Plasma Derived from Human Umbilical Cord Blood Modulates Mitogen-Induced Proliferation of Mononuclear Cells Isolated from the Peripheral Blood of ALS Patients

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by degeneration of motor neurons in the spinal cord and brain. This disease clinically manifests as gradual muscular weakness and atrophy leading to paralysis and death by respiratory failure. While multiple interdependent factors may contribute to the pathogenesis of ALS, increasing evidence shows the possible presence of autoimmune mechanisms that promote disease progression. The potential use of plasma derived from human umbilical cord blood (hUCB) as a therapeutic tool is currently in its infancy. The hUCB plasma is rich in cytokines and growth factors that are required for growth and survival of cells during hematopoiesis. In this study, we investigated the effects of hUCB plasma on the mitogen-induced proliferation of mononuclear cells (MNCs) isolated from the peripheral blood of ALS patients and apoptotic activity by detection of caspase 3/7 expression of the isolated MNCs in vitro. Three distinct responses to phytohemagglutinin (PHA)-induced proliferation of MNCs were observed, which were independent of age, disease duration, and the ALS rating scale: Group I responded normally to PHA, Group II showed no response to PHA, while Group III showed a hyperactive response to PHA. hUCB plasma attenuated the hyperactive response (Group III) and potentiated the normal response in Group I ALS patients, but did not alter that of the nonresponders to PHA (Group II). The elevated activity of caspase 3/7 observed in the MNCs from ALS patients was significantly reduced by hUCB plasma treatment. Thus, study results showing different cell responses to mitogen suggest alteration in lymphocyte functionality in ALS patients that may be a sign of immune deficiency in the nonresponders and autoimmunity alterations in the hyperactive responders. The ability of hUCB plasma to modulate the mitogen cell response and reduce caspase activity suggests that the use of hUCB plasma alone, or with stem cells, may prove useful as a therapeutic in ALS patients.

Keywords

Human umbilical cord blood plasma (hUCBP)Autoimmunity Phytohemagglutinin Nonresponders Caspase

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease involving both upper and lower motor neuron damage in the spinal cord and brain. This disease clinically manifests as muscular weakness and atrophy, which lead to paralysis and death of patients by respiratory failure within 3 to 5 years (34). Most cases of ALS are sporadic; the familial (FALS), or genetically linked, form of ALS represents only 10–13% of all cases (14,30). About 20% of FALS cases are the result of mutations in the gene for Cu/Zn superoxide dismutase (SOD1) that are associated with a change in SOD1's activity. Over 140 different SOD1 gene mutations have been reported (2). Available treatments for this disease lack the capacity to arrest disease progression or repair motor neuron function. Cell therapy may be a promising new treatment for ALS.

Human umbilical cord blood (hUCB) may be preferable to other cell sources, such as bone marrow, due to hUCB cells' low pathogenicity and immune immaturity. The mononuclear cell fraction from hUCB (MNC hUCB) is relatively rich in multipotent progenitors and has extensive proliferation capacity (26,37). A number of studies have shown that intravenously administering MNC hUCB (Saneron's proprietary fraction U-CORD-CELL™) into the jugular vein of G93A SOD1 mice delayed the progression of disease and prolonged life span, increased motor neuron survival in the cervical/lumbar spinal cord, decreased proinflammatory cytokines [interleukin (IL)-1α, IL-1β, tumor necrosis factor (TNF)-α], and restored leukocyte profiles in these mice (15–17). While multiple interdependent factors may underlie the pathogenesis of ALS, increasing evidence supports a role for autoimmune mechanisms (1,3,10,28,29). We hypothesized that MNC hUCB may provide neuroprotective and/or trophic effects for motor neurons by modulating the host immune inflammatory system through release of various growth or anti-inflammatory factors. Additionally, hUCB plasma (hUCBP) is a rich source of cytokines and other proteins, such as insulin-like growth factor-1 (IGF-1), transforming growth factor (TGF)-β, and vascular endothelial growth factor (VEGF) required for growth and survival of hematopoietic stem cells (8,21,23). Moreover, it has been shown that hUCB serum contains more neurotrophic factors [substance P, IGF-1, nerve growth factor (NGF)] compared to the peripheral blood serum effectively used for the treatment of persistent corneal epithelial defects (38), neurotrophic keratitis (40), and recurrent corneal erosion (39). hUCBP has also been used as a replacement for fetal bovine serum in in vitro studies including the expansion of endothelial colony-forming cells (18), mesenchymal stromal cells (MSCs) (4,11), T cells (21), and dental stem cells (24), demonstrating that it can exert a favorable influence on stem cells. These results suggest that hUCBP may be effective as an additive to, or substitute for, cells in developing clinically useful protocols for cell-based ALS therapies. Including hUCBP with hUCB cells may add significant therapeutic benefits, and plasma alone may also be a useful treatment approach.

