Parkinsonian Brain Repair Using Human Stem Cells
Parkinsonian Brain Repair Using Human Stem Cells
Implantation of Celavie human stem cells (OK99) is intended to address the underlying
 pathology of the disease by replacing damaged/destroyed cells of the brain, and/or
 stimulating the patient's brain to repair itself.
This trial is a longitudinal, prospective, interventional, uncontrolled study designed to
 test, firstly, the safety and secondly, the potential efficacy of intraputaminal grafting of
 undifferentiated hfSC for the treatment of PD. Patients were monitored carefully for any
 adverse effects. All patients underwent baseline and 6- and 12-month post-surgery
 neurological, neuropsychological, MRI, and PET evaluations.

 Procurement, isolation, expansion, and characterization of hfSC, as well as assessment of
 patients' immune response to hfSC grafting, were performed at Celavie Biosciences, LLC
 (Oxnard, CA USA). Patient selection, pre- and post-surgery neurological, neuropsychological,
 and MRI evaluations, as well as stereotactic surgery and post-surgery care, were performed at
 Hospital Angeles del Pedregal (Mexico City, Mexico). Synthesis of radiopharmaceuticals and
 PET imaging were carried out at the Radiopharmacy-Cyclotron Unit of the Faculty of Medicine,
 Universidad Nacional Autonoma de Mexico (Mexico City, Mexico). Eight patients with moderate
 to advanced PD were selected for this trial. One of the patients was lost to follow up due to
 reasons unrelated to this study. Subjects that completed follow up were 2 females and 5
 males, with ages ranging between 43-74 years (mean age 56 years).

 Procurement and expansion of hfSC

 Human fetal brain tissue was procured via routine sterile manual aspiration methods with
 informed consent from the donor in accordance with NIH guidelines for use of fetal tissue as
 well as federal, and state laws. Tissue donor and hfSC recipients remained unknown to each
 other.

 Maternal blood samples (sera) were tested for: HIV (Abbott Laboratories, Abbott Park IL, USA)
 hepatitis A, B and C (Abbott); HTLVI (Abbott); VDRL (Baxter Agglutination Slide Test and
 reflex FTA), and cytomegalovirus (Quest, Oxnard CA). Women with a history of genital herpes,
 cancer, asthma, lupus, rheumatoid arthritis, allergies, vasculitis of autoimmune origin, and
 drug abuse were excluded. Gestation was determined according to Carnegie stages. Fetal tissue
 was harvested at the sixth week of gestation after elective abortion. Fetal brain was
 dissected, minced and triturated to a single cell suspension. Cells were cultured in flasks
 incubated at 37°C under hypoxic conditions (5% O2 and 5% CO2) through 4 doublings. At the
 second doubling (D2) cell culture was tested for sterility (USP <71>) and at D4 culture was
 karyotyped and PCR tested for presence of adventitious agents: HTLV-1, HTLV-2, HIV-1 (A, B,
 D, F, H, N), hepatitis A, B and C, T. p. pallidum, CMV, HSV-1, HSV-2, HPV. Cells were then
 transferred to a closed bioreactor system (GE WAVE Bioreactor 2/10 System, Uppsala SWE),
 operating under the same physical and chemical conditions. The bioreactor was used to create
 the Master Cell Bank (MCB), which was harvested, tested, characterized and rate-control
 cryopreserved after a total of seven doublings (D7).

 After the MCB was safety tested and characterized, a portion of the batch was thawed and used
 to seed the bioreactor for the Working Cell Bank (WCB) production. Cells were cultured in the
 bioreactor until they reached D13. They were harvested (Centritech LAB-III, Carr Centritech
 Separation System, Rancho Cucamonga, CA, USA), aliquoted (Fill-It; TAP Biosystems,
 Wilmington, DE, USA), and cryopreserved to create a WCB. The WCB was subjected to release
 testing for safety and characterization assays. Safety testing included sterility (USP <71>),
 mycoplasma (USP <63), endotoxin (USP <85>), and karyotyping (Cell Line Genetics, Madison WI,
 USA). Characterization included flow cytometry testing for: Oct-4 >90% (10H11.2, EMD
 Millipore, Billerica, MA, USA; AF488 conjugated), Sox-2 >90% (Btjce, eBioscience, San Diego
 CA, USA; AF488 conjugated), MHC-I <10% (A4, eBioscience; APC conjugated), MHC-II <10% (CVS20,
 Novus Biologicals, Littleton CO, USA; AF488 conjugated), CD105 <10% (SN6, eBioscience; PE-Cy7
 conjugated), and tyrosine hydroxylase <10% (EP1532Y, Abcam, Cambridge, UK; FITC conjugated
 goat anti-rabbit IgG; Abcam; polyclonal). Both MCB and WCB were stored in gas phase LN2 at
 -196°C.

