Tavassoli | Structure and function of sinusoidal endothelium of bone marrow[END_REF].

Recently, the importance of hematopoietic stem cell populations defined by other molecules (e.g., adhesive or chemokine receptors) has increased. Such populations can play an important role during stem cell homing to the BM and during permanent hematopoiesis reconstitution.

One previously identified but frequently discussed hematopoietic stem cell marker is the molecule CD133 [START_REF] Summers | AC133+ G0 cells from cord blood show a high incidence of long-term cultureinitiating cells and a capacity for more than 100 million-fold amplification of colonyforming cells in vitro[END_REF]. CD133 is reported to be a structural homolog of murine promin-1. It is a marker for stem cells with pluripotent plasticity, and CD133+ cells have been shown to be capable of induced differentiation to endothelial cells, neural cells, hepatocytes, myocytes, and osteoblasts [START_REF] Kuçi | Efficient in vitro generation of adult multipotent cells from mobilized peripheral blood CD133+ cells[END_REF].

Providing patients with a pool of cells that is capable of invoking both short-term and longterm hematopoiesis reconstitution is the most important phase of both autologous and allogeneic BM transplantation.

This study is focused on comparing eight populations of hematopoietic progenitors obtained using magnetic separation: CD133+ and CD34+ cells from both BM (CML Ph+, CML Phand healthy volunteers) and PBSCs. Flow cytometry was used for that purpose.

The first part of the study examined the proliferation potential of all studied populations. Cell expansion was monitored together with the differentiation potential of individual populations.

The main reasons for performing these experiments were 1) to provide an overview of the populations' ability to grow, expand, and proliferate and 2) to determine the share of nondividing -quiescent cells in these populations. Quiescent stem cells are defined as stem cells not currently undergoing repeated cell cycles, but that might be stimulated in the future. The presence of such a cell type is very important for long-term hematopoiesis reconstitution. The distribution of the CD38-population was measured during the isolation of individual populations as well as during short-term cultivation [START_REF] Goussetis | Kinetics of quiescent cord blood stem/progenitor cells with high proliferative potential in stem-cell expansion culture[END_REF].

Another topic studied in this article is the distribution of both markers (CD133 and CD34) in individual populations. We sought to determine the degree of overlap between these populations. Recent studies [START_REF] Hooper | Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells[END_REF] aimed at regeneration of sinusoidal BM cells have shown that endothelial progenitor cells (EPCs) are very important for graft engraftment. The determination of the EPC perceptual distribution was thus also included in this study. This determination was achieved by monitoring the distribution of cells expressing a VEGFR2 marker. The expression of this particular marker in the hematopoietic progenitor population defines the subpopulation of EPCs [8,[START_REF] Peichev | Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors[END_REF].

In order to study the percentage of progenitor cells, the following lineage commitment markers were analyzed: CD71 for the erythroid lineage, CD33 for the myeloid lineage, and CD10 for the lymphoid lineage. The presence of those progenitors in grafts is very important from the perspective of short-term hematopoiesis reconstitution. The ability of individual populations to reconstitute hematopoiesis was also proven using CFU-GM and CFU-E experiments.

Materials and Methods

Patients and samples

BM samples were taken from 12 patients diagnosed with chronic myeloid leukemia (CML) and 7 healthy volunteers. The CML samples included seven patients with de novo CML and five patients in complete molecular remission (i.e., with no leukemia cells detected by PCR and FISH). Molecular emission was evaluated according to the recommendations of European Leukemia Net. Regular evaluation of BM-treated CML patients at exact intervals was used to monitor the treatment success rate. Cytogenetic monitoring was performed at months 3, 6, 12 and 18. Molecular monitoring was performed every 3 months. Patients were analyzed on month 3 (hematologic response [HR]), month 6 (HR or cytogenetic response [CgR]), month 12 (CgR [Philadelphia chromosome-positive (Ph+) < 35%]) and month 18 (complete CgR, major molecular response [MMolR]) [START_REF] Baccarani | Chronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNet[END_REF]. CML de novo diagnosed patients played a very important role in the study: large amounts of both CD34+ and CD133+ cells were obtained from their BM samples (see results). Thus, most of the experiments mentioned in the results section were done simultaneously using one sample and even multiple repetitions.

Twelve samples of mobilized PBSCs were taken from volunteers. All samples were taken after obtaining written, informed consent. All samples were processed immediately after collection.

