Abstract

Introduction: Stem cell-based therapies for Parkinson's disease (PD) represent a promising frontier in regenerative medicine. This study assesses the efficacy of regenerative treatments, specifically platelet-rich plasma (PRP) and peripheral blood-derived very small embryonic-like (PBD-VSEL) stem cell therapy, in managing PD.

Methods: A quasi-experimental study design was employed, involving 50 PD patients divided into two cohorts. Group A (n = 25) received only standard therapy, while Group B (n = 25) received the addition of PRP and PBD-VSEL stem cell therapy to the standard treatment. The primary outcomes measured were changes in the Unified Parkinson's Disease Rating Scale (UPDRS) and the Parkinson's Disease Questionnaire-39 (PDQ-39) scores.

Results: The average age was 61.40 years in Group A and 62.16 years in Group B. At baseline, both groups exhibited comparable UPDRS and PDQ-39 scores. However, at the 6-month follow-up, Group B demonstrated greater improvement in PDQ-39 scores (64.16 ± 5.44) compared to Group A (71.64 ± 10.68). After one year, Group B presented significantly reduced UPDRS (60.80 ± 5.85) and PDQ-39 (59.88 ± 5.12) scores relative to Group A (69.44 ± 9.67 and 66.52 ± 5.52), highlighting superior enhancements in motor function and quality of life for Group B.

Conclusion: The findings suggest that PRP and PBD-VSEL stem cells from peripheral blood may offer a viable adjunctive treatment for Parkinson's disease. Nonetheless, additional research is essential to elucidate the mechanisms underlying cell therapy and substantiate its clinical application. Cell transplantation appears to be a safer and more efficacious strategy for treating Parkinson's disease. The management of advanced PD patients requires careful decision-making, including the introduction of new pharmacotherapies and adjustments to levodopa dosages. Larger studies with extended follow-up periods are necessary to confirm these results.

Introduction

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of mesencephalic dopaminergic neurons within the substantia nigra, leading to symptoms such as bradykinesia, resting tremor, and rigidity predominantly affecting the body's central nervous system (CNS) related to motor control1. Over the past 25 years, the prevalence of PD has increased twofold. On a global scale, PD-related disability and mortality rates are rising more rapidly compared to other neurological conditions1, 2. PD can be classified into juvenile, early, or late-onset categories. Juvenile cases are rare, usually familial, and often associated with mutations in the Parkin gene3. Generally, PD impacts approximately 1% of the population over the age of 60 and 4% of individuals over the age of 804. The clinical manifestations of PD are a consequence of dopamine deficiency and the progressive degeneration of dopaminergic neurons (DAn)5, 6. The neurodegenerative effects of PD may extend beyond motor functions, affecting both motor and non-motor regions of the body7. The primary pathological feature of this neurodegeneration is the loss of dopaminergic neurons in the substantia nigra pars compacta, resulting in reduced dopamine levels in the neostriatum8, 9.

L-DOPA (L-3,4-dihydroxyphenylalanine), a precursor to dopamine, serves as the primary therapeutic agent for PD, providing immediate symptomatic relief. However, it often leads to motor fluctuations after 5-10 years of use 10. Although levodopa remains the gold standard treatment for symptom relief, developing neuroprotective strategies is essential for creating therapies that more specifically target the underlying pathology11. As neurodegeneration advances, significant alterations occur in both brain and body, with organs, tissues, and cells communicating these changes via systemic signaling pathways12. Despite L-DOPA administration, PD patients may experience refractory dyskinesia. Newer treatments, such as dopamine agonists and deep brain stimulation, help address these challenges but do not prevent disease progression or alleviate symptoms resulting from non-dopaminergic pathologies13.

Numerous clinical trials have demonstrated that plasma administration can enhance cognitive function in aging rodents14, 15, 16. This suggests that circulating plasma components can ameliorate cognitive and disease-related symptoms17. This has encouraged the exploration of blood products and their active components for treating various disorders, particularly those related to dementia or neurodegeneration. The limitations of pharmaceutical interventions have spurred interest in developing alternative treatments18. Human embryonic dopaminergic neurons have been investigated in clinical trials for cell replacement therapy with some success19, 20. However, promising clinical results and evidence of graft survival remain limited. Intracerebral xenografts require strict immunosuppression and pose a risk of transmitting animal viruses21. The loss of midbrain neurons implies that regenerating a single brain region with new cells may mitigate these deficits, positioning PD as a pioneer in cell-based therapy22. Due to the non-targeted and non-physiological distribution of dopamine in the brain, current therapies, although alleviating pyramidal symptoms, have significant side effects18, 23. As dopamine deficiency underlies PD symptoms, cell transplantation offers potential symptomatic relief without the pyramidal and neuropsychiatric side effects associated with dopaminergic drugs24. Over the last twenty years, regenerative cell therapy has opened unprecedented avenues for innovative strategies to combat neurodegenerative diseases25.

