cerebrovascular disorder , mainly including ischemic and hemorrhagic stroke , is the third cause of death after cardiovascular disease and cancer   and the leading cause of long - term disability in adults in china .
although stroke mortality has declined in the past decades , its prevalence and morbidity increased dramatically because of enhanced stroke survival .
about 40% of stroke survivors have certain degree of impairment and 1530% are severely disabled .
up to date , many targets within the cascade of neuronal death have been identified . however , neuroprotective strategies in treating poststroke with sequela are controversial .
since neural progenitor and stem cells were discovered in the adult brain for the first time in 1992 , different experimental stroke models indicated that neural progenitor and stem cells with activities of migration and differentiation are activated by hypoxia in brain [ 7 , 8 ] .
increasing evidence from basic studies indicates that cord blood mononuclear cells , bone marrow mononuclear cells , and bone marrow stromal cells can survive in postischemia tissue and reduce neuronal damage when transplanted into rodents subjected to cerebral infarction [ 912 ] .
bone marrow - derived cd34 positive cells have previously been used to reconstitute the hematopoietic system after radiation or chemotherapy . recently
, cd34 positive cells have been widely reported to induce therapeutic angiogenesis in animal models of myocardial , peripheral , and cerebral ischemia [ 1315 ] . circulating bone marrow - derived immature cells , including cd34 positive cells ,
in addition , the level of circulating cd34 positive cells was decreased in patients with vascular - type cognitive impairment .
recently , the positive correlation between the number of circulating cd34 positive cells and patients with acute stroke stress has been demonstrated , which suggests that increased numbers of circulating cd34 positive cells might benefit neurologic function recovery .
although previous clinical data have demonstrated a positive benefit in patients with stroke , there are no clinical reports on autologous cd34 positive precursor cells in the treatment of poststroke . in our ongoing clinical study
, we designed a protocol to determine the safety and potential efficacy of g - csf mobilization of bone marrow - derived cd34 positive cells in the treatment of patients with a history of stroke . in the current study , we focused on the investigation of the safety of cd34 positive cell administration .
the study was approved by the institutional review boards of 323 hospital of pla ( xi'an , china ) .
the procedure of our ongoing clinical study was under steps criteria [ 20 , 21 ] .
eight male patients with a history of stroke and left sequela were the target population in this study . the inclusion and exclusion criteria
all patients were treated subcutaneously with 5  g / kg body weight g - csf for five consecutive days .
leukapheresis was performed using a cobe spectra cell separator ( cobe bct , lakewood , co ) .
blood pressure and heart rate were monitored , and a physician was available for any emergencies during the performance .
cd34 positive cells were extracted from leukapheresis products by immunoselection using an automated cell separation system ( miltenyi biotec , bergisch gladbach , germany ) based on the mechanism of selectivity of a monoclonal cd34 antibody conjugated to a magnetic particle [ 22 , 23 ] . for purity analysis of immunoselected cd34 positive cells ,
cells were evaluated by flow cytometry using cd34-pe - conjugated monoclonal antibody . then the immunoselected cd34 positive cell components were divided into multiunits stored in liquid phase nitrogen at 196c and will be infused later .
0.83.3  10 ( varying from different patients ) in 2  ml medium was used per time .
the cells from the good manufacturing practice ( gmp ) workshop were ensured to have been fully screened against potential pathogens and meet the eligible criteria for clinical use .
patients ' x - rays , computed tomography scans , and magnetic resonance images of spine were reviewed prior to administration .
intrathecal injection by lumbar puncture was repeated four or five times depending on the patient 's condition .
patients were given local anesthesia , and lumbar puncture was performed in the lateral decubitus position , prepped , and draped in sterile fashion , and the needle was placed in the lumbar cistern .
2  ml of cerebrospinal fluid was removed and replaced by 2  ml of immunoselected cd34 positive cell suspension .
clinical chemistry monitoring , including tests of blood count , liver function , renal function , lipids , and glucose , was performed before , 1 day , and 7 days after cell treatment , respectively .
in addition , nih stroke scale and barthel index , which are widely used in evaluation of functional disability , were documented at baseline and at 1st , 6th , and 12th months in the 12-month followup .
as shown in table 2 , 8 male patients ( age : 2258  yrs , median : 42  yrs ) with a history of stroke ( range : 17  yrs , median : 2  yrs ) were enrolled .
g - csf and leukapheresis were well tolerated in all patients during the clinical treatment course .
a mean of 1.8  10 of cd34 positive cells ( range 0.83.3 ) was collected by immunoselection .
