Bilateral transplantation of allogenic adult human bone marrow-derived mesenchymal stem cells into the subventricular zone of Parkinson’s disease: a pilot clinical study.

A clinical study was designed to determine the safety, feasibility, and efficacy of allogenic adult bone-marrow-derived mesenchymal stem cells in Parkinson's disease (PD) patients. According to the national guidelines, Institutional Ethics Committee (IEC) approval was obtained for conducting the study. Necessary approvals for isolation, culturing, and transplantation of stem cells were also taken. Each patient who participated in the study was counseled on the procedure and informed consent obtained. The patient was screened for HIV, HBV, HCV, CMV, and VDRL by a nationally certified testing laboratory before being included in the study. All deviations to the protocol, drop outs, and adverse events were documented and informed to the IEC. 2.1.

Study Design and Randomization

The study was conducted as a prospective, uncontrolled, one year, single centre safety, and efficacy clinical study of allogenic BM-MSCs bilaterally transplanted in patients diagnosed with PD. 2.2.

Isolation of Mesenchymal Stem

Cells from Healthy Adult Donors 2.2.1.

Selection of Healthy Donors

Healthy donors were selected according to the donor inclusion criteria and as per the guideline of International Society of Cell Therapy (ISCT). Healthy donors were either male or female in the age group of 18–30 years of age, able to understand the voluntary donation program, and ready to provide voluntary written informed consent. Donors were excluded if they have illness such as autoimmune disorders, tuberculosis, malaria and any other infection, any illness which precludes the use of general anesthesia, history of malignancy, diabetes, hypertension, significant heart disease, genetic or chromosomal disorders, history of any inherited disorders, hemoglobin less than 10, and pregnant women. At the time of obtaining informed consent they were screened for infection with human immunodeficiency virus (HIV), hepatitis B (HBV), hepatitis C (HCV), cytomegalovirus (CMV), and syphilis (VDRL) using reverse transcriptase-polymerase chain reaction (RT-PCR) method and excluded if found positive. They were also tested for complete blood count (CBC), renal function test (RFT), liver function tests (LFT), blood glucose, chest X-ray, Echocardiogram, and Electrocardiogram (ECG). 2.2.2.

Isolation of Mesenchymal Stem Cells

BM-MSCs were isolated from healthy screened donors between the age of 18–30 years with informed consent. 60 milliliter of bone marrow was aspirated aseptically under local anesthesia from iliac crest of the healthy screened donors. The sample will be transported appropriately to the processing lab consisting of a class 1000 cGMP facility, and all sample processing was done in a class 100 biosafety cabinet. Bony spicules and particles were removed using a cell strainer and further diluted with DMEM-KO, centrifuged at 1800 rpm for 10 minutes at 20°C. The cells were resuspended with DMEM-KO and gently layered onto a density gradient solution (Lymphoprep, Axis Shield PoC AS, Oslo, Norway) to obtain an enriched mononuclear fraction. This was washed with DMEM-KO and centrifuged to collect the cells. The cells obtained were resuspended and plated in MSC complete culture medium consisting of Dulbecco's modified Eagle Medium (DMEM-KO) 10% fetal bovine serum (FBS) from preselected lots, and glutaMAX as described elsewhere [ 20 ]. The culture was maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO 2 and subcultured prior to confluency. 2.3. Subculturing and Expansion Once 80% confluent these cells were dissociated with 0.25% trypsin/0.53 mM EDTA (Invitrogen) and further upscaled and expanded in order to provide the required number of cells to the patient. Briefly, trypsinized cells were reseeded at a density of 1000 cells per cm 2 in cell stacks (Corning). After 14 days in culture, the cells reached 80% confluency and were ready for transplantation. 2.4.

Quality Control Testing

MSCs were tested for quality control parameters such as Mycoplasma, Endotoxin, sterility and cell surface markers such as CD73, CD90, CD105, CD166, CD34, and CD45 markers using flow cytometry. They should be more than 80% positive for CD73, CD90, CD166, and CD105 but negative [