Long term outcomes of intracarotid arterial transfusion of circulatory-derived autologous CD34 + cells for acute ischemic stroke patients-A randomized, open-label, controlled phase II clinical trial

Abstract

Background: This phase II randomized controlled trial tested whether the intracarotid arterial administration (ICAA) of autologous CD34 + cells to patients within 14 ± 7 days after acute ischemic stroke (IS) could be safe and further improve short- and long-term outcomes.

Methods: Between January 2018 and March 2022, 28 consecutive patients were equally randomly allocated to the cell-treated group (CD34 + cells/3.0 × 10⁷/patient) or the control group (receiving optimal medical therapy). CD34 + cells were transfused into the ipsilateral brain infarct zone of cell-treated patients via the ICAA in the catheterization room.

Results: The results demonstrated 100% safety and success rates for the procedure, and no long-term tumorigenesis was observed in cell-treated patients. In cell-treated patients, the angiogenesis capacity of circulating endothelial progenitor cells (EPCs)/Matrigel was significantly greater after treatment than before treatment with granulocyte colony-stimulating factor (all p < 0.001). Blood samples from the right internal jugular vein of the cell-treated patients presented significantly greater levels of the stromal cell-derived factor 1α/EPC at 5, 10 and 30 min compared with 0 min (all p < 0.005). The National Institute of Health Stroke Scale scores were similar upon presentation, but a greater response was observed by Days 30 and 90 in the cell-treated group than in the control group. Tc-99 m brain perfusion was significantly greater at 180 days in the cell-treated group than in the control group (p = 0.046). The combined long-term end points (defined as death/recurrent stroke/or severe disability) were notably lower in the control group compared with the cell-treated group (14.3% vs. 50.0%, p = 0.103).

Conclusion: Intracarotid transfusion of autologous CD34 + cells is safe and might improve long-term outcomes in patients with acute IS. Trial registration ISRCTN, ISRCTN15677760. Registered 23 April 2018- Retrospectively registered, https://doi.org/10.1186/ISRCTN15677760.

Keywords: Acute ischemic stroke; Angiogenesis; Endothelial progenitor cells; Intracarotid transfusion of CD34 + cells; Neurological outcomes.

Fig. 1

Circulatory EPC levels in the cell-treated group and control group after G-CSF treatment A1–A4 Flow cytometric analysis for the identification of CD34 + KDR + CD45^dim cells. A5 Analytical result of the number of CD34 + KDR + CD45^dim cells, * vs. other groups with different symbols (†, ‡), p < 0.0001. B1–B4 Flow cytometric analysis for the identification of CD34 + CD133 + CD45^dim cells. B5 Analytical result of the number of CD34 + CD133 + CD45^dim cells, * vs. other groups with different symbols (†, ‡), p < 0.0001. C1–C4 Flow cytometric analysis for the identification of CD31 + CD133 + CD45^dim cells. C5 Analytical result of the number of CD31 + CD133 + CD45^dim cells, * vs. other groups with different symbols (†, ‡), p < 0.0001. D1–D4 Flow cytometric analysis for the identification of CD34 + CD133 + KDR + cells. D5 Analytical result of the number of CD34 + CD133 + KDR + cells, * vs. other groups with different symbols (†, ‡), p < 0.0001. E1–E4 Flow cytometric analysis for the identification of CD133 + cells. E5 Analytical result of the number of CD133 + cells, * vs. other groups with different symbols (†, ‡), p < 0.0001. F1–F4 Flow cytometric analysis for the identification of CD34 + cells. F5 Analytical result of the number of CD34 + cells, * vs. other groups with different symbols (†, ‡), p < 0.0001. All the statistical analyses were performed via one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 14 for each group). Symbols (*, †, ‡) indicate significance (at the 0.05 level). EPC = endothelial progenitor cell; G-CSF = granulocyte colony-stimulating factor. G1 = the control group; G2A (cell-treated group) = pre-G-CSF treatment; G2B = post-G-CSF treatment. G3 = plasma containing isolated CD34 + cells