 All procedures were performed under aseptic conditions in an ISO 8 clean room, utilizing ISO
 5 bio-safety cabinets and laminar airflow hoods, according to validated protocols. Cells were
 shown to have a normal karyotype and did not produce teratomas in immunocompromised rodents
 (unpublished data).

 Pharmacotherapy

 Immunosuppression via cyclosporine A at a dose of 15 mg/kg/day was started 10 days prior to
 surgery and continued for one month thereafter. Patients also received Indomethacin
 225mg/day, starting at 10 days prior to implantation and for six months postoperatively
 thereafter. Wide spectrum antibiotic (Zannat 700mg) was given preoperatively and 48 hours
 post-operatively. Antiparkinsonian medications were adjusted to patient's requirements.

 Stereotactic surgery

 MRI-guided stereotactic intraputaminal cell implantation into PD patients was performed using
 a Leksell Stereotactic System and Stealth Station Surgical Navigation System (Fridley,
 Minnesota, USA). Ropivacaine was used as a local anesthetic for frame placement. For target
 locations, measurements were made using CT-scan images fused with previous MR images (both in
 DICOM format, in axial sections 1.0 mm thick). With the patient under general anesthesia, the
 stereotactic frame was fixed to the operating table with a Mayfield head holder. Bilateral
 parasagittal incisions and corresponding 14 mm burr holes (one for each hemisphere) were made
 in preparation for cell suspension injections. Two different needle tracks through the same
 burr hole were selected for each side. Target locations were determined by height and length
 of putaminal nuclei. The lowest Z-coordinates of each track were located in the dorsal
 putamen and spaced 4mm apart in the X-direction. Each needle track received 1X106 cells in 1
 cc of culture medium. To ensure complete cell suspension delivery, injections were carried
 out slowly for 2 min with reciprocal withdrawal of the delivery needle to avoid both damage
 to stem cells and brain tissue, as well as to avert reflux or bubble formation. After
 surgery, patients were kept in a conventional post-operative care unit for 1 h. The day
 following surgery, MR images were obtained to confirm correct placement of cell suspensions.
 All patients were discharged 24 h after surgery.

 Neurological evaluations

 Neurological endpoints of this study included evaluation of the number and severity of
 adverse events after cell grafting, and the efficacy in the improvement of motor responses,
 as assessed on the UPDRS part I (mentation, behavior and mood), part II (motor activities of
 the daily living), part III (motor performance), and part IV (complications of therapy), as
 well as the modified Höehn and Yahr Scale, and the modified Schwab and England Activities of
 the Daily Living Scale. Patients were clinically evaluated with these scales at screening (as
 baseline, before surgery), and then after the procedure at six and 12 months. At every visit,
 patients were asked to discontinue antiparkinsonian medications at least 12 hours before the
 UPDRS assessment (for practical purposes, "OFF" state was defined as overnight drug
 withdrawal) in order to be rated in their "OFF" state and then, UPDRS "ON" medication
 evaluations were performed 1 hour after receiving their usual dose of levodopa. Each patient
 received the same preoperative levodopa dose for each assessment. Adverse events, including
 those reported by the patients spontaneously and those observed during the evaluations were
 recorded. After obtaining signed informed consent from all patients that completed the study,
 all "OFF" and "ON" UPDRS evaluations, were videotaped.