flow cytometry analysis showed that cd34 positive cells in the immunoselected fraction were above 85% .
no allergic or immunological reactions were noted at the time of injection or while under observation . no chest pain and abnormal level of cardiac enzymes were observed .
in addition , there is no statistical difference of blood tests ( tables 3 and 4 ) before treatment , 1 day , and 7 days after treatment .
therefore , the above data indicated that cell infusion into the subarachnoid space was a safe procedure in patients with poststroke .
as shown in table 5 , we observed that the daily activities had a gradual improvement tendency in the 12-month followup .
mean of nih stroke scale and bi scores shifted from 7.5 and 43.8 at baseline to 4.4 and 64.4 in 12 months later , respectively .
the improvements of muscle tone , rigidity , and motor power were observed in all patients .
however , we must emphasize that patients with stroke do gradually improve their functions , and here it is not possible to define whether the therapy itself or the natural history of the disease is responsible for the improvements noted .
in the current study , we observed that g - csf administration , leukapheresis , and intrathecal administration of cd34 positive cells were safe and well tolerated in a group of patients with past cerebral infarction with 12-month followup .
g - csf has been used extensively in the last decades to mobilize cd34 positive hematopoietic stem cells in neutropenic patients and for reconstitution of bone marrow [ 24 , 25 ] . recently , g - csf as a promising drug candidate can reduce neuroinflammation and potentiate both neurogenesis and angiogenesis after ischemic stroke by promoting bone marrow cell migration into the ischemic brain [ 26 , 27 ] .
many years of clinical experience with g - csf have shown the safety of this agent in the general population .
increasing clinical studies indicated mobilization of cd34 positive precursor cells in patients with acute stroke by g - csf is safe and effective [ 2931 ] . in the current study ,
patients with past cerebral infarction were treated by g - csf ; subsequently , leukapheresis was performed for cd34 positive cells collection by immunoselection .
there were no emergencies , and adverse events , such as hyperviscosity syndromes , occurred in the whole process .
previously , it has been reported that intrathecal administration was safe with a variety of stem cells including adipose tissue - derived mesenchymal stem cells , embryonic stem cell - derived hematopoietic stem cells , and autologous bone marrow - derived hematopoietic stem cells in the treatment of neurological disorders [ 32 , 33 ] . in this study ,
intrathecal injection of the cd34 positive cells into subarachnoid space was performed , thereby migrating more efficiently into the injured area related to stroke through cerebrospinal fluid compared to the intravenous route .
what is more , there were no side effects including headache , low - grade fever , and meningism and vomit occurring in eight patients .
cd34 positive cells comprise a population enriched with endothelial progenitor cells whose contribution to neovasculature includes both direct participation in forming the neovessel and regulatory roles and the sources of growth / angiogenesis factors including vegf , hgf , and igf-1 , contributing to the maintenance of the microvasculature . the positive correlation between levels of circulating cd34 positive cells and neovascularization in patients with postcerebral ischemia
given the variance of age , transplantation cell numbers , and stroke history , the discrepant response to the treatment is apprehensible .
the current data showed a trend that cd34 positive cells might achieve clinical benefits in the treatment of patients with poststroke .
however , improvements do occur naturally in patients with stroke , and it is not possible to define whether the therapy itself or the natural history of the disease was responsible for the improvements noted . in conclusion , this study indicates that intrathecal administration of cd34 positive cells in patients with past stroke is a relatively safe and simple procedure with no long - term adverse effects .
however , further larger clinical studies with multiple groups including blinding and placebo controls will be required to confirm our findings in the current study .
 regenerative strategies in treatment of stroke have great potential . 
 the goal of the current study was to investigate safety of intrathecal administration of autologous cd34 positive cells in treatment of patients with poststroke . 
 a total of eight male patients with a history of stroke were enrolled . 
 the patients were treated subcutaneously with 5  g / kg body weight rhg - csf for 5 consecutive days , and then leukapheresis was performed to concentrate cells for cd34 positive immunoselection . 
 all patients underwent intrathecal administration of cd34 positive cells via lumbar puncture . 
 the primary outcome was safety evaluation for 12-month followup . 
 in addition , behavioral function was evaluated with nih stroke scale and barthel index 1 , 6 , and 12 months after the last treatment , respectively . 
 there were no major adverse events , and abnormal changes of blood tests during the whole treatment process included intrathecal administration and 12-month followup . 
 the main message from the current study was that administration of g - csf - mobilized autologous cd34 positive cells in patients with poststroke was safe . 
 future studies with larger population and control group are needed to confirm the safety and investigate the efficacy . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion