United States

Background: Hydrodynamic transfection was recently demonstrated to deliver recombinant arylsulfatase A, the enzyme deficient in metachromatic leukodystrophy, to diverse tissues in murine models of this lysosomal storage disorder (LSD). Late-infantile neuronal ceroid lipofuscinosis (LINCL) is another LSD, caused by the deficiency of the lysosomal protease tripeptidyl-peptidase 1 (TPP1), whose existing therapy could be improved by endogenously synthesizing and trafficking extra-chromosomally-encoded recombinant TPP1 (rTPP1) across the blood-brain barrier (BBB). We sought to determine if hydrodynamic transfection could target therapeutic quantities of rTPP1 across the BBB in mouse models of LINCL.

Methods: Previously-designed rTPP1 constructs (wild-type human TPP1 [WT TPP1], TPP1R465G W470G, and TPP1R465G W470G-(GS)5-D10-(GS)5-K16ApoE [TPP1R465G W470G DKA]) were cloned onto plasmid vectors: (1) N1, a multipurpose vector and (2) CAG, a vector optimized for hydrodynamic transfection. Four plasmid constructs were synthesized: N1 WT TPP1, N1 TPP1R465G W470G, N1 TPP1R465G W470G DKA, and CAG WT TPP1. Plasmid constructs were purified from bacterial cultures and LINCL model mice lacking TPP1 activity were injected with 50 μg of plasmids in 2.5 mL of phosphate-buffered saline (PBS). Blood plasma samples were drawn at 6, 24, and 48 hours post-transfection, after which point the mice were sacrificed and brains and livers were harvested. Plasma samples and brain and liver homogenates were assayed for TPP1 activity, which was compared with non-transfected LINCL mice. Activity was normalized to that of non-injected WT mice. Welch’s ANOVA test was used to determine enzymatic activity differences between non-transfected LINCL mice and those transfected with synthesized constructs, and the resulting p-values were adjusted for multiple comparisons using Dunnett’s T3 test.

Results: All constructs exhibited high levels of expression in murine hepatocytes (p&lt;0.0001) and were successfully secreted into the bloodstream (p&lt;0.0001). No TPP1 activity was detected in non-injected LINCL mice and PBS-injected LINCL mice. N1 TPP1R465G W470G (mean [95% confidence interval]: 5.0% [2.1%, 8.0%], p=0.0352) and CAG WT TPP1 (3.8% [1.9%, 5.6%], p=0.0198) exhibited increased brain delivery at the 0.05 level of significance, while brain delivery of N1 WT TPP1 (4.8% [0.9%, 8.6%], p=0.1712) and N1 TPP1R465G W470G DKA (1.5% [1.0%, 2.0%], p=0.0518) were not statistically-significant.

Conclusion: N1 TPP1R465G W470G and CAG WT TPP1 exhibited modest ability to cross the BBB. Although this result is encouraging, further study is required to evaluate the therapeutic efficacy of these constructs. In addition, it will be necessary to determine the potential to translate these findings to humans.

2023 AMA Research Challenge

Evaluation of Hydrodynamic Transfection for Brain Enzyme Replacement Therapy for Late-Infantile Neuronal Ceroid Lipofuscinosis

Background: Hydrodynamic transfection was recently demonstrated to deliver recombinant arylsulfatase A, the enzyme deficient in metachromatic leukodystrophy, to diverse tissues in murine models of this lysosomal storage disorder (LSD). Late-infantile neuronal ceroid lipofuscinosis (LINCL) is another LSD, caused by the deficiency of the lysosomal protease tripeptidyl-peptidase 1 (TPP1), whose existing therapy could be improved by endogenously synthesizing and trafficking extra-chromosomally-encoded recombinant TPP1 (rTPP1) across the blood-brain barrier (BBB). We sought to determine if hydrodynamic transfection could target therapeutic quantities of rTPP1 across the BBB in mouse models of LINCL.