Human stem cells, specifically multipotent region-specific and pluripotent embryonic stem cells, are considered potential sources of dopaminergic neurons for PD treatment through cell transplantation26. These transplanted neurons must meet certain criteria, including proper synthesis and release of dopamine, reinnervation of the striatum, and mitigation of motor symptoms27. Very small embryonic-like cells (VSELs), identified as putative pluripotent stem cells in human umbilical cord blood (HUCB) and mouse bone marrow, hold significant promise for regenerative medicine applications28, 29. Recent studies suggest that effective dopaminergic neurons should express the G-protein-coupled inward rectifying K+ channel subunit (Girk2), predominantly found in the substantia nigra pars compacta30. Stem cell-based therapies offer a promising pathway for developing effective regenerative medicine benefiting a broad population14, 31. Despite limited research on the use of PRP and PBD-VSEL stem cells for PD treatment, stem cells are likely to find clinical application soon. While progress remains gradual, several promising clinical trials are underway, indicating that stem cell therapy may soon be viable for PD management. The current study aims to assess the efficacy of PRP and stem cell therapies in managing PD.

× Figure 1 . Patient’s enrolment and follow-up . Patient enrollment in the consort diagram is shown at baseline, 6 th month, and 1 year of treatment. Figure 1 . Patient’s enrolment and follow-up . Patient enrollment in the consort diagram is shown at baseline, 6 th month, and 1 year of treatment. Methods Patient Enrollment with Inclusion and Exclusion Criteria

This quasi-experimental study received approval from the Research Ethics Committee at The University of Lahore, Pakistan (IRB-SPRM-2020-2), with input from a review committee comprised of expert neurologists and neuropathologists from the University of Jeddah and King Abdulaziz University in Jeddah, Saudi Arabia. Informed written consent was obtained from either the patient or the patient's legal guardian if the patient was unable to communicate. The study is registered at ClinicalTrials.gov (NCT06142981), which can be accessed at https://clinicaltrials.gov/study/NCT06142981. All participants, clinically diagnosed with Parkinson's Disease (PD), were referred from the Institute of Neurosciences, Lahore, Pakistan (Figure 1). The sample size calculation, using a 21.7% prevalence rate of PD, a margin of error of 8%, an 80% confidence level, and a 10% dropout rate, resulted in an estimated sample size of 50 participants32. Patients were comprehensively briefed about the treatment procedures, inclusive of potential risks, benefits, complications, and long-term effects. Data on each patient's medical history, PD stage, symptom characteristics, previous treatments, and comorbidities were collected through interviews and retrospective chart reviews. Following treatment, participants were monitored over two years. Inclusion criteria focused on individuals with a PD diagnosis spanning 4 to 10 years, aged 50 to 70, of either gender, and on stable treatment regimens. Exclusion criteria ruled out individuals with gout, congestive heart failure, renal failure, uncontrolled atrial fibrillation, stroke, anaphylaxis, coagulopathies, and those with clinical suspicion or confirmed diagnosis of atypical Parkinsonism or essential tremor.

Clinical Assessment of PD Treatment Response

Neurologists conducted clinical assessments using recognized scales to evaluate the response to PD treatment. The primary tool utilized was the Unified Parkinson's Disease Rating Scale (UPDRS), a prominent rating scale from the 1980s for tracking PD progression2. Additionally, the self-report Parkinson's Disease Questionnaire–39 (PDQ-39) was employed, which is a specific health status questionnaire for PD encompassing 39 items, asking participants to rate the frequency of experienced difficulties related to their PD on a five-point scale33.

