Inraventricular transplantation of autologous umbilical cord blood (UCB)-derived cells for global hypoxic-ischemic brain injury; case report

Inraventricular transplantation of autologous umbilical cord blood (UCB)-derived cells for global hypoxic-ischemic brain injury; case report

Inraventricular transplantation of autologous umbilical cord blood (UCB)-derived cells for global hypoxic-ischemic brain injury; case report

Domanska-Janik K, Kotulska K, Habich A, Kmiec T, Sarnowska A, Janowski M, Oldak T, Kropiwnicki T, Jurkiewicz E, Litwin M, Boruczkowski D, Walecki J, Roszkowski M, Lukomska B, Jozwiak S

INTRODUCTION
Hypoxic-ischemic encephalopathy remains one of the most devastating conditions in children, resulting in brain atrophy and persisted functional neurological impairment. Current therapeutic strategies include rehabilitation and antiepileptic therapy when needed, seem not to be effective enough once the injury has occurred. Transplantation of neural stem/precursor cells e.g. human umbilical cord blood derived cells, are broadly proposed as an alternative method to repair damaged brain. Double-blinded clinical trials have proven cell therapy to be safe and feasible but no significant clinical benefits were so far reported. Apart of this neurotransplantation is broadly offered to patients in several centers worldwide not covered by public health system as it still does not meet the criteria for evidence based medicine. Basing on our previous data showing that indeed cord blood could be a source of neural stem cells and could be effective in partial structural and functional recovery in vitro or in vivo in animal stroke models, we feel entitled to hypothesize that cord blood cell transplantation may be helpful also for treatment of global brain ischemia. Accordingly, with informed consent of patient, we were authorized by the Ethical Commission of Ministry of Health in our country to perform the first clinical study to determine whether the therapeutic benefit could be reached in child after autologous cord blood cell transplantation directly into lateral cerebral ventricle in the case of generalized brain damage caused by cardiac arrest's evoked global cerebral ischemia.

SUBJECT
Eighteen months old child at 5 month after the onset of cardiac arrest-induced cerebral hypoxicischemic injury resulting in a permanent vegetative state, received 3 monthly infusions of autologous UCB-derived-neurally-committed cells (UCB-NC) directly into lateral ventricles. This treatment as a supplementary to unsuccessful conventional broad rehabilitation program, has been approved and monitored by Ethics Committee at The Children's Memorial Health Institute, Warsaw. Transplantation material & method The autologous, nucleated UCB cells stored in the Polish Stem Cell Bank in Warsaw (PBKM SA) were cultured by 10 days in neurogenic conditions (Habich et al 2006). Resulting UCB-NCs were partly pre-labeled by SPIO (supraparamagnetic iron oxide; Guerbet SA-Endorem) for in vivo MR monitoring. The unlabeled UCB-NC were divided on 3 equal samples of 12 x 106 cells each and then frozen until transplantation. The first transplantation procedure consisting abut 30 % of SPIOlabelled cells was performed by means of neuronavigation. Radiological MR examination in vivo and in vitro was performed on the Siemens 1.5 T Sonata Vision equipped with clinical head receiver coil. For imaging the T2/SWI (Susceptibility Weighted Image) sequence has been employed. MRI was performed at 24 hours after each surgery and 4 months after the last one. For scaling sensitivity of MR signal different concentrations of SPIO labeled UCB-NC pellets were spun down in PCR tubes and scanned in the phantom consisting of agar and copper sulfate. Imaging of SPIO-labeled UCB-NC cells in vitro The cells fixed in 4% glutaraldehyde were stained by Psrl's Prussian blue method and counterstained for cell nuclei with neutral red. Parallel cell samples were analyzed immunocytochemically for their neural characteristic.

Fig.1 Phase contrast images of UCB-NC cultured for 10 days (A). SPIO-stained cells for 48 hours (B), insert with higher magnification. Specific immunostaining of UCB-NSC cultured in vitro for 10 days (C-H) visualizing the double staining for NF200 (red) and GFAP (green) markers with insert depicting higher magnification of these cells (C); TUJ1-positive cells (D); double staining for Nestin (red) and S100â markers (green) (E); double staining for ß-tubulin III (green) and Ki67 antigens (red) (F); doublecortin-positive cells (G); CXCR4-expressed cells (H). Cells stained with Alexa Fluor 546 (red) and Alexa Fluor 488 (green) were detected simultaneously and together with their nuclei revealed by use of Hoechst 33252 staining (blue). Scale bars 100μm.

Fig. 2 RT-PCR analysis of UCB-NC cells cultured in vitro for 10 days.

Fig. 3 MR examinations of a child 5 months following global ischemia performed the day before (A) and the day after the first transplantation of autologous umbilical cord blood stem cells into the right cerebral hemisphere (B). A: Note widened sulci between gyri of the cerebral cortex and widened supratentorial ventricular system with 12- mm- wide III ventricle as well as thinning of corpus callosum. No focal changes were seen. B: Labeled, transplanted UCB-NC cells visible as a small (3.6 mm in diameter) area of hypointense signal (arrow), extending along ependyma from the cornu posterius through corpus to cornu anterius of the right lateral ventricle.

Fig. 4 Longitudinal MR imaging study of cornu posterius of right lateral ventricle. Upper and lower rows present different axial sections through the brain. Transplanted cells are detected as an area of hypointense signal (dotted line and arrows). Gradual disappearance of this signal over time has been found.

Fig. 5 In vitro MR imaging of SPIO-tracked UCB-NC cells in phantom experiment. Left column presents the cells incubated with SPIO for 12h, right column - for 48h. Increasing cell density from 103 to 4 x 106 in consecutive rows from up to bottom has been shown. White arrow indicates the optimal cell detection.

Fig. 6 Intraventricular transplantation of UCB-NC cells into newborn rats revealed their presence in lateral ventricle (A, B) and in chorioid plexus (C) 24 hours after grafting.

Results
MRI revealed the labeled UCB-NC cells visible as a small area of hypointense signal extending along ependyma of unilateral ventricle from the right cornu posteriori through plexus chorioideus, to cornu anteriori. Gradual lowering of the signal intensity was observed up to 4 months after the first surgery. No adverse events or abnormal reactions, except of transient increases in body temperature immediately after each tx has been noticed.
Neurological status of the child was assessed before and at 6 moths after the last tx. The patient, despite still severely impaired was no longer justified to be diagnosed as in a vegetative state. He started to respond to his mother's voice by smiling. The reduction of seizures by 50% has been achieved. The spasticity and nystagmus, although still present, was less pronounced than at baseline examination and the child achieved head and neck control good enough to move in certain position to dampen the frequency and the amplitude of the nystagmus. However, the only one case described herein does not justify us to conclude either UCB-NC cell delivery itself or the pharmaceutically aided or spontaneous neuroregeneration had been decisive for observed clinical improvement.

Conclusions
The intraventricular UCB-NC transplantation was found safe and generally well tolerated. The SPIO labelled cells engraftment was detected noninvasively with the use of MRI under neuronavigation. Subsequent MRI data proved that autologous UCB-NC cells could survive in the host brain for at last 4 months and thus may present promising tool in management of brain hypoxic-ischemic injury.