Methods: Previously-designed rTPP1 constructs (wild-type human TPP1 [WT TPP1], TPP1R465G W470G, and TPP1R465G W470G-(GS)5-D10-(GS)5-K16ApoE [TPP1R465G W470G DKA]) were cloned onto plasmid vectors: (1) N1, a multipurpose vector and (2) CAG, a vector optimized for hydrodynamic transfection. Four plasmid constructs were synthesized: N1 WT TPP1, N1 TPP1R465G W470G, N1 TPP1R465G W470G DKA, and CAG WT TPP1. Plasmid constructs were purified from bacterial cultures and LINCL model mice lacking TPP1 activity were injected with 50 μg of plasmids in 2.5 mL of phosphate-buffered saline (PBS). Blood plasma samples were drawn at 6, 24, and 48 hours post-transfection, after which point the mice were sacrificed and brains and livers were harvested. Plasma samples and brain and liver homogenates were assayed for TPP1 activity, which was compared with non-transfected LINCL mice. Activity was normalized to that of non-injected WT mice. Welch’s ANOVA test was used to determine enzymatic activity differences between non-transfected LINCL mice and those transfected with synthesized constructs, and the resulting p-values were adjusted for multiple comparisons using Dunnett’s T3 test.

Results: All constructs exhibited high levels of expression in murine hepatocytes (p<0.0001) and were successfully secreted into the bloodstream (p<0.0001). No TPP1 activity was detected in non-injected LINCL mice and PBS-injected LINCL mice. N1 TPP1R465G W470G (mean [95% confidence interval]: 5.0% [2.1%, 8.0%], p=0.0352) and CAG WT TPP1 (3.8% [1.9%, 5.6%], p=0.0198) exhibited increased brain delivery at the 0.05 level of significance, while brain delivery of N1 WT TPP1 (4.8% [0.9%, 8.6%], p=0.1712) and N1 TPP1R465G W470G DKA (1.5% [1.0%, 2.0%], p=0.0518) were not statistically-significant.

Conclusion: N1 TPP1R465G W470G and CAG WT TPP1 exhibited modest ability to cross the BBB. Although this result is encouraging, further study is required to evaluate the therapeutic efficacy of these constructs. In addition, it will be necessary to determine the potential to translate these findings to humans.

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Brain network organization varies between individuals, and individual differences in intrinsic brain networks have been linked to differences in cognitive ability. Individual differences in brain connectivity may be especially evident in brain networks that support executive functions such as cognitive control and attention such as the frontoparietal and salience networks. The frontoparietal network is theorized to play a role in cognitive control, which refers to the selection of thoughts and behaviors in context of task demands. Specifically, the frontoparietal network is necessary to flexibly coordinate behavior in a goal-driven manner. Meanwhile, the salience network has been hypothesized to modulate the relationship between the default mode network, which contributes to self-oriented cognition, and the frontoparietal network.
Prior work suggests that the maturation of executive functions during development parallels the integration of the salience networks with the frontoparietal network. Additionally, increased frontoparietal and salience intranetwork connectivity has been associated with development from periadolescence to adulthood. However, associations between frontoparietal or salience intranetwork connectivity and executive functions abilities have not been tested in periadolescent children. Here we tested whether intranetwork connectivity of frontoparietal and salience networks was correlated with executive function measures in periadolescent children. Participants in the NIA-funded Polygenic Risk of Alzheimer’s disease in Nebraska Kids (PRANK) study (N = 94) completed the NIH toolbox cognition battery as well as a functional MRI (fMRI) scan including resting-state data collection. Intranetwork connectivity was measured using the Human Connectome Project’s connectome workbench software. Results of the data analysis indicated a positive correlation between scores on fluid cognition measures and frontoparietal intranetwork connectivity, r(73) = .29, p = .01. A positive correlation was also observed with flanker task scores and frontoparietal intranetwork connectivity, r(73) = .25, p = .03. Finally, we observed a positive correlation with salience intranetwork connectivity and age-adjusted dimensional card sorting task scores, r(91) = .22, p = .03. Our preliminary findings show an association of executive function measures with intranetwork connectivity of two key intrinsic functional networks, and they suggest that frontoparietal and salience intranetwork connectivity may reflect increased efficiency of cognitive control during development.

Associations between salience and frontoparietal intranetwork connectivity and executive functions tasks in periadolescent children.