Fig. 2

Estimation of angiogenesis using the Matrigel assay with and without G-CSF treatment in both groups of patients A An example of how to identify angiogenesis variables, including tubular length (blue line), cluster formation (red circle) and network formation (blue triangle). B–E Matrigel assay for identification of angiogenesis capacity in G1 (i.e., the control group) B and G2 (the cell-treated group) at the time points of pre-G-CSF treatment C and post-G-CSF treatment D as well as at the time of plasma isolation from CD34 + cells E. The pink arrows indicate tubular length. Red arrows indicate cluster formation. The black dotted line circle indicates network formation. F Analytical result of the number of tubules, * vs. other groups with different symbols (†, ‡), p < 0.001. G Analytical result of total tubular length, * vs. other groups with different symbols (†, ‡), p < 0.001. H Analytical result of the mean tubular length, * vs. other groups with different symbols (†, ‡), p < 0.0001. I Analytical result of cluster formation, * vs. other groups with different symbols (†, ‡), p < 0.0001. J Analytical result of network formation, * vs. other groups with different symbols (†, ‡), p < 0.0001. All the statistical analyses were performed using one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 14 for each group). Symbols (*, †, ‡) indicate significance (at the 0.05 level). G-CSF = granulocyte colony-stimulating factor; G1 = the control group. G2A (cell-treated group) = pre-G-CSF treatment; G2B = post-G-CSF treatment. G3 = plasma containing isolated CD34 + cells

Fig. 3

Serial changes in SDF-1α levels and the EPC/HPC ratio in the RIJV in14 cell-treated patients A ELISA findings for identification of the time courses of stromal cell-derived factor (SDF)-1α in cell-treated patients; analytical result of SDF-1α, * vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. B Flow cytometric analysis of the levels of CD34 + KDR + CD45^dim cells at baseline and over time; analytical result of the number of CD34 + KDR + CD45^dim cells; * vs. other groups with different symbols (†, ‡, §), p < 0.0001. C Flow cytometric analysis of the levels CD34 + CD133 + CD45^dim cells at baseline and over time; analytical result of the number of CD34 + CD133 + CD45^dim cells; * vs. other groups with different symbols (†, ‡, §), p < 0.0001. D Flow cytometric analysis of CD34 + CD133 + KDR + cells at baseline and over time; analytical result of the number of CD34 + CD133 + KDR + cells; * vs. other groups with different symbols (†, ‡), p < 0.0001. E Flow cytometric analysis of CD31 + CD133 + CD45^dim cells at baseline and over time; analytical results of the number of CD31 + CD133 + CD45^dim cells; * vs. other groups with different symbols (†, ‡, §, ¶, α), p < 0.0001. F Flow cytometric analysis of CD34 + cells and the analytical results of the number of CD34 + cells at baseline and over time; * vs. other groups with different symbols (†, ‡, §), p < 0.0001. G Flow cytometric analysis of CD133 + cells and the analytical results of the number of CD133 + cells at baseline and over time; * vs. other groups with different symbols (†, ‡, §), p < 0.0001. All the statistical analyses were performed using one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 14 for each group). Symbols (*, †, ‡) indicate significance (at the 0.05 level). G-CSF = granulocyte-colony stimulating factor; RIJV = right internal jugular vein; EPCs = endothelial progenitor cells

Fig. 4

Longitudinal evaluation of serial changes in the NIHS and Barthel index in each group of patients A Time courses of the National Institute of Health Stroke Scale (NIHSS) scores in the cell-treated group and control group. The results were analyzed via repeated-measures ANOVA, with p < 0.0001 in cell-treated group and p < 0.001 in control group. B Time courses of the Barthel index in the cell-treated group and control group. The results were analyzed via repeated-measures ANOVA, with p < 0.0001 in the cell-treated group and p < 0.0001 in the control group

Fig. 5

Images from brain perfusion single-photon emission computed tomography (SPECT) studies using 99mTc-labeled radiopharmaceuticals A and B Images indicating the baseline (A) prior to and 6 months (B) after CD34 + T-cell therapy in the cell-treated group (G1). C and D Baseline (C) and 6-month follow-up (D) data after acute ischemic stroke without CD34 + cell therapy in the control group (G2) (i.e., the control group). In the cell-treated group, the results of Tc-99 m ECD brain perfusion SPECT revealed that, compared with that at baseline, brain perfusion (white arrows) was notably increased by 6 months after CD34 + T-cell therapy. On the other hand, in the control group, compared with that at baseline, brain perfusion (white arrows) was markedly decreased at 6 months after acute ischemic stroke