Neurological and functional recovery in human stroke are associated with peripheral blood CD34+ cell mobilization
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A spontaneous mobilization of Peripheral Blood-Mononuclear CD34+ Cells (PB-MNC-CD34+) has recently been reported in human myocardial infarction and found to be related to improved heart function and survival. However, nothing is known regarding a possible relation between PB-MNC-CD34+ mobilization and neurological recovery in human acute cerebral ischemia.
Methods and Results
PB-MNC-CD34+ were determined daily after an acute cerebral ischemic attack for 14 days in 25 patients with acute ischemic stroke and compared with controls. Results indicated that stroke was followed by large and bursting mobilizations of PB-MNC-CD34+. The amplitude of the mobilizations was similar to those observed in Granulocyte Colony Stimulating Factor (G-CSF) conditioned aplastic patients following myeloablative therapy before leukapheresis and autologous bone graft. The extent of PB-MNC-CD34+ mobilization in each patient was directly related to neurological and functional recoveries as assessed by NIH Stroke Scale, and modified Rankin Scale respectively.
The mobilization of PB-MNC-CD34+ cells might be predictive of neurological and functional recovery.
Keywordsangiogenesis neurological recovery functional recovery stem cell plasticity stroke
Recent studies suggest that CD34+ Peripheral Blood-Mononuclear Cells (PB-MNC-CD34+) might play a role in ischemic diseases [2, 4, 7, 31]. Asahara et al.  first reported the presence of endothelial progenitor cells derived from PB-MNC-CD34+ in adult peripheral blood and showed that in animals these cells are mobilized during vascular endothelial growth factor (VEGF)-induced neovascularization. VEGF is well known to be secreted in response to a hypoxic stress both in vivo and in vitro [4, 20, 25]. Moreover, transplanted CD34+ cells have been shown to regenerate ischemic brain or heart tissue in vivo . Shintani et al.  further reported a PB-MNC-CD34+ mobilization in patients with acute myocardial infarction. In vitro stimulation of human PB-MNC-CD34+ by VEGF leads to the release of cells that express macrophage, endothelial or neuronal markers and properties [6, 17]. More recently, Taguchi et al.  have shown that the administration of human CD34+ cells after stroke in a mouse model of middle cerebral artery occlusion, enhances neurogenesis: human CD34+ cells were shown to secrete some angiogenic factors including VEGF, HGF and IGF; these factors would contribute to the development of a pattern of newly formed vascularization which in turn would provide an optimal environment for neurogenesis.
Taken together these findings suggested that PB-MNC-CD34+ might contribute to tissue repair following acute ischemic stroke (AIS) in humans.
The objective of this study was 1) to confirm data on the hypothesis that AIS induces a mobilization of PB-MNC-CD34+; 2) to elucidate the kinetic of mobilization of CD34+ cells daily after ischemic stroke and 3) to assess whether the extent of this mobilization correlates with neurological and functional recovery.
Materials and methods
Patient with AIS
Inclusion criteria were: moderate to severe acute atherothrombotic ischemic stroke, involving the middle cerebral artery (MCA) territory in patient aged up to 80 years and with National Institute of Health Stroke Scale (NIHSS) between 8 and 24. Non inclusion criteria were defined to exclude any condition that might have led to changes in PB-MNC-CD34+ counts. Among these aer: administration of unfractioned heparin or statins and/or medical history of stroke, angina pectoris, cerebral trauma, peripheral arteriopathy, neoplasm. Exclusion criteria were previous ischemic or haemorrhagic stroke as assessed by clinical history and CT (cerebral tomodensitometry), lacunar stroke, history or sign of myocardial infarction, diabetes, major organ insufficiency, anemia, any hypoxic disease, any sign of infection or inflammatory biological pattern. We excluded any menstruating or possibly pregnant women. Twenty five voluntary atherothrombotic AIS patients (45–75 years) admitted in our stroke unit met the inclusion criteria and were enrolled consecutively. Ischemic stroke was confirmed by cerebral CT or MRI on admission, and at 24 to 48 hours. Infarction concerned at least 1/3 of the MCA territory.(NIHSS and modified Rankin Scale (m-RS) were used to assess neurological and functional recovery respectively. NIHSS and m-RS were measured at admission, at one and three months by a certified neurologist blinded for PB-MNC-CD34+ counts. All patients had low molecular weight heparin (LWMH) for deep vein thrombosis prevention and acetylsalicylic acid treatment for secondary prevention of stroke. None of the patients was eligible for being treated by thrombolysis. This prospective study has been approved by the local ethic committee. Written informed consent was obtained from all patients.
Three control groups were enrolled: (i) healthy controls (25–45 years) with no suspected or known disease and free from any medication for at least one year (n = 25); (ii) patients matched for sex and age with a confirmed diagnosis of neuro-degenerative disease (Alzheimer: n = 10, Parkinson: n = 15) but free from any vascular disease. Forty percent of these patients (bedridden) received (LWMH) for deep vein thrombosis prevention. (iii) G-CSF-conditioned aplastic patients following myeloablative therapy before leukapheresis and autologous bone graft (n = 25).