The aim of this preclinical study was to determine the efficacy of hUCBP on the functional activity of lymphocytes from the peripheral blood of ALS patients. First, we analyzed hematological profiles in the peripheral blood of ALS patients. Second, we investigated the mitogen-induced proliferation response of MNCs isolated from the peripheral blood of ALS patients in vitro when cultured with hUCBP. Finally, we examined the effect of hUCBP upon the apoptotic cell death response in ALS patients.

Materials and Methods

ALS Patients and Healthy Volunteers

A total of 12 ALS patients and six healthy volunteers were used in this study (IRB Study #103861). Patients were referred to the USF clinic after confirmed diagnosis of “Definite ALS” by a non-USF board-certified neurologist (primary neurologist). The healthy volunteers were gender- and age-matched to ALS patients and had no neurological, autoimmune, systemic, or psychiatric diseases. Each participant signed an informed consent form prior to enrolling in the study. The patient care database form and medical history form were completed by each patient and healthy volunteer. A neurological exam was performed upon each study participant. Each study participant was graded on the ALS functional rating scale (ALSFRS; maximum score 40) and ALSFRS-revised (ALSFRS-R; including pulmonary/respiratory function; maximum score 48) using the online ALS C.A.R.E. Program (http://www.outcomes-umassmed.org/als/alsscale.aspx) from data collected by the same neurologist at USF.

Hematological Analysis

Peripheral blood (~80 ml) from ALS patients and healthy volunteers was obtained via venipuncture by a nurse at the USF clinic. Hematological analysis [complete blood cell (CBC) and white blood cell (WBC) differential counts] was performed for each blood sample (performed by Quest Diagnostics).

Isolation of MNCs From the Peripheral Blood

Fresh peripheral blood from ALS patients and healthy volunteers was collected in sterile tubes with heparin (10 units of heparin per 1 ml of blood; BD, Franklin Lakes, NJ, USA) and was then diluted (1:1) with sterile phosphate-buffered saline (PBS) without Mg²⁺ and Ca²⁺ (Sigma-Aldrich, St. Louis, MO, USA) at 37°C. Then 12.5 ml of Ficoll (Histopaque-1077, Cat. No.10771; Sigma-Aldrich) was added into 50-ml sterile centrifuge tubes (Cat. No. 352074; BD Falcon, Bedford, MA, USA). Blood samples diluted in PBS were overlaid on the Ficoll and centrifuged at 400 × g for 40 min at 26°C. The MNC layer was transferred with plasma to new 50-ml tubes by using 10-ml serological pipettes (Cat. No.13-678-11E; Fisherbrand, Waltham, MA, USA). The MNCs were washed twice in 30 ml of PBS at 440 × g for 13 min at 21°C. The cell numbers and viability were determined using a Vi-CELL Viability Analyzer (Beckman Coulter, Brea, CA, USA). The MNCs were frozen in Cryopreservation Media (Saneron CCEL Therapeutics, Inc. Tampa, FL, USA) at 2 × 10⁶ cells per vial and stored in liquid nitrogen.

hUCB Plasma

The hUCBP was obtained during isolation of the MNC hUCB (n = 4; one male, three females; Saneron CCEL Therapeutics Inc.) as described above for peripheral blood and stored at −20°C. The hUCBP was diluted in PBS 1:1 during processing.

Mitogen-Induced Proliferation of MNCs Isolated From the Peripheral Blood

Cryopreserved MNCs were thawed rapidly at 37°C and then transferred slowly with a pipette into a 15-ml centrifuge tube containing sterile PBS. The cells were centrifuged (400 × g/7 min), the supernatant discarded, and the process repeated. After the final wash, viability of cells was assessed using the 0.4% trypan blue dye exclusion method prior to culture. The cells (25 × 10³) were plated in triplicate in 96-well plates (Fisher Brand) with Roswell Park Memorial Institute (RPMI)-1640/10% fetal bovine serum (FBS; Medium 1; all from Sigma-Aldrich) or RPMI-1640/10% hUCBP ABO Rh matched (Medium 2). After 24 h of incubation, phytohemagglutinin (PHA; Sigma-Aldrich) was added to the culture at 1 μg/ml or 10 μg/ml. The cell colonies in the entire well were counted at 24, 48, and 72 h of incubation. The index of stimulation (IS) was determined as the number of induced colonies/number of spontaneous colonies in the control wells.