Cocaine use disorder (CUD) is a prevalent neuropsychiatric disorder with few existing treatments. Thus, there is an unmet need for the identification of new pharmacological targets for CUD. Previous studies using environmental enrichment versus isolation paradigms have found that the latter induces increased cocaine self-administration with correlative increases in the excitability of medium spiny neurons (MSN) of the nucleus accumbens shell (NAcSh). Expanding upon these findings, we sought in the present investigation to elucidate molecular determinants of these phenomena.

To identify potential new drug targets for CUD, we mined a previous large-scale differential transcriptomic dataset derived from the NAcSh of rats in environmentally enriched or isolated conditions that self-administered either saline or cocaine. Bioinformatic analyses revealed that fibroblast growth factor 13 (FGF13) mRNA, which codes for a prominent auxiliary protein of the voltage-gated Na+ (Nav) channel, is differentially expressed in the NAcSh by cocaine versus saline self-administration. Based upon this finding, we used in vivo genetic silencing to study the functional and behavioral consequences of knocking down FGF13 in the NAcSh. Functional studies revealed that knockdown of FGF13 in the NAcSh augmented excitability of MSNs by increasing the activity of Nav channels. Behaviorally, these electrophysiological changes were shown to correlate with knockdown of FGF13 in the NAcSh decreasing cocaine demand elasticity. 


FGF13-mediated Regulation of Medium Spiny Neuron Excitability and Cocaine Self-administration

Background
Sensorimotor disruption can lead to many neurological disorders, thus the investigation of these cortical areas in animal models is paramount to learn more about their function. Here, we used optogenetics a biological tool to control activity of neurons and other cell types with light and a whisker discrimination task in mice. This invoked localized silencing of the Somatosensory (S1) and Motor (M1) cortices to demonstrate their involvement in the whisker discrimination task and thus deepen our understanding of cortical involvement in behavior. 

Methods
We used transgenic VGat-Chr2 mice, which expressed channelrhodopsin in inhibitory neurons. These inhibitory neurons can then be activated by light leading to localized silencing in targeted areas. The animals underwent a thinned-skull surgical procedure, where cranial windows were implanted over S1 and M1. The mice were then trained in a whisker-based go/no-go task that consisted of a direction discrimination, where one rotor direction is a distractor and the second is the important stimulus (Direction task). Optogenetic stimulation was applied during the behavioral sessions at different time points. Three cortical targets were examined: M1, S1, and V1 (control site).

Results
During the Direction task, optogenetic stimulation impaired performance, particularly during the Stimulus phase. M1 stimulation led to a reduction in correct rejections (CRs) and an increase in false alarms (FAs) during direction 2. S1 stimulation decreased the number of hits and CRs. The results for V1 were inconclusive due to limited data.

Conclusion
The impact of optogenetic inhibition of S1 suggests a disruption in the encoding of sensory properties and a decline in the animals' ability to select the correct response. Surprisingly, M1 inhibition resulted in an increase in false alarms (FAs) instead of suppressing motor planning and execution, indicating that M1 may play a role in the integration of sensory information rather than being directly involved in the motor response of licking. Overall, inhibiting S1 impairs the encoding of sensory properties and decision-making processes, while inhibiting M1 affects the integration of sensory information. This research provides valuable insights into the specific roles of cortical regions in whisker discrimination tasks, emphasizing the potential of optogenetic modulation to enhance our understanding of sensorimotor pathways.

Investigating cortical involvement in sensorimotor whisker discrimination task in mice

Stroke is a leading cause of disability in the United States and the general population worldwide. Currently there are no successful treatments to restore normal function to stroke patients once brain damage has occurred. Our laboratory has pioneered antibody therapy directed at the neurite inhibitory protein Nogo-A to promote neuroplasticity and improve post-stroke recovery. Recent work from our laboratory has showed that Nogo-A blocks nerve growth factor (NGF)-dependent TrkA phosphorylation and activates a ceramide-dependent pathway, which negatively influences neuronal survival and neurite outgrowth. These findings suggest that the upregulation of Nogo-A following ischemic injury may interfere with the intrinsic neurotrophin-dependent plasticity that mediates recovery. Our central hypothesis is treatments that circumvent or block Nogo-A inhibition of TrkA to stimulate TrkA-mediated neurotrophin signaling pathways will enhance neuroplasticity and improve recovery following ischemic stroke. We conducted a pilot study, where we examined the effect of a TrkA agonist on Nogo-A signaling and functional recovery after experimental stroke in vivo. Baseline behavioral tests including skilled forelimb reaching task and horizontal ladder walk task were conducted on 8 male Long Evans, black hooded rats. The animals were then subjected to ischemic damage in the sensorimotor cortex by permanent middle cerebral artery occlusion. We continued behavior analysis 1 week after stroke and the animals were treated either with novel selective TrkA agonist gambogic amide or vehicle. Behavior was repeated 8 weeks later to determine functional recovery. In addition, we will also inject BDA for tracing of axonal pathways. The preliminary data from the behavior tests suggests that animals treated with agonist have better functional recovery as compared to control animals. This work will provide new treatments and strategies for enhancing stroke recovery in addition to advancing the knowledge of the function of Nogo-A as a negative regulator of neurotrophin signaling following brain injury.

