Plasma derived from human umbilical cord blood: Potential cell‐additive or cell‐substitute therapeutic for neurodegenerative diseases

Abstract Limited efficacy of current therapeutic approaches for neurodegenerative disease has led to increased interest in alternative therapies. Cord blood plasma (CBP) derived from human umbilical cord blood (hUCB) may be a potential therapeutic. Benefits of CBP injection into rodent models of aging or ischaemic stroke have been demonstrated, though how benefits are elicited is still unclear. The present study evaluated various factors within the same samples of CBP and human adult blood plasma/sera (ABP/S). Also, autologous CBP effects vs. ABP/S or foetal bovine serum supplements on mononuclear cells from hUCB (MNC hUCB) in vitro were determined. Results showed significantly low concentrations of pro‐inflammatory cytokines (IL‐2, IL‐6, IFN‐γ, and TNF‐α) and elevated chemokine IL‐8 in CBP. Significantly higher levels of VEGF, G‐CSF, EGF and FGF‐basic growth factors were determined in CBP vs. ABP/S. Autologous CBP media supplements significantly increased MNC hUCB viability and decreased apoptotic cell activity. We are first to demonstrate the unique CBP composition of cytokines and growth factors within the same CBP samples derived from hUCB. Also, our novel finding that autologous CBP promoted MNC hUCB viability and reduced apoptotic cell death in vitro supports CBP's potential as a sole therapeutic or cell‐additive agent in developing therapies for various neurodegenerative diseases.

Alzheimer's disease (AD) mouse model by injection of a specific fraction from cord blood serum compared to adult blood serum. Additionally, umbilical cord serum has being effectively employed for the treatment of corneal defects 8,9 and neurotrophic keratitis 10 in humans.
In a relatively recent study, 11 we showed the ability of CBP to modulate mitogen-induced in vitro proliferation of mononuclear cells (MNC) isolated from the peripheral blood of amyotrophic lateral sclerosis (ALS) patients. Interestingly, three distinct cell responses to the mitogenic factor phytohemmagglutinin were noted, suggesting altered lymphocyte functionality in ALS patients. MNC responses were shown to be regulated by CBP treatment in vitro. Additionally, the apoptotic activity of MNCs isolated from ALS patients was significantly reduced by supplementing media with CBP. Thus, these study results have not only broadened the therapeutic application of CBP for ALS, but also further expanded its potential for treatment of other neurodegenerative disorders with immunological aspects.
It has been shown that CBP contains high amounts of various growth factors, such as vascular endothelial growth factor (VEGF), insulin-like growth factor-1 and transforming growth factor (TGF)-β, that are required for cell maintenance during hematopoiesis. 3,12 Although CBP can exert a favourable effect on hematopoietic stem cells, whether CBP elicits therapeutic benefit as an additive to, or substitute for, cells must be determined before developing clinically relevant CBP-based therapies for various neurodegenerative diseases.
The aim of this study was to characterize the composition of factors in CBP derived from hUCB, which might mediate therapeutic benefit. First, cytokine and growth factor profiles were analyzed in the same CBP samples. Second, the efficacy of autologous CBP on MNC hUCB viability in vitro was investigated. Finally, the effect of autologous CBP upon the apoptotic MNC hUCB response in vitro was examined. These study results provide a basis for further establishment of CBP as a potential self-contained therapeutic or as a supportive diluent for MNC hUCB infusion in treatment of neurodegenerative diseases.

| Ethics statement
The human umbilical cord blood (hUCB) units were collected by Texas Cord Blood Bank (TCBB, GenCure, West San Antonio, TX, USA) and provided to Saneron CCEL Therapeutics, Inc. for research purposes. The cord blood units were obtained from full-term pregnancies by vaginal delivery. The umbilical cord blood units were received within 48 hours of collection. Maternal blood samples, collected as the same time as the cord blood, were tested by TCBB for infectious disease markers of HIV, hepatitis B and C, syphilis, CMV and HTLV I&II, and test results were provided for validation of the cord blood units. Each cord blood unit in the study was negative for all infectious disease markers as determined in maternal blood.
Human adult blood plasma or sera (ABP/S) was obtained from a commercially available source (Sigma-Aldrich, St. Louis, MO, USA).
Upon receipt of ABP/S, samples were aliquoted and stored at −20°C.