Caspase 3/7 Cell Activity

MNCs isolated from the peripheral blood of ALS patients and healthy volunteers were plated and incubated in Medium 1 as described above for 5 days, after which the cells were incubated in Medium 2 for 24 h. Caspase 3/7 activities were determined in these cells using a Magic Red Caspase 3/7 kit (Immunochemistry Technologies, LLC, Bloomington, MN, USA). Briefly, 10 μl of the 31X Magic Red (aspartate–glutamate–valine–aspartate)₂, [MR-(DEVD)₂] solution was added to each cell well and incubated for 1 h. Then Hoechst dye (nuclei staining; Sigma-Aldrich) was added at 1 μl/well and incubated for an additional 5 min. Immediately after incubation, five representative photomicrographs were produced, and counts of caspase 3/7- and Hoechst-positive cells were performed using ImagePro Software. Apoptotic caspase 3/7 cells were expressed as the percentage of the total Hoechst cells.

Statistics

Data are presented as mean ± SEM. The results were evaluated using ANOVA and Tukey's post hoc test or a paired Student's t-test (Excel; Microsoft, Redmond, WA, USA). A value of p < 0.05 was considered significant.

Results

Subject Demographics

Twelve clinically definite ALS patients (11 males and 1 female, mean age 53 ± 2.7 years; range 39–69) and six healthy controls (three males and three females, mean age 61.3 ± 4.8 years; range 38–69) entered our study (Table 1). Eleven patients were Caucasian, and one patient was African-American. The ALS patients were divided into three groups based on their ALSFRS assessment scores with four patients in each: Group A (late stage; ALSFRS < 20; 17.75 ± 0.9), Group B (intermediate; 20 < ALSFRS < 30; 22 ± 0.7), and Group C (early stage; ALSFRS score ≥ 30; 32.5 ± 1.0). The three groups are significantly different based on ALSFRS and ALSFRS-R scores (p < 0.05) but not age, disease duration, or time from diagnosis. All healthy control patients scored 40/48 on the ALSFRS/ALSFRS-R assessments.

Table 1.

ALS Patient Demographics

	All ALS Patients (n = 12)	Patients Grouped by ALSFRS Score			Healthy Volunteers (n = 6)
	All ALS Patients (n = 12)	Group A (n = 4)	Group B (n = 4)	Group C (n = 4)	Healthy Volunteers (n = 6)
Age (years)	53 ± 2.7 (39–69)	51.3 ± 6.4 (39–69)	54.0 ± 5.2 (39–63)	53.8 ± 3.1 (45–59)	61.3 ± 4.8 (38–69)
Sex (male/female)	11/1	4/0	3/1	4/0	3/3
ALSFRS	24.1 ± 1.9 (15–35)	17.8 ± 0.9 (15–19)	22 ± 0.7 (21–24)	32.5 ± 1.0 (30–35)	40.0 ± 0.0
ALSFRS-R	30.7 ± 2.1 (21–41)	24 ± 1.5 (21–27)	28.5 ± 1.6 (25–32)	39.5 ± 1.0 (37–41)	48.0 ± 0.0
Disease onset (months)	42.5 ± 7.8 (11–96)	53.5 ± 13.9 (26–88)	47 ± 16.8 (20–96)	27 ± 8.4 (11–49)	NA
Months since diagnosis	21.5 ± 4.6 (5–53)	26 ± 6.9 (13–43)	25.8 ± 9.9 (7–53)	12.8 ± 7.1 (5–34)	NA

Values are mean ± SEM. The range is shown in parentheses. ALSFRS-R, amyotrophic lateral sclerosis functional rating scale-revised.

CBC and WBC Differentials

CBC profiles and WBC differentials were analyzed for the ALS and control patients (Fig. 1). CBC and WBC differential results showed no significant differences between ALS patients and controls except for monocytes, which were significantly higher in all ALS patients (8.98% vs. 7.3%; p < 0.05).

Figure 1.

Hematological analysis of the peripheral blood. No significant differences in the hematological analysis of the peripheral blood were observed between amyotrophic lateral sclerosis (ALS) patients (n = 10) and healthy volunteers (n = 5), except for a significant increase in monocyte number (*p < 0.05).