Removal of Nogo-A Inhibition by Neurotrophin Signaling Pathway Activation Improves Sensorimotor Function After Stroke 


Background
Alzheimer's disease (AD) is the only top 10 cause of death that cannot be prevented, cured, or even slowed. We are developing attenuated furoxans− thiophilic NO donors that have neuroprotective activity, cross the blood brain barrier (a common obstacle in CNS drug discovery), and improve memory in mice− with the goal of alleviating the symptoms of AD and understanding its pathophysiology. Although Furoxans have been studied extensively, their mechanistic chemistry and protein targets have not been explored; in order to identify the subset of relatively reactive cellular thiols we will need to incorporate binding partners.

Methods
In order to identify the cellular targets responsible for furoxan bioactivation, we will synthesize furoxan analogs incorporating a photoaffinity labeling group and a terminal alkyne to covalently modify target proteins. We used OCF3 due to previously seen activity and incorporated diazirine, a main photoreactive group used in identifying biomolecule targets, to help facilitate a covalent bond between the compound and its target. Mainly diazirines present reactive and alkynyl sites are crucial in understanding cellular interactions with attenuated furoxans. The PAL group will attach the probe to binding partners upon irradiation with UV light. Following the synthesis of the compounds, we performed photolysis analysis via HPLC to determine the affects on photoreactivity.

Results
The tautomeric form of the furoxan being studied (3-aryl versus 4-aryl) does impact photoreactivity. The 4-Aryl OCF3 analog had a half-life of 7.7 minutes at 312 nm and 4.9 minutes and 365 nm. The 3-Aryl OCF3 analog had a half-life of 27.0 minutes at 312 nm and 5.6 minutes and 365 nm.The 4-aryl has a shorter half-life than the 3-aryl at 312 nm, however, 3-aryl has a shorter half-life at 365 nm in comparison to the 4-aryl.

Conclusion
Based on the non-reactive nature of the compounds, utilization of molecular probes will aid in understanding the mechanism of action. Incorporating a photoaffinity labeling cap group to probes that possess the efficacy of our active furoxan library would help to identify the cellular targets responsible for furoxan bioactivation and address the reactivity of our series of compounds. The purpose of the work is to use various molecular probes to broaden our understanding of our furoxan library, how it functions in the biological system, and also to use the obtained information to enhance affinity, efficacy, and metabolic effects.

Synthesis of 3 & 4 Aryl Furoxan Photoaffinity Probes

Background: Ovarian cancer (OC) is a deadly disease, with the 3rd highest mortality to incidence ratio of all cancers. OC’s asymptomatic nature early in the disease causes identification at advanced stages in 70+% of patients, at which point the 5-year survival rate can be as low as 20%. To date, no single biomarker has been able to detect early OC with high sensitivity and specificity. While elevated levels of Cancer Antigen 125 (CA-125) have been used to monitor patients pre-/post-operatively, CA-125 is limited in detecting early OC, and other benign ovarian or nongynecological conditions also have shown elevated CA-125. There is a critical need for novel biomarkers that can be used for screening, early diagnosis of OC and distinction of benign vs malignant masses. Our objective is to develop a novel sensitive and specific biomarker panel for OC early diagnosis where prognosis can be improved.