Isolation of CD34+ and CD133+ cells

Mononuclear cells (MNCs) were isolated from the BM and PBSCs using Histopaque-1077 density gradient centrifugation (Sigma-Aldrich, St. Louis, USA). After washing three times with phosphate-buffered saline (PBS), the MNCs were incubated with an erythrocyte lysis buffer (ammonium chloride lysis buffer) to remove residual erythrocytes.

MNCs from the BM and PBSCs were divided into two equal parts: the first one was separated into CD34+ cells, and the second one was separated into CD133+ cells. For CD34+ and CD133+ selection, the MNCs were subjected to immunomagnetic separation using magneticactivated cell sorting (MACS) with a CD34 or CD133 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Positive selection of CD34 and CD133 was performed according to the instructions of the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were labeled with anti-CD34 or anti-CD133 antibodies, directly coupled to magnetic microbeads, and incubated for 30 min at 4°C. The cells were washed with PBS and then counted in a Bürker counting chamber using a Countess Automated Cell Counter (Invitrogen, Carlsbad, USA).

We obtained eight different cell populations: three of BM CD34+ (source: CML Ph-, CML Ph+ and healthy volunteers) three of BM CD133+ (source: CML Ph-, CML Ph+ and healthy volunteers), one of PBSC CD34+ and one of PBSC CD133+ cells. These cell populations were compared with each other.

Lymphocyte depletion

The mononuclear portion of leukocytes of the BM of healthy donors or naive de novo CML patients consists of immature myeloid cells, lymphocytes and monocytes in various percentages [START_REF] Racil | The assessment of human organic cation transporter 1 (hOCT1) mRNA expression in patients with chronic myelogenous leukemia is affected by the proportion of different cells types in the analyzed cell population[END_REF]. To enrich the primitive hematopoietic cells in the mononuclear cell fraction for FACS analysis, we depleted the lymphocytes by magnetic labeling. CD2 Microbeads (MACS © Cell Miltenyi Biotec, Bergisch Gladbach, Germany) were used to deplete the T cells and NK cells. CD19 Microbeads (MACS © Cell Miltenyi Biotec, Bergisch Gladbach, Germany) to deplete the B cells.

Cell viability

Cell viability was evaluated through the trypan blue exclusion test using the Countess Automated Cell Counter. A single sample measurement using this counter provided the following data: live and dead cell concentration/ml, total cell concentration/ml, viability, mean diameter, and cell images.

For identification of apoptotic and necrotic cells, we also used an annexin V-FITC Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and propidium iodide (PI) (Sigma-Aldrich, St. Louis, USA) according to the manufacturer's directions. The viability of CD34+ and CD133+ cells just after isolation was 99 ± 0.5%.

CFSE staining

CD34+ and CD133+ cells were stained with 1 µM 5(6)-carboxyfluorescein diacetate Nsuccinimidyl ester (CFSE; Sigma-Aldrich, St. Louis, MO, USA) as described previously [START_REF] Holyoake | Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia[END_REF]. These cells were cultured for six days in StemSpan Serum-Free Expansion Medium (SFEM; StemCell Technologies, Köln, Germany) with a cytokine mixture. After three and six days, the cells were harvested, washed once in PBS, and stained with anti-CD34-PE or anti-CD133-PE antibodies for flow cytometric analysis. We also cultured the cells in the presence of 100 ng/ml demecolcine (Sigma-Aldrich, St. Louis, MO, USA), which arrests the cell cycle. The cultures containing demecolcine were used to establish a non-dividing cell population (CFSE maximum) during the six-day interval.

Multicolor flow cytometric analysis

The percentage of human CD133, CD34, CD38, VEGF R2, CD10, and CD33 cells was determined using antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC). The anti-CD133 antibody conjugated to PE (clone: AC133/2) and the anti-CD38 antibody conjugated to APC were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). The anti-CD34 antibody conjugated to PE or FITC was from Becton-Dickinson (Heidelberg, Germany). The anti-human VEGR R2/KDR conjugated to FITC was from RD Systems (Minneapolis, MN, USA). The anti-CD10 antibody conjugated to PE and the anti-CD33 antibody conjugated to FITC were obtained from Immunotech (Prague, Czech Republic). The immunofluorescent staining was performed according to the manufacturer's instructions. The cells were incubated with appropriate antibodies for 30 min at 4°C in the dark. Subsequently, the cells were washed once with PBS.

Flow cytometric analysis was performed on the BD FACS Canto II flow cytometer (Becton-Dickinson, Heidelberg, Germany) using BD FACSDiva software (Becton-Dickinson, Heidelberg, Germany). A minimum of 15,000 events were analyzed in each sample. In the tables, the medians and range were used to describe data collected in flow cytometry. For flow cytometric analysis, the cells were taken from cell culture on days 0, 3, and 6.

In vitro cultivation

Liquid culture: CD34+ or CD133+ cells were maintained in SFEM supplemented with the StemSpan CC100 cytokine mixture. The concentration of cytokines in the CC100 cocktail used for cultivating enriched HSCs was 100 ng/mL for SCF and FLT-3 ligand (Flt-3-L), and 20 ng/mL for IL-3 and IL-6.

(StemCell Technologies, Köln, Germany) at 37°C in 5% CO 2 and 100% humidity. The cells were cultivated for 6 days in 4-well multidishes from Nunclon (Thermo Fisher Scientific, Langenselbold, Germany), with 2.0×10 4 -10 5 cells/ml added to each well. The medium was changed after three days.

Semi-solid culture: CD133 and CD34 cells were resuspended in SFEM at 5x10 3 cells/ml and mixed with 4 volumes of methylcellulose-based semi-solid culture medium (Human MethoCult; StemCell Technologies, Köln, Germany) containing stem cell growth factor (SCGF; 20 ng/ml) and granulocyte colony-stimulating factor (G-CSF; 6000 U/ml) for generating CFU-GM and SCGF (20 ng/ml) and erythropoietin (EPO; 1.5 U/ml Sigma-Aldrich, St. Louis, USA) for generating CFU-E. Aliquots of the mixtures (10 3 cells/1 ml) were plated in dishes and incubated for 15 days at 37°C in a humidified atmosphere with 5% CO2 at 37°C.

Statistical analysis

Data are presented as medians. To determine statistical significance, the nonparametric Mann-Whitney U test was applied. The criterion for statistical significance was defined as p< 0.05.

The software available at http://faculty.vassar.edu/lowry/utest.html was used for the computation of statistical values.

Results

In vitro expansion

Cell growth, absolute cell counts in the culture and population viability was monitored in parallel. At the beginning of cultivation, a given amount of cells was added to the culture. Cell growth measurements for individual populations are shown in Table 1. The median values for each group were based on 7 CML Ph+, 5 CML Ph-and 7 healthy volunteer samples for the BM and 12 samples for PBSC groups. There were no significant differences in proliferation activity among all of the studied sources (CML Ph+, CML Ph-and healthy volunteers) of the CD133 cells. The same was observed for the CD34 cells.

Although the concentrations of cells (cell/ml) added to the cultures differed at the beginning of each experiment, this difference did not have a statistically significant effect on the proliferation and viability of the resulting culture. A significant increase in proliferation activity was observed in cultures of CD133+ cells from the BM (p=0.006). The increase was more than two times higher (as seen in Table 1) than that observed in all CD34+ cultures. Interestingly, the differences among populations of the same cell type were insignificant.

CFSE experiments

Using the CFSE method, we were able to monitor changes in the number of divisions in individual cultures. As clearly shown in Table 2 and Figure 1, it was easy to determine the number of cell divisions and the number of cells remaining in the non-dividing state (i.e., division 0).

From the perspective of cell counting, it was obvious that there was a high percentage of CD133+ cells from the BM that were not dividing, even after 6 days of culture. Despite this fact, those cells highly expressed the CD133 marker. Such cells were also found in the CD34+ BM and the CD34+ PBSC populations but at much lower concentrations (as seen in Table 2). It can thus be assumed that the distribution of non-dividing cells (which are assumed to be primitive progenitors) in the CD133+ BM population is significantly higher than in other populations.

To confirm this assumption, we performed an independent experiment to monitor the CD133+/38-and CD34+/38-populations in parallel. Particular divisions were monitored on culture day 5. We found 70% of cells were viable in the CD133+ BM population and 68% were viable in the CD34+ BM population.

The most of cells in the D0 phase were CD133+/38-cells, while the populations of CD133+ and CD38-were minor. In subsequent generations, there were less CD133+ cells. The distributions of the individual cell types in the D0-D3 divisions on culture day 5 are clearly visible in Figure 2.