Effects of the Mitogen PHA on MNC Populations In Vitro

MNCs were isolated from peripheral blood collected from ALS patients and healthy individuals and were cultured in vitro with the mitogen PHA (Fig. 2A). While the typical time- and dose-dependent increase in PHA stimulation was observed in healthy control volunteers (p < 0.05; n = 5), the dose-dependent (p < 0.05), but not the time-dependent, increase was observed in all ALS patients. At 72 h and a dose of 10 μg/ml, the control patients' IS was significantly higher than that for the ALS MNCs. There were three distinct profiles that emerged when the isolated MNCs were incubated with PHA (Fig. 2B). The IS for some ALS patients was similar to that of controls showing the typical time-dependent response but also a nonsignificant dose-dependent response (Group I; n = 5). However, abnormal extensive proliferation (an increased stimulation with a decreasing trend over time) was observed in one patient (Group III; this is neither the female patient, the African-American, nor the patient with the lowest ALS score, though it is the oldest patient), while noninducible proliferation was observed with MNCs isolated from other ALS patients (Group II; n = 6). Group II also showed a significant dose-dependent response at each time point (p < 0.05) and was significantly reduced compared to Group I and controls at 48 and 72 h for both concentrations of PHA. Additionally, MNCs isolated from hUCB showed little to no cell proliferation with either concentration of PHA used (data not shown). The normal, abnormal extensive proliferation, and nonresponding patients did not correlate with the three ALSFRS-designated groups. Reanalysis of the previous parameters using this grouping also did not reveal any significant differences. Since Group III only contained one patient, no statistics could be performed using this group.

Figure 2.

PHA-induced proliferation of MNCs isolated from peripheral blood in Medium 1 (containing FBS) and Medium 2 (containing hUCBP). (A) The response profile of mononuclear cells (MNCs) from healthy controls (n = 5) to phytohemagglutinin (PHA; 10 μg/ml) stimulation when the cells were incubated with Medium 1 [fetal bovine serum (FBS) only containing] showed a normal increasing index of stimulation (IS) with time. However, in ALS patients (n = 12) this was not observed. A smaller, but similar, effect was seen with the lower dose (1 μg/ml). The 10-μg/ml PHA IS was significantly higher than the 1 μg/ml at all time points for both ALS and controls (p < 0.05), and the 72 h 10 μg/ml was significantly higher in controls. (B) Examination of the responses to PHA stimulation revealed that there were three different response profiles for the ALS patients' cells. The IS of MNCs from some ALS patients was similar to controls (Group I; n = 5), but abnormal extensive proliferation (increased stimulation with a decreasing trend over time; Group III; n = 1) and noninducible proliferation were also observed (Group II). Group II (n = 6) was significantly different from both Group I and controls at both concentrations (*p < 0.05), and the 10 μg/ml PHA IS was significantly higher than the 1 μg/ml at all time points for Group II ALS and control only (p < 0.05). (C) When MNCs were cultured in Medium 2 containing hUCB plasma, the proliferation response of cells to PHA (10 μg/ml) of ALS patients remained significantly reduced compared to controls (*p < 0.05). (D) Splitting the ALS patients into the previous three groups based on their response to PHA in Medium 1 demonstrated that the proliferation response of cells to PHA (10 μg/ml) was blunted in cells exhibiting abnormal extensive proliferation (Group III) when cultured in Medium 1. An insignificant increase in cell proliferation was observed in cultures with a “normal” response to PHA (Group I), and no significant differences between Medium 1 and Medium 2 were found in cell cultures with noninducible proliferation (Group II). Group II remained significantly different from control and Group I with Medium 2 (*p < 0.05). (E) Images show decreased numbers of colonies in Medium 2 (Group III; abnormal extensive cell proliferation). Scale bars: 100 μm.

Isolated MNCs were also cultured with media supplemented with plasma collected from hUCB (Medium 2) to determine whether this might effect PHA-induced proliferation. Cells from control individuals showed no significant increase in the IS after incubating with Medium 2 at each point (Fig. 2C). Cells from all ALS patients appeared to exhibit a mild time-dependent IS response, which was significantly lower than that for the control MNCs at 48 and 72 h. A closer examination of the ALS patients revealed that the cells that exhibited abnormal extensive proliferation (Group III) using Medium 1 showed a blunted expansion with Medium 2 (Fig. 2D). Insignificant increases were observed in cultures with a standard response to PHA (Group I; n = 5), while no differences between Medium 1 and Medium 2 were observed from cell cultures that exhibited noninducible proliferation (Group II). Group II MNCs had a significantly lower index of stimulation than Group I and controls at both 48 and 72 h with regard to Medium 2. Again, no differences were observed when the patients were grouped by ALSFRS, and no correlations were evident. An example of the abnormal extensive proliferation is shown in Figure 2E.