Methods: 288 plasma samples from women with OC (at various stages), benign gynecological conditions, other cancer types, and healthy non-cancer controls were analyzed with a curated panel of 8 biomarkers: CA-125, Growth Differentiation Factor 15 (GDF-15), Human Epididymis Protein (HE-4), Hepatocyte Growth Factor (HGF), Interleukin-6 (IL-6), Leukemia Inhibitory Factor (LIF), Osteopontin (OPN), and Prolactin (PRL). Receiver-operating characteristic (ROC) analysis was performed to assess sensitivity and specificity of single and multi-biomarkers amongst various subgroups.

Results: Univariate ROC analysis identified HE-4 and GDF-15 as having the highest area under the curve (AUC) (AUC=0.868, AUC=0.850, respectively) when comparing all stages of OC to healthy controls, a value greater than CA-125 (AUC=0.762). GDF-15 had the highest AUC when differentiating between early OC and healthy controls (AUC=0.772), a value greater than CA-125 (AUC=0.607). Amongst univariate markers, LIF was able to best differentiate between several other types of cancer (peritoneal, pancreatic, lung, and stomach) and OC. Multivariate analysis identified the panel containing CA-125, GDF-15, HE-4, IL-6, OPN, and PRL as having the highest AUC when comparing OC to healthy controls (AUC=0.956 for all stages, AUC=0.896 for early stages) and to benign conditions (AUC=0.826 for all stages). Adding LIF to this model allowed for best differentiation between OC and other cancer types (AUC=0.805 for all stages).

Conclusion: These results validate a novel biomarker panel for early detection of OC as well as distinction of benign masses and other malignancies from OC. Our results are expected to have an important positive impact by accurately detecting premalignant changes or early-stage OC in asymptomatic individuals.

Novel Biomarker Panels for Ovarian Cancer Screening

Purpose: Age-related macular degeneration (AMD) is characterized by pathologic extracellular lipid-rich deposits below and above the retinal pigment epithelium (RPE), respectively termed drusen and reticular pseudodrusen (RPD). Given apolipoprotein E (apoE) is one of the major components in drusen and RPD, the polarity of apoE secretion by the RPE likely contributes to whether RPD, drusen, or both dominate in an AMD patient. The mechanism dictating the polarity of apoE secretion in the RPE remains unknown. However, ATP-binding cassette A1 (ABCA1), a lipid transporter expressed in the RPE, is required for maintaining extracellular apoE levels in hepatocytes, where ABCA1 and apoE are both polarized to the sinusoidal side of the cell. We therefore investigated whether localization of ABCA1 in the RPE dictates the polarity of apoE secretion and if modulation of ABCA1 levels or function can alter extracellular apoE levels.

Methods: Human fetal (hf) RPE were plated on Transwells to allow for separate manipulation of the apical and basolateral sides of the RPE. To induce preferential apical apoE secretion, we applied two treatments we have shown will alter apoE secretion polarity: the addition of serum to only one chamber and aging cultures for more than a year. Immunocytochemistry (ICC) was then used to assess the localization of ABCA1 in hfRPE. ABCA1 was colocalized to phalloidin, an apical marker, and MCT3, a basolateral marker. Accumulation of secreted apoE was evaluated by Western Blots of the apical and basolateral media. ABCA1 expression was determined by Western Blot after treatment with Liver X Receptor (LXR) agonist, which upregulates ABCA1 expression, and after treatment with probucol, a ABCA1 inhibitor.

Results: ICC of hfRPE under conditions that favored apical apoE secretion did not show a corresponding shift in apical localization of ABCA1. In general, whether apoE secretion was polarized or not, ABCA1 had a fairly even distribution between the apical and basolateral RPE membranes. Treatment of hfRPE with LXR agonist upregulated ABCA1 but did not increase or switch the polarity of apoE secretion. Treatment with probucol caused a decrease in ABCA1 expression but no consistent effects on apoE secretion.

Conclusions: We demonstrated that the localization of ABCA1 in hfRPE is not related to the polarized secretion of apoE. Further experiments will probe other candidate mechanisms governing the polarity of apoE secretion in the RPE.

ABCA1 is Not Responsible for Polarized ApoE Secretion in the RPE

Purpose: To test the hypothesis that rod photoreceptor mitochondria function in vivo progressively declines over time.