| Human umbilical cord blood processing and plasma isolation
Human umbilical cord blood (hUCB) units (n = 20), with maternal blood samples negative for all tested infectious markers, were processed to obtain an autologous CBP fraction and mononuclear cell population (MNC hUCB, U-CORD-CELL ™ , Saneron CCEL Therapeutics, Inc., Tampa, FL, USA) as detailed below. Upon receipt, the cord blood units were diluted

| Growth factor profile in human umbilical cord blood plasma
A human growth factor 4-plex panel (Invitrogen; Cat No. LHC0007) was employed to determine various growth factor levels within CBP (n = 20) and ABP/S (n = 6) samples in triplicate, following the manufacturer's protocol. All measurements were performed by an investigator blinded to the source of the samples. Levels of VEGF, granulocyte colony-stimulating factor (G-CSF), epidermal growth factor (EGF) and fibroblast growth factor basic (FGF-basic) were determined using the Bio-Rad Bio-Plex ® Luminex 200 multiplex assay (BioRad Laboratories Inc., Hercules CA, USA). The Bio-Rad Bio-Plex ® 200 software (BioRad Laboratories Inc., Hercules CA, USA) was used to calculate the sample growth factor concentrations accordingly to a standard curve and results were presented as pg/mL.

| Viability of MNC hUCB cultured with autologous CBP
Cryopreserved MNC hUCB cells (n = 4 units) were quickly thawed at 37°C, washed with PBS, and centrifuged at 400 g for 5 minutes.
Cell quantity and viability were determined using a haemocytometer.

| Apoptotic activity of MNC hUCB cultured with autologous CBP
Cryopreserved MNC hUCB cells (n = 6 units) were quickly thawed at 37°C, washed with PBS, and centrifuged at 400 g for 5 minutes.
Cell quantity and viability were determined using a haemocytometer.
Cells were then re-suspended with phenol-free RPMI-1640 media and plated in a 96-well culture plate at a density of 2 × 10 4 cells/ well. Pre-designated wells were supplemented with 10% of either autologous CBP, ABP/S, or FBS upon initial plating in duplicate. Cells were incubated at 37°C with 5% CO 2 for 5 days. Media was chan-

| Statistical analysis
Data was presented as mean ± SEM Statistical analysis was performed using GraphPad Prism Software version 5 (GraphPad Software, Inc.). The results for MNC hUCB viability and apoptotic activity were evaluated using a one-way ANOVA with Tukey's Multiple Comparison post-hoc test. The results for cytokine and growth factors in CBP were analyzed with a two-tailed t test using same software. A value of P < 0.05 was considered significant.

| Cord blood plasma cytokine profile
Samples of CBP and ABP/S were assayed to determine cytokine profiles using an ultrasensitive human cytokine 10-plex panel. Results   Figure 1J). Although the levels of IL-1β, IL-4 and IL-10 were slightly reduced in CBP compared to ABP/S, these reductions were not statistically significant (P > 0.05) (Figure 1A,C,G). While anti-inflammatory IL-4 and IL-10 cytokine concentrations were not significantly different between CBP and ABP/S, it is important to note that most of the pro-inflammatory cytokines within CBP were present at lower concentrations than their anti-inflammatory counterparts. Concentrations of cytokines in CBP and ABP/S are provided in Table 1A.