Caspase Activity in MNCs From ALS Patients

Caspase 3/7 activity was determined in MNCs isolated from the peripheral blood of ALS patients to determine the potential of these cells to undergo apoptosis. MNCs, isolated from ALS patients, cultured in Medium 1 showed many caspase 3/7-positive cells with significantly more pronounced expression in cells compared to controls (p < 0.05) (Fig. 3A). Caspase activity of the ALS patients generally showed more activity in patients that exhibited abnormal extensive proliferation or noninducible proliferation compared to MNCs that showed a normal response to PHA. Using Medium 2 supplemented with hUCB plasma resulted in significantly lower apoptotic activity after a 24-h incubation for all ALS patients (p < 0.05). However, group analysis suggested that only the Group A (ALSFRS < 20) and Group C (ALSFRS > 30) patients had significantly reduced levels of caspase 3/7 (p < 0.05; data not shown). When grouped by their response to PHA, only MNCs from patients that exhibited an abnormal response to PHA stimulation showed decreased apoptotic activity (p < 0.05) when cultured in Medium 2 for Groups II and III (Fig. 3B).

Figure 3.

Caspase 3/7 activity in MNCs isolated from the peripheral blood of ALS patients. (A) Many caspase 3/7-positive cells were found in the MNCs of ALS patients cultured for 5 days in Medium 1, which was significantly different from that in controls (*p < 0.05). When Medium 1 was changed to Medium 2 containing hUCB plasma for 24 h, the apoptotic activity of cells in the ALS patients was significantly lower than in Medium 1 (p < 0.05). (B) More caspase 3/7-positive cells were found in patients with abnormal extensive proliferation (Group III) and noninducible proliferation (Group II) compared to patients with “normal” response to PHA (Group I), though this was not significant. Cultured MNCs in Medium 2 showed significantly decreased apoptotic activity in patients with an abnormal response to PHA stimulation (p < 0.05). (C) Images show the decreased numbers of caspase 3/7-positive cells in Medium 2 (Group III) (red, asterisks). The nuclei are stained with Hoechst. Magnification: 20×.

Discussion

As we have shown previously, intravenous administration of hUCB cells delayed the progression of disease and prolonged life span in the G93A SOD1 mouse model of ALS (15–17). These results were further supported by observations of increased motor neuron survival in both the cervical and lumbar regions of the spinal cord. Also, restored WBC profiles and decreased proinflammatory cytokine production were determined. While these results have yet to be replicated in the clinic, here we demonstrate the therapeutic potential of using plasma derived from hUCB to mitigate the mitogen-induced proliferation response of MNCs isolated from the peripheral blood of ALS patients in vitro.

ALS patients differed in lymphocyte functionality, possibly due to differences in the immune response as a consequence of the disease state. The patient with an abnormally extensive cell proliferation in response to mitogen (PHA) in vitro (Group III) may result from an autoimmunity impairment, while the noninducible proliferation patients (Group II) may indicate immune deficiency. Repetition in a larger group of patients (also containing more females) should be considered to determine whether the abnormally extensive cell proliferation is an “anomaly” or a “real” response in ALS. This suggests the use of therapies that affect the immune system may not be effective in all patients, indicating that a more personalized medicine approach may be necessary. A recent clinical study of autologous MSCs as a treatment therapy for ALS suggested that not all patients responded to treatment (20). A higher secretion of biological markers such as VEGF, angiopoietin, and TGF-β was observed from the MSCs of those patients who responded to the treatment, and this could be explored further with regard to our observations.