Quantification of PB-MNC-CD34+
The circulating PB-MNC-CD34+ absolute counts were quantified every day (8 AM) for 14 consecutive days. Sampling started on the first day following stroke. Heparinized PB samples were treated immediately and assayed for the absolute PB-MNC-CD34+ counts using standardized procedures. Briefly, blood samples were stained with a phycoerythrin-conjugated anti-CD34 monoclonal antibody (HPCA2, Becton Dickinson, CA, USA). Erythrocytes were then lysed on a TQ-Prep workstation (Beckman- Coulter, Fl, USA). Absolute PB-MNC-CD34+ counts were quantified using a calibrated flow cytometer with a standardized absolute flow-count method (Flow-Count Fluorospheres, Beckman Coulter, Fl, USA) and expressed as number of absolute CD34+ cells per ml of blood. Analyses were performed using a MCL-XL flow cytometer (Beckman Coulter, Fl, USA). CD34+ standardized reference samples (Fluotrol, Bioergonomic, Min, USA) were analyzed every day for intra-laboratory quality assurance. In control subjects, PB-MNCCD34+ counts were quantified on admission for patients with neuro-degenerative disease and at day 1, 2, 3, 4 and 5 for G-CSF-conditioned aplastic patients.
We used the non-parametric test of Kruskal-Wallis Rank and Mann-Whitney U test. Since there was multiple testing, we used Bonferroni correction. To assess age as potential confounding factor regarding recovery, we used two-factor analysis of variance. All results are expressed as mean ± standard deviation.
Neurological and functional recovery
We then assessed functional recovery, defined as the difference in m-RS between admission and 3 months after stroke. This difference was larger in the high mobilizers group (3.14 ± 1.06 vs. 1.42 ± 1.04, p < 0.03). The amplitude of PB-MNC-CD34+ mobilization was also correlated with the m-RS improvement at 3 months (r = .69, p < 0.05).
It is noteworthy that the two patients (60 and 43 year old) who showed the largest increase in PB-MNC-CD34+ counts ( > 80 000 cells/ml) completely recovered from stroke while their baseline NIHSS was among the most severe (19 and 22 respectively).
Age analysis showed that high mobilizers were slightly younger (60.5 ± 2.5 vs. 66.8 ± 3.4, p < 0.05) and it is well known that PB-MNC-CD34+ counts decrease with age [6, 23]. We therefore used a two-factor analysis of variance to determine whether age could be a confounding factor. The positive correlation between the population CD34+ cells and the neurological recovery is retained (r = 0.67) even if the youngest patients are excluded from the calculation. Results indicated statistically significant difference in neurological and functional recovery between high and poor mobilizers.
PB-MNC-CD34+ represent a small subset of human PB mononuclear cells which are well known to be mobilized in response to exogenous cytokines such as G-CSF, but also to drugs such as cyclophosphamide . Several studies have reported increases in PB-MNC-CD34+ in patients who suffered cardiovascular acute ischemic events [19, 24, 26]. PB-MNC-CD34+ mobilization in AIS is less documented [20, 27].
Already in 2002 we had shown that an ischemic event such as stroke induced an important release of CD34+ cells in the peripheral blood . Now we can confirm these findings, showing that PB-MNC-CD34+ cells are mobilized after ischemic stroke, but we also show that these cells are not mobilized in a constant pattern. Rather PB-MNC-CD34+ are released in short lived bursts that can be easily overlooked if counts are not assessed on a daily basis. A bursting pattern of PB-MNC-CD34+ is well known to hematologists who monitor G-CSF stimulated aplastic patients . In AIS patients the bursts mainly appear 1–3 days and 7–10 days after the acute event. The importance of the mobilization is comparable with those of G-CSF stimulated patients. Moreover, we observed that patients who mobilize their PB-MNC-CD34+ to larger extent show better neurological and functional recovery assessed at 1 and 3 months.
The origin of PB-MNC-CD34+ cells and their fate are not known. It is of interest to note that Zhao et al.  recently identified pluripotent stem cells from a PB monocyte-subset in healthy subjects. This in vitro M-CSF stimulated-cell subset expresses CD34+ hematopoietic stem cell markers. They can be induced to acquire endothelial or neuronal phenotypes in response to nerve growth factor or VEGF . Slevin et al.  reported that AIS is followed by sustained increases in plasma VEGF levels which is known to induce a mobilization of PB-MNC-CD34+ in mice . It has also been proposed that bone marrow derived stem cells can differentiate in vitro into neurons, cardiomyocytes, smooth muscle cells or endothelial cells [1, 9, 10, 13, 22, see, however, ref. 3, 16, 18 for a different interpretation].
Considering that human CD34+ stem cells can acquire both neuronal or endothelial cell phenotypes, one could suggest that human PB-MNC-CD34+ spontaneously mobilized in AIS patients might contribute to cerebral tissue repair. This hypothesis is fully consistent with recent results obtained in murine models [8, 11, 12, 21, 28]. Haematopoietic CD34+ progenitor cells are a rich source of several pro-angiopoietic factors which contribute to neovascularization and therefore to neurogenesis after stroke.
As a result we suggest that PB-MNC-CD34+ mobilization might explain differences in recovery between patients presenting the same initial clinical picture. Furthermore, the better recovery displayed by young patients might be related to the fact that their levels of PB-MNC-CD34+ cells are higher than in older patients in whom the CD34+ cell are mobilized to a lower extent. These data suggest that a large mobilization of PB-MNC-CD34+ is a positive and independent marker for better and faster neurological recovery. Future investigations are now required (i) to document the PB-MNC-CD34+ mobilization bursting pattern (ii) to define the pluripotent stem cell plasticity of these cells and (iii) to evaluate the possibility to expand them for cell therapy.
Authors are very thankful to: Dr Frederic Berthier (from the “Département d’Information Médicale, CHU de Nice) for his valuable contribution in biostatistics; Mr Julien Nivet, Clinical Research Associate; the National Institute of Science and Medical Research and the University Hospital of Nice. We are also very thankful to the “UEFCT’s technicians”.
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