| Cord blood plasma growth factor profile
The levels of several common growth factors were measured in CBP and ABP/S using a human growth factor four-plex assay. The concentrations of VEGF were significantly (P < 0.01) higher in CBP, more than two-fold, vs. ABP/S (Figure 2A). The concentrations of G-CSF, a bone marrow stem cell stimulating growth factor, were also significantly (P < 0.05) higher in CBP compared to ABP/S ( Figure 2B).     14 and a mouse model of Alzheimer's disease (AD). 15 Middeldorp et al 15 demonstrated that parabiosis of young wild-type mice with AD mice for 5 weeks effectively improved learning and memory while also reducing inflammation in AD mice. Additionally, the authors noted increased synaptic activity in the hippocampus of AD mice. Based on these study results, clinical trial (NCT02256306) investigated the safety of 4-weekly infusions of young blood plasma from donors aged between 18 and 30 years of age into patients with AD. Although no serious adverse reactions occurred, the study found no significant effect on patient cognition but did show significant improvements in daily living skills.
Although results of using young blood are promising, it is still unclear which constituents of "young" blood are providing beneficial effects.
Potentially, paracrine actions are involved in positive outcomes for treatment of an age-related disease such as AD. Also, hormonal status of donors should be investigated due to the wide age range (18-30 years) of donors. Alternatively, plasma derived from hUCB could be a more beneficial therapeutic due to its unique and uniform molecular composition.
It has been shown that in addition to a high concentration of growth factors (reviewed 16  ing that the delivery method impacts oxidative stress. 21 In our study, the low concentration of GM-CSF found in CBP together with the low concentrations of pro-inflammatory cytokines provide further evidence of anti-inflammatory hUCB content. Thus, low levels of pro-inflammatory and immunomodulatory cytokines in CBP provide a favourable microenvironment for cellular content in hUCB. It has been shown that transplantation of MNC derived from hUCB even from unrelated donors into patients with haematologic malignancies causes a low incidence of graft-versus-host disease compared to bone marrow or peripheral blood cell administration. 22,23 Our study results also demonstrated similar amounts of antiinflammatory IL-4 and IL-10 cytokines in CBP and ABP/S, However, it is important to note that these anti-inflammatory cytokines were present at a greater concentration than the pro-inflammatory constituents of CBP, suggesting a favourable cytokine composition towards developing CBP as potential therapeutic agent. Since IL-10 is an important cytokine for downregulation of Th1 inflammatory cytokines and MHC class II antigens, a decrease of this cytokine is mainly associated with altered cell-mediated immunosuppression and induction of complications during pregnancy. 24 In contrast, increased cord blood IL-10 was determined in preterm infants compared to full-term newborns. 25,26 In our study, hUCB units were used from healthy infants delivered naturally, so IL-10 levels Exclusively, VEGF has been studied for potential therapeutic efficacy in animal models of ALS 38,39 and its use in clinical settings has been discussed (reviewed 40 ). Nevertheless, CBP containing high levels of EHRHART ET AL.
| 6163 IL-8 and VEGF might be a beneficial treatment for repair of the damaged blood-brain barrier and/or blood-spinal cord barrier in patients with ALS, [41][42][43][44] AD, 45 Parkinson's disease 46  been observed from intravenous administration of CBP into rats modelling acute ischaemic stroke 5 or into an animal model of ageing. 6 In these studies, multiple injections of CBP were performed and this therapeutic approach needs to be considered. In agreement with this approach, repeated deliveries of CBP could provide ongoing trophic support for damaged cells and/or tissues. Our study showed that CBP is a potential therapeutic due to its unique composition. We are planning in the near future to determine the effect of CBP alone and in combination with MNC hUCB for treatment of ALS using a symptomatic animal model of disease for a translational perspective.
In conclusion, our study results demonstrate uniquely protein These findings further support the potential of CBP as an independent therapeutic or cell-additive agent in clinical applications for various neurodegenerative diseases.

ACKNOWLEDGEMENTS
This study was supported in part by the University of South Florida's Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, and by Saneron CCEL Therapeutics, Inc.

CONFLI CT OF INTEREST
PRS is a co-founder and SGD is a consultant for Saneron CCEL Therapeutics, Inc. JE is the Director of Research and Development for Saneron CCEL Therapeutics, Inc. PRS and SGD have patents for the application of hUCB as a cell therapy for several disorders.

AUTHOR CONTRI BUTIONS
JE and SGD designed the studies and wrote the manuscript. JE performed all assays and data analysis. PRS participated in study design and discussion of results. All authors reviewed the manuscript.