 Neuropsychological evaluations

 Cognitive performance was evaluated using the following instruments: Mexican adaptations of
 Beck and Steer's anxiety [25] and depression [26] inventories; our brief neuropsychological
 (NEUROPSI)[27,28] and computerized neuropsychological test batteries, and the Mini-mental
 Parkinson State Examination (MMPSE)[29]. Patients reported on their quality of life as
 related to their daily living activities; physical and mental well-being (health status);
 cognition and communication, and one summary index.

 Immunogenicity testing

 Patient's blood was evaluated for increase in titers of hfSC specific antibodies and for
 increase in antibody-dependent cell-mediated cytotoxicity after implantation as compared to
 baseline values. Samples were drawn one month prior to cell implantation, and then one month
 and six months after surgery. Whole blood specimens were collected from each patient and
 processed as serum one and six months post-grafting using the lipophilic membrane dye PKH67
 (Sigma-Aldrich) as described by the manufacturer for cell tracking in immune response and
 cytotoxicity assays by flow cytometry [30,31]. Cytotoxicity assay was performed with 100:1,
 50:1, and 25:1 effector to target ratios.

 MRI

 MR images were obtained before surgery (baseline) and three times after cell implantation at
 24 h, and six and 12 months post-surgery. They were acquired with a 3 Tesla magnet, MR
 Systems Achieva release 2.6.3.8 Philips (Best, The Netherlands).

 PET molecular imaging

 Patients underwent PET molecular imaging at baseline, at six months (data not presented), and
 at one year after hfSC implantation. Radiopharmaceuticals utilized were
 (+)-alpha-[11C]Dihidrotetrabenazine (DTBZ), 6-[18F]Fluoro-L-DOPA (FDOPA), and [11C]Raclopride
 (RAC). All patients underwent DTBZ-PET scans and one additional study with either FDOPA or
 RAC, at least one week apart. Patients were asked to discontinue antiparkinsonian medications
 at least 12h before each study. Scans were acquired on a Siemens Biograph 64 PET/CT.
 Thirty-minute brain emission scans were acquired 20 minutes post-injection of DTBZ or RAC,
 while 15 min scans were acquired for FDOPA 75 min post-injection. All patients studied with
 FDOPA were pre-medicated with 150 mg of carbidopa to prevent peripheral decarboxylation.
 Images were reconstructed using an OSEM-2D algorithm and analyzed with the software
 Statistical Parametric Mapping (SPM v.12). Each individual PET brain image was normalized on
 an anatomical MRI atlas to be evaluated within a standard space. Following normalization, FSL
 structural atlases were used for definition of the regions of interest. To facilitate the
 quantitative analysis, specific uptake ratios (SUR) in the caudate and putamen were
 calculated by subtracting the background signal of a reference region with nonspecific uptake
 from striatal activity and dividing by reference region activity [(target uptake - reference
 uptake)/reference uptake], using occipital cortex (DTBZ and FDOPA) and cerebellum (RAC), as
 reference regions.

 Statistical analysis

 Comparisons between baseline and 12-month follow-up measurements for anxiety, depression,
 NEUROPSI, MMPSE examinations, right and left finger tapping in "ON" and "OFF" medication
 states were performed using the Wilcoxon test. All other end measures were reported as
 individual results for each patient.
Inclusion Criteria:

 -  Diagnosis of idiopathic PD with tremor, rigidity or hypokinesia as major symptoms.

 -  A two to twenty-five-year history of PD with significant medical management or
 difficulty in medical management.

 -  A definite response to levodopa compounds with inadequately relieved of symptoms, or
 severe secondary effects of the drug.

 -  Good general health.

 -  A strong will or desire to have the procedure after being fully informed of its
 experimental nature.

 Exclusion Criteria:

 -  History of repeated strokes with stepwise progression of parkinsonian features

 -  History of repeated head injury

 -  History of definite encephalitis

 -  Oculogyric crises (unless drug-induced)

 -  Neuroleptic treatment at onset of symptoms

 -  Supranuclear gaze palsy

 -  Cerebellar signs

 -  Babinski sign

 -  Presence of cerebral tumor or communicating hydrocephalus on CT scan

 -  Sustained remission or negative response to an adequate dose of levodopa

 -  Patients with parkinsonism other than Idiopathic PD