Innate and adaptive immune responses clearly play an important role in ALS. Infiltration of microglia and T cells is evident, and it has been suggested that these cells may initially be protective (5,6,9), but some studies have also observed lymphopenia in ALS patients or G93A SOD1 symptomatic mice (5,22,31). However, the precise roles of the immune responses, whether causative and/or a consequence of the disease, still need to be determined [reviewed in Murdock et al. (27) and Rodrigues et al. (33)]. While there is no doubt that the immune system is involved in ALS, it is worth noting that immunosuppressive therapies for ALS are not very effective (29). There is evidence for autoimmunity being a component of ALS, though it is unclear whether it is causative or an epiphenomenon (1,3,10,28,29,33), with some suggestion that autoimmunity could be beneficial in chronic neuroinflammation [reviewed in Schwartz and Baruch (36)]. Serum, CSF, and immune cells from ALS patients have also been shown to contain increased levels of IL-17 and IL-23, which may be a sign of T helper 17 (Th17) cell activation—a cell type that may play a crucial role in destructive autoimmunity (13,32,35).

While our study demonstrated impairment of mononuclear cells obtained from the peripheral blood of ALS patients via mitogen induction, Bossolasco et al. (7) have detected impaired functionality of bone marrow stem cells (BMSCs) from ALS patients in the ability to proliferate and differentiate into adipogenic and osteoblastic tissue, though Ferrero et al. (12) noted no significant differences in the proliferation potential of bone marrow mesenchymal stem cells from ALS patients. Liu and Martin (25) showed a similar impairment of neural stem cells (NSCs) in the subventricular zone of symptomatic G93A SOD1 mice. These studies suggested that some cell populations, such as the peripheral blood lymphocytes and possibly the BMSCs, undergo changes in their ability to proliferate and/or differentiate in ALS patients. However, no reports exist to confirm any abnormal cell function, though Kang et al. (19) have detected enhanced proliferation of nonstimulated oligodendrocytic progenitors in the G93A SOD1 transgenic mouse.

Our other study findings demonstrated that cord blood plasma was effective at modulating the cell response to PHA in the patient with abnormally extensive cell proliferation (Group III) as well as the patients who responded similarly to controls (Group I), but not the patients with noninducible cell proliferation (Group II). Also, hUCBP decreased apoptotic caspase 3/7 activity in MNCs isolated from the peripheral blood of patients with both abnormal extensive or noninducible cell proliferation to the mitogen (PHA). Additionally, when standard media (Medium 1) was replaced with media containing hUCBP (Medium 2), the apoptotic activity of the MNCs in culture tended to decrease. These findings reinforce the current anti-inflammatory observations that have been made of hUCB cells (15) and also demonstrate that plasma derived from cord blood could be an effective treatment in ALS patients with immune dysfunction as an immune-modulator and/or antiapoptotic factor.

ALSFRS/ALSFRS-R scoring of ALS patients is a well-recognized and widely used standard in ALS clinics to validate patient disease stage. Although the testing methodology might be subjective, all the data were collected by the same neurologist in order to minimize the potential for bias. The scores were then calculated using the online ALS C.A.R.E. program (http://www.outcomes-umassmed.org/ALS/alsscale.aspx).

Although the patient sample size in our study was modest, it was sufficient to provide a valid analysis of hUCB plasma effects on mitogen-induced proliferation of MNCs isolated from the peripheral blood of ALS patients. Additionally, the significant reduction of apoptotic activity of these cells via hUCB plasma is an important study finding. However, one consideration is the imbalance in gender (only one female patient) within this study. Using a gender-balanced cohort of patients would help confirm variances in cell response to mitogen, especially with regard to the single patient that showed abnormally extensive proliferation of peripheral blood mononuclear cells. Even if this patient had an anomalous cell proliferative reaction, the heterogeneous nature of cellular response to mitogen noted in the other two patient groups suggests the likely necessity of a personalized therapeutic approach.

In conclusion, the results of this present study illustrate the therapeutic potential of using hUCBP to modulate immune cell response to stimulation with the mitogen PHA. Also, hUCBP might be a novel therapy that potentially corrects any immunological issues that arise from ALS and should be further investigated as a potential therapy for this disease. This therapy could also be combined with hUCB cell (or other cell) transplants to potentially help provide a more supportive environment for the transplanted cells.

Footnotes

Acknowledgments

This study was, in part, supported by the Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, and Saneron CCEL Therapeutics, Inc. P.R.S. is cofounder of, and S.G.D. is a consultant for, Saneron CCEL Therapeutics, Inc., and they both hold patents in the use of hUCB as a cell therapy for a number of disorders. S.G.D. is funded by NIH grant (R01 NS090962-01). Neither D.J.E., as associate editor of Cell Transplantation, nor any member of the editorial office or editorial board affiliated with the author's institution were involved in the review process or the decision making with regard to publication for this manuscript.

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