CD38+ antigen determination

The CD38-population was monitored in order to determine the distribution of primitive HSCs in individual populations. Numbers of the CD133+/38-and CD34+/38-cells from the BM and PBSCs were monitored using flow cytometry on days 0, 3 and 6 during cultivation. The results are shown in Table 3. The differences between the BM populations are also documented in Figure 3. During CD38+ antigen determination in both types of CML samples (Ph+ and Ph-), it was observed that the CD133+ population generally contained more CD38cells than the CD34+ population. The measurements on samples from healthy volunteers confirmed the same trend: the CD133+ population contained 9.2% CD38-cells and the CD34+ population contained only 3.5% on day 0.

Mutual distribution of individual populations

The amounts of cells isolated from 5 ml of BM or PBSCs were quantified. The numbers of CD133+ cells isolated from the BM of de novo diagnosed CML (CML Ph+) patients were 268,000-873,000 (median 634,000), and the numbers of CD34+ cells isolated from the BM of de novo diagnosed CML patients were 658,000-1,600,000 (median 1,197,000).

The cell counts for the patients in molecular remission (CML Ph-) were lower: the numbers of CD133+ cells from the BM were 43,000-112,000 (median 76,000) and the numbers of CD34+ cells were 105,000-256,000 (median 183,000).

The cell counts for the healthy volunteers were also different: the numbers of CD133+ cells from the BM were 180,000-320,000 (median 221,000) and the numbers of CD34+ cells were 225,000-550,000 (median 413,000).

The numbers of CD133+ cells isolated from PBSCs were 78,000-288,000 (median 108,000) and the numbers of CD34+ cells isolated from PBSCs were 122,000-608,000 (median 495,000).

For both BM and PBSCs, the cell yields during CD133+ antigen isolation were about 50% of that during CD34+ antigen isolation.

To validate the presence of two times more CD34+ cells in both bone marrow and PBSC, we performed FACS analysis directly on the harvested samples. Because the distributions of both CD133+ and CD34+ cells are between 0.3% and 1.2%, the significance of the FACS analysis would not be sufficient. Therefore, we decided to deplete the lymphocyte fraction in order to obtain an enriched population. The measurements of the CD133+ and CD34+ distributions were performed on BM samples of one de novo CML (one sample), one CML in molecular remission, and one healthy donor and two samples of PBSC. The distributions in the enriched population were 25.3% ± 4.8% for CD34+ and 13.1% ± 3.4% for CD133+.

By comparing the expression of the CD133 and CD34 antigens using multicolor flow cytometry, we observed that CD133+ cells were clearly not in the subgroup of CD34+ cells [START_REF] Peterkova | The in vitro proliferation potential of CD133+ versus CD34+ hematopoietic stem cells[END_REF]. For example, the BM sub-population sorted from healthy volunteers using the CD133 antigen contained only 72% of CD34 cells. Table 4 shows the changes in the individual markers (CD133 and CD34) immediately after isolation and during cultivation.

From the discussion above, it is obvious that BM cells and PBSCs differ significantly. The CD133+ and CD34+ PBSC populations demonstrate a relatively large overlap; however, for the BM, the CD133+/34-subpopulation represents more than 30% of the entire CD133+ population (Table 4).

Analysis of the distribution of lineage-committed progenitors

Flow cytometry was used to study the distributions of the following antigens: CD71 for erythroid progenitors, CD10 for lymphocytic progenitors and CD33 for myeloid progenitors.

For the CD71 antigen, only day 0 was included in the study because EPO was not included in the cultivation medium; therefore, differentiation into the erythroid line was not supported.

The average median of these populations was 5.4% ± 1.3% (data not shown). The distribution was thus sufficient in all populations to permit the successful reconstitution of erythropoiesis in the presence of the relevant cytokines. Significant differences among the populations were not observed during analysis of the myeloid and lymphoid line markers. The p-values for comparisons between the individual populations were between 0.625 and 0.961. During cultivation, similar increases were observed across all populations (Figure 4).

The CFU-GM and CFU-E formations were used to compare the colony-forming capacities of CD133 and CD34 cells derived from the BM of the same healthy volunteer. In five separate experiments, cells were cultured for 15 days in methylcellulose-based semi-solid medium.

CD133 and CD34 cells displayed non-significant differences in CFU-GM formation (median number of colonies 43, cf. 39).