Treatment Method

The study enrolled 50 patients, split into two equal groups. Group A (n = 25) received standard Parkinson's disease therapy, while Group B (n = 25) was treated with Standard Therapy plus Regenerative Medicine (PRP and PBD-VSEL Stem Cell Therapy). Post initial clinical assessment by neurologists, patients were enrolled in a treatment plan and, following cognitive and laboratory testing, scheduled for treatment infusion within two weeks (Figure 1). Group A received a daily dose of 300 mg levodopa, administered over three doses and adjusted based on individual response, alongside lifestyle modification therapy. Group B received PRP injections at four acupuncture points (ST36 and GB 34, bilaterally) 34, 35, 36, followed by PBD-VSEL stem cell administration at these points post-PRP sessions, with some patients receiving intra-discal injections for targeted spinal intervention to enhance regenerative outcomes. Following PRP sessions, patients were re-evaluated by neurologists, with PBD-VSEL stem cell therapy administered at the 90-day mark. Evaluations continued at 3-month intervals, with PRP booster doses provided at 6 and 9 months post-cell therapy, followed by annual evaluations and monitoring for side effects.

Single Syringe Method for PRP Isolation from Blood

Venous blood drawn was centrifuged at 2800 rpm for 10 minutes using a soft spin, dividing into 60 mL and 140 mL portions. From the 60 mL portion, 10–12 mL PRP was extracted and combined with the remaining 140 mL autologous blood. During sessions, 4 mL of PRP mix was injected at four acupuncture sites.

Peripheral Blood-Derived Very Small Embryonic-Like (PBD-VSEL) Stem Cells Preparation and Regimen

120 mL of peripheral venous blood was drawn for processing. 30 mL was used for VSEL isolation, with remaining blood discarded after plasma separation or reserved for further analysis. VSELs isolated via centrifugation at 600×g and 1200×g steps, were purified, exposed to monochromatic light to boost regenerative potential before re-administration. Post-isolation, sterility tests were done to ensure the absence of contamination before injection, with evaluations on cell viability using a Muse Cell Analyzer (Merck, Millipore, USA). Internal tests showed stable cell count and viability for 24 hours at 2-8°C. The corrected mention aligns with study goals. Injection procedures adhered to sterile standards with live C-arm fluoroscopic guidance and consistent physician oversight. Post-procedural follow-up assessed infection risk and pain management efficacy37, 38.

Primary Outcome and Statistical Analysis

Follow-ups for both groups were conducted at baseline, the 6th month, and 1-year intervals. Primary outcomes measured improvements via UPDRS, as the main scale for PD progression, and PDQ-39 health questionnaire responses. Data analysis was done using SPSS 25.0 with differences assessed via independent sample t-tests, with significance set at p ≤ 0.05. ANOVA analysis contributed to statistical validation.

× Figure 2 . Gender-based comparison of UPDRS and PDQ-39 at different follow-ups . Figure 2 . Gender-based comparison of UPDRS and PDQ-39 at different follow-ups . Results

The study involved 50 patients, equally divided into Group A (n = 25) and Group B (n = 25). The mean age was 61.40 years in Group A and 62.16 years in Group B. The average duration of Parkinson's disease was comparable between the groups, with 7.32 years in Group A and 6.76 years in Group B, indicating that both groups had experienced the disease for similar periods. Gender distribution was also similar, with Group A comprising 60% males and 40% females, whereas Group B included 64% males and 36% females. A notable difference was observed in family history, with 28% of Group A reporting a family history of Parkinson's disease compared to 60% in Group B. In terms of motor subtype dominance, tremor-dominant patients were more prevalent, especially in Group B (80%) as opposed to Group A (64%). In contrast, Group A had a higher proportion of patients with postural instability and gait difficulty (36%) compared to Group B (20%) (Table 1).

Table 1.

Demographics and clinical assessment of patients

Group A Group B Age (Mean +SD) 61.40+4.70 62.16+6.61 Duration of disease (years) 7.32+1.99 6.76+1.66 Gender f (%) f (%) Male 15(60.0) 16(64.0) Female 10(40.0) 9(36.0) Family history Yes 7(28.0) 15(60.0) No 18(72.0) 10(40.0) Motor Subtype dominant Postural instability and gait difficulty (PIGD) 9(36.0) 5(20.0) tremor-dominant (TD) 16(64.0) 20(80.0) Specific Area of Motor Symptoms Head 7(28.0) 8(32.0) Arm 14(56.0) 13(52.0) Leg 4(16.0) 4(16.0)

Table 2.

Comparison of UPDRS and PDQ-9 at different follow-ups

Variable