At the beginning of the experiment, all BM populations showed equal distributions of the CD71 marker. However, they showed different numbers of colonies during CFU-E experiments. Even though the differences in the number of colonies generated from CD133 and CD34 cells were not significant (at p < 0.05), larger numbers of these colonies were found in CD133 cells (median number of colonies was 36, cf. 25)

Analysis of VEGF distribution

As stated above, the presence of sinusoidal progenitor cells seems to be one of the most important factors for successful transplantation. The VEGFR antigen was used to monitor this subpopulation. As shown in Table 5 and Figure 5, the amount of cells bearing this antigen was significantly (p=0.0093) higher in the CD133+ BM populations compared to the CD34+ BM population. Furthermore, these results indicate that the concentration of these cells is not high enough to negatively affect the reconstitution of hematopoiesis.

Discussion

Currently, the transplantation of hematopoietic stem cells is one of the most extensively used treatments for multiple types of leukemia.

The transplantation of hematopoietic stem cells depends on four main factors: homing, which is influenced by the presence of adhesive molecules in the graft; graft engraftment, which is an immunological process; reconstitution of the blood cell population; and reconstitution of the stem cell population, which is related to the presence of stem cells in the graft.

Unfortunately, although these four factors have been extensively studied, the probability of non-relapse mortality is still far from zero. This problem provides the motivation for further investigation of the suitability of the most frequently used source of hematopoietic stem cells, CD34+ cells from PBSCs. Many studies that aimed to define the total amount of CD34+ cells [START_REF] Yoon | Higher infused CD34+ hematopoietic stem cell dose correlates with earlier lymphocyte recovery and better clinical outcome after autologous stem cell transplantation in non-Hodgkin's lymphoma[END_REF][START_REF] Siena | Therapeutic relevance of CD34 cell dose in blood cell transplantation for cancer therapy[END_REF] can be found in the literature.

This study focused on the CD133 and CD34 populations from BM and PBSCs. These populations were compared in terms of their mutual distributions. An attempt was made to evaluate the distributions of cells for the fast and permanent reconstitution of hematopoiesis and the presence of cells capable of reconstituting sinusoidal endothelial cells.

The first aim of this article was to determine the amount of cells that could be obtained from a given volume of BM or PBSCs and the expansion potential of the individual populations.

Because the sample used for isolation was split into two identical portions before MACS separation, it was possible to determine the population distributions in the samples. As described in the Results section, the cell yields for the CD133+ antigen were always about 50% of those for the CD34+ antigen in both the BM and PBSCs. Thus, this population is minor compared with the CD34+ population. A comparison of the relationships between these two populations resulted in the hypothesis that there is a functional hierarchy between CD133 and CD34 [START_REF] Goussetis | A functional hierarchy among the CD34+ hematopoietic cells based on in vitro proliferative and differentiative potential of AC133+CD34(bright) and AC133(dim/)-CD34+ human cord blood cells[END_REF]. This hypothesis was also based of the expression profiles for the two populations [START_REF] He | Differential gene expression profiling of CD34+ CD133+ umbilical cord blood hematopoietic stem progenitor cells[END_REF]. A comparison of the expansion potentials was also very interesting.

CD133+ cells had a more than two-fold higher expansion potential than the CD34+ cells, even for a short-term 6-day cultivation. Furthermore, it was clear that the CD133+ population also contained a high percentage of CD34-cells that are capable of expressing CD34+ during cultivation. Therefore, those cells can be regarded as a more primitive cell type. Some authors have suggested that the CD34-cell subpopulation is fully capable of reconstituting hematopoiesis [START_REF] Zanjani | Reversible expression of CD34 by adult human bone marrow long-term engrafting hematopoietic stem cells[END_REF][START_REF] Verfaillie | Kinetics of engraftment of CD34-and CD34+ cells from mobilized blood differs from that of CD34-and CD34+ cells from bone marrow[END_REF]. It is widely accepted that the more primitive population of HSCs does not express the CD38 antigen [START_REF] Mckenzie | Reversible cell surface expression of CD38 on CD34-positive human hematopoietic repopulating cells[END_REF]. This antigen is expressed even on lineagecommitted progenitors and especially on immune system cells, in which it is responsible for cell-to-cell interactions [START_REF] Deaglio | Human CD38: a (r)evolutionary story of enzymes and receptors[END_REF]. In this study, we demonstrated that the CD133 population (especially from the BM) contained a significantly higher amount of CD133+/CD38-cells compared with the CD34+ population. In the CD38-population, quiescent stem cells should also be present. Over long periods of time, those cells remain in the BM in a quiescent, nondividing state and are localized in the stem cell niches. They are also responsible for hematopoietic homeostasis and the creation of a stem cell pool hierarchy by asymmetric selfrenewal, which produces lineage-restricted, short-term repopulating cells and long-lasting repopulating cells (LTRCs). Recent studies have also shown that these cells have very important functions in repopulation dynamics, turnover, hierarchical structure, and the cell cycle status of human HSCs in the recipient BM microenvironment [START_REF] Yahata | Quiescent human hematopoietic stem cells in the bone marrow niches organize the hierarchical structure of hematopoiesis[END_REF]. Using the CFSE experiments, we demonstrated that a high percentage of cells in the CD133+ BM population neither divided nor were differentiated, even under the pressure of growth factors. They remained fully viable and retained CD133+ antigen expression. The presence of these cells is very important from the perspective of permanent hematopoiesis reconstitution after transplantation.

An equally important prerequisite for successful transplantation is the presence of lineagecommitted progenitors in the graft. This study demonstrated that lymphoid, erythroid, and myeloid progenitors were similarly present in all populations. Subsequent CFU-GM experiments also did not show any significant differences in the numbers of colonies generated. However, the CFU-E experiments showed differences: the CD133+ population was able to generate a slightly larger number of colonies. Finally, the content of those cells was sufficient for high quality, short-term hematopoiesis reconstitution.

CD133 is a marker of pluripotent plasticity. CD133-positive cells are capable of differentiating into various cell and tissue types, including a wide variety of hematopoietic and non-hematopoietic types [START_REF] Goussetis | A functional hierarchy among the CD34+ hematopoietic cells based on in vitro proliferative and differentiative potential of AC133+CD34(bright) and AC133(dim/)-CD34+ human cord blood cells[END_REF][START_REF] Loges | Identification of the adult human hemangioblast[END_REF][START_REF] Quirici | Differentiation and expansion of endothelial cells from human bone marrow CD133(+) cells[END_REF]. This characteristic could indicate that this cell pool is not appropriate for transplantation. However, this population also contains endothelial progenitors that play an important role during successful transplantation [START_REF] Hooper | Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells[END_REF]. High dose chemotherapy to a large extent destroys the architecture of the BM. When transplanted cells do not find relevant niches in the BM or cells capable of reconstituting the BM architecture are not present in the graft, the graft itself is not able to engraft [START_REF] Li | Bone marrow sinusoidal endothelial cells undergo nonapoptotic cell death and are replaced by proliferating sinusoidal cells in situ to maintain the vascular niche following lethal irradiation[END_REF]. The CD133+ population contains a higher percentage of EPCs than the CD34+ population. EPCs support the creation of sinusoidal endothelial cells. Endothelial cells are a mandatory component of the BM niche and play an important role in neoangiogenesis [START_REF] Peichev | Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors[END_REF]. During the evaluation of the VEGFR antigen (EPC marker) on BM cells, significant differences were not observed among the CD34 sub-populations (CML Ph+/Ph-, healthy volunteers). Significant differences were also not observed among CD133 sub-populations (CML Ph+/Ph-, healthy volunteers) [START_REF] Krauth | Immunohistochemical detection of VEGF in the bone marrow of patients with chronic myeloid leukemia and correlation with the phase of disease[END_REF].

The number of EPCs was measured in the CD34+ and CD133+ populations, and a significant difference was observed. The high pluripotent plasticity of CD133+ VGFR+ and CD34+ VEGFR+ cells was demonstrated using cultivation experiments. These cells did not proliferate into endothelial cells in the presence of relevant growth factors. During cultivation, the VEGFR antigen disappeared. Because an increase in apoptotic cell death corresponding to the percentage of EPCs was not observed during culture (97% viability), we suggest that the cultivation environment induced proliferation into the hematopoietic pathway.

Many recent studies have reported that the CD133+ cell population is either a subpopulation of CD34 cells [START_REF] Matsumoto | In vitro proliferation potential of AC133 positive cells in peripheral blood[END_REF][START_REF] Lang | Transplantation of a combination of CD133+ and CD34+ selected progenitor cells from alternative donors[END_REF][START_REF] Freund | Comparative analysis of proliferative potential and clonogenicity of MACSimmunomagnetic isolated CD34+ and CD133+ blood stem cells derived from a single donor[END_REF][START_REF] Hemmoranta | Transcriptional profiling reflects shared and unique characters for CD34+ and CD133+ cells[END_REF] or an independent population that is fully capable of supporting or reconstituting hematopoiesis for both patients and SCID mice [START_REF] Gordon | Large-scale isolation of CD133+ progenitor cells from G-CSF mobilized peripheral blood stem cells[END_REF].

There are articles in which CD133 was reported to be a marker for a new selection strategy for the purification of autologous grafts and transplantation [START_REF] Koehl | Autologous transplantation of CD133 selected hematopoietic progenitor cells in a pediatric patient with relapsed leukemia[END_REF]. In an article by Isidori [START_REF] Isidori | Positive selection and transplantation of autologous highly purified CD133(+) stem cells in resistant/relapsed chronic lymphocytic leukemia patients results in rapid hematopoietic reconstitution without an adequate leukemic cell purging[END_REF], the capacity of positively selected autologous CD133+ hematopoietic stem cells (HSCs) to reconstitute lymphomyelopoiesis in chronic lymphocytic leukemia is described. The authors state that the reinfusion of highly purified CD133+ HSCs facilitated a rapid and sustained recovery of hematopoiesis after myeloablative treatment in resistant/relapsed CLL patients.

The aim of the study by Hess et al. [START_REF] Hess | Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells[END_REF] was to identify an alternative HSC population to the standard CD34+ marker and show that cell selection based on ALDH activity and CD133 expression provides a novel method for purification of HSCs with long-term repopulating capacity Even when samples from patients with CML (Ph+, Ph-) were compared with samples from healthy volunteers, significant differences were observed in both cellular proliferation and the expression of relevant CD antigens in the individual populations.

In the PBSC populations, significant differences between CD133 and CD34 were not observed. The statistical significance (p) values of the monitored parameters were near 0.06.

In contrast, the differences for the BM were significant at p=0.015. This work demonstrates that the CD133+ cell population of BM shows many differences compared with the other populations included in the study. During the experiments described above, the CD133+ BM population had better cultivation and expansion potentials than the CD34+ population.

The population also contained a higher percentage of non-dividing CD38-cells. Those cells can be assumed to be more primitive component of the bone marrow, which may be important for long term hematopoiesis reconstitution. During analysis of the CFU-GM and CFU-E experiments, we did not observe significant differences between the CD133+ and CD34+ cells; therefore, from the perspective of short-term hematopoiesis, both populations are comparable.

Furthermore our study confirmed that the CD133+ BM population contains a significantly higher percentage of the so called endothelial progenitors in the CD133 population. Those cells play a crucial role in the reconstitution of the BM architecture and thus in engraftment.

In summary, the CD133+ selected population has many advantages over other studied populations.

There are many studies available in which the CD133+ enriched population was used for transplantation. For example, a report by Lang et al. showed the transplantation of a combination of CD133+ and CD34+ selected progenitor cells from alternative donors [START_REF] Lang | Transplantation of a combination of CD133+ and CD34+ selected progenitor cells from alternative donors[END_REF].

The patients showed rapid platelet recovery, whereas T cell regeneration was variable. The implication of these results is that CD133+ selection can be feasibly used for transplantation from alternative donors, and further trials should be conducted with total CD133+ separated grafts.

The results presented in this work confirm that the CD133+ population is suitable for transplantation. However, an important limiting factor is also presented. The content of the CD133+ cells in bone marrow is significantly lower than that of CD34+ cells. The logical solution therefore is the ex-vivo expansion, which should enable the application of the mentioned method for transplantation. Tables Table 1. The amount of input cells in the culture and fold increase of cell amount after three and six days of cultivation. The measured population viability in Day 0 was 99% in all cases.

Figure 1 .

 1 Figure 1. The flow cytometric analysis of experiments with CFSE. A) CD34+ cells from BM

Figure 2 .

 2 Figure 2. The distribution of analysed populations in individual divisions after five days of

Figure 3 .

 3 Figure 3. The flow cytometric analysis of CD133+/CD38-and CD34+/CD38-populations

Figure 4 .

 4 Figure 4. The visualization of CD33+ and CD10+ cells in the analyzed populations on day 0

Figure 5 .

 5 Figure 5. The graphic visualization of VEGFR+ cells from healthy volunteers' BM in all

  Figure 1 Click here to download high resolution image

Table 2 .

 2 The amount of cells bellow 30,000 was not included in the calculation of fold increase. The percentage of cells (median) in individual divisions after a six-day cultivation *healthy volunteers -H.V.

					Day 3	Day 6
		Type of isolation	Day 0 Cell count median (cell/ml)	Fold increase (median) Average population	Fold increase (median) Average population
					viability (%)	viability (%)
			CML Ph +	65,000 (44,000-85,000)	4.5 (2.5-6.0) 94%	24 (18.0 -32.0) 91%
		CD133+	CML Ph -	29,000 (10,000*-32,000)	4.0 (1.5-5.0) 94%	19 (12.0 -29.0) 91%
			healthy	60,000	4.5 (2.5-5.0)	21 (16.0 -30.0)
	BM		volunteers CML Ph +	(40,000-80,000) 97,000 (80,000-110,000)	94% 4.8 (3.0-4.3) 96%	91% 11 ( 5.0-16.0) 88%
		CD34+	CML Ph -	37,000 (30,000-60,000)	3.2 (1.2-4.0) 96%	9 ( 5.0-14.0) 88%
			healthy	90,000	4.8 (3.0-4.3)	10 ( 7.0-15.0)
			volunteers	(80,000-100,000)	96%	88%
	PBSC	CD133+ PBSC CD34+ PBSC	32,000 (10,000*-94,000) 41,000 (30,000-85,000)	3.9 (2.2-4.8) 92% 3.8 (1.9-4.7) 93%	13( 8.0-19.0) 90% 11 (9.0-14.0) 90%

*

Table 3 .

 3 The percentage of CD38 in analyzed populations on day 0 and after 3 and 6 days of cultivation. "H.V." stands for healthy volunteers

	Content of			BM (median %)			PBSC (median %)
	CD 38 -in individual populations	CD133+/CD38 -CML Ph + CML Ph -H.V.*	CD34+/CD38 -CML Ph + CML Ph -H.V.*	CD133+/ CD38 -	CD34+/CD3 8 -
	Day 0	8.4	6.6	9.2	3.1	2.9	3.5	5.4	1.2
	3	5.1	3.8	4.2	2.1	1.5	2.5	1.6	1.2
	6	2.6	2.3	2.4	1.6	1.4	1.2	0.7	0.6

*

Table 4 .

 4 The percentage of mutual distribution of CD133+ and CD34+ antigens of analyzed populations on day 0 and after 3 and 6 days of cultivation. For example cells isolated using CD133 antigen from BM of CML Ph+ patients contained only 52% of CD34+ cells in day 0.

	* "H.V." stands for healthy volunteers
			BM		PBSC
		CD133+ isolation	CD34+ isolation
			(median)		(median)	CD133+	CD34+
						isolation	isolation
	CD34+ antigen	CML Ph +	CML Ph -H.V.*	CML Ph +	CML Ph -H.V.*	(median)	(median)
	Day 0	52% 64% 72% 98% 95% 98%	74%	97%
	3	56% 59% 61% 74% 69% 59%	62%	68%
	6	51% 54% 47% 42% 52% 45%	56%	42%
	CD133+
	Antigen
	Day 0	94% 97% 96% 45% 51% 53%	97%	62%
	3	65% 62% 67% 28% 29% 34%	67%	46%
	6	42% 48% 29% 19% 19% 18%	39%	29%

Table 5 .

 5 The percentage of VEGFR-positive cells in all analyzed populations on day 0 and after 3 and 6 days of cultivation.

	* "H.V." stands for healthy volunteers
			BM (median %)			PBSC (median %)
	VEGF+
			CD133+		CD34+
	antigen
		CML	CML		CML	CML		CD133+	CD34+
				H.V.*			H.V.*
		Ph +	Ph -		Ph +	Ph -
	Day 0	17.1 13.8 12.1	6.6	5.2	7.4	11.8	10.6
	3	2.4	2.1	1.9	1.3	1.6	2.3	1.2	1.5
	6	0.5	0.7	0.3	0.8	0.9	0.7	0.5	0.5

Acknowledgments

This work was generously supported by the Ministry of Education of the Czech Republic grant MSM-0021622430, NPVII (2B06052). We would like to thank the Dept. of Internal Medicine -Hematooncology, Hospital Brno, Jihlavska 20, 625 00 Brno, Czech Republic, especially Zdenek Koristek M.D. and Zdenek Racil M.D. for the collection of blood and bone marrow samples.