Methods: 2, 12, and 24 mo dark- and light-adapted C57BL/6J (B6J) mice were examined by OCT. We measured i) an index of mitochondrial configuration within photoreceptors measured from the profile shape aspect ratio (MCP/AR) of the hyperreflective band posterior to the external limiting membrane (ELM)], ii) a proxy for energy-dependent pH-triggered water removal, the thickness of the ELM-retinal pigment epithelium (ELM-RPE), and its correlate iii) the hyporeflective band (HB) signal intensity at the photoreceptor tips. Visual performance was assessed by optokinetic tracking.

Results: In 2 mo and 24 mo mice, MCP/AR in both inferior and superior retina were smaller in light than in dark; no dark-light differences were noted in 12 mo mice. Dark-adapted inferior and superior, and light-adapted superior, ELM-RPE thickness increased with age. The dark-light difference in ELM-RPE thickness remained constant across all ages. All ages showed a decreased HB signal intensity magnitude in dark relative to light. In 12 mo mice, the dark-light difference in HB magnitude was greater than in younger and older mice. Anatomically, outer nuclear layer thickness decreased with age. Visual performance indices were reduced at 24 mo compared to 2 mo mice.

Conclusions: Whilst the working hypothesis was not supported herein, the results raise the possibility of a mid-life adaptation in rod mitochondrial function during healthy aging in B6J mice, a plasticity that occurred prior to declines in visual performance. 


Do Multiple OCT Biomarkers Indicate Age-related Decline in Rod Mitochondrial Function in C57BL/6J Mice? 

Purpose:
Multipotent adult stem cells (MASCs) isolated from human neonatal foreskin have been previously shown to differentiate into multiple mesodermal phenotypes. In vivo bone regeneration studies indicated that MASCs differentiated to osteoclasts, which are derived from monocytes. We tested the ability of MASCs isolated from human neonatal foreskin to differentiate into hematopoietic phenotypes in culture.

Methods:
Human tissues were obtained from the National Disease Research Institute under an IRB. MASCs isolated from human neonatal foreskin were isolated using an established protocol, enriched by freeze thawing, and expanded in culture. The MASCs were obtained by trypsinizing confluent adherent cultures, then aliquoting these cells into suspension or adherent cultures. The cells were fed with media with logarithmic concentrations of dexamethasone, a non-specific differentiator, from 0 M to 10-6 M, for 4 weeks. One culture method was the use of 15 mL centrifuge tubes. The centrifuge tubes were placed in the incubator after feedings at a 45 degree angle. Another method was suspending the MASCs into methylcellulose. Adherent cultures were done in 24 well plates. The presence of hematopoietic phenotypes was tested by immunohistochemical staining with primary antibodies to CD45, CD11b, CD33, and CD4. Alexa Flour 488 conjugated goat anti-mouse was used as a secondary antibody, and DAPI was used to stain nuclei. Staining was then visualized and images were captured.

Results:
MASCs from human neonatal foreskin differentiated into hematopoietic phenotypes. Nucleated hematopoietic cells (anti-CD45), myeloid lineages (anti-CD33), leukocytes (CD11b), and lymphocytes (CD4) were visualized in the final suspension cultures. Phenotypes were absent in control cultures (no dexamethasone), and found in greater numbers cultures treated with 10-8 to 10-6 M dexamethasone. 

Conclusion:
These preliminary results indicate that human MASCs are capable of differentiating into cells of the hematopoietic lineage. CD45 is a marker for all nucleated hematopoietic cells, CD33 is specific for monocytes/macrophages, CD11b is specific for leukocytes, and CD4 is specific for lymphocytes. Further research is needed to confirm these in vitro results, and then test the ability of MASCs to differentiate to hematopoietic phenotypes in vivo.

Differentiating Human Multipotent Adult Stem Cells toHematopoietic Phenotypes in Vitro

Background
Our group has shown that high density mesenchymal stromal cell (MSC) sheet implantation improves bone healing in a critical-sized bone defect model; however, these cells exhibit increased aging and cellular senescence in prolonged sheet culture, diminishing their therapeutic potential. Promisingly, rapamycin has emerged as a potential agent to rejuvenate aged MSCs. This study aimed to confirm rapamycin’s ability to prevent senescence and isolate specific pathways/genes responsible.

Methods
hPMSCs were seeded at high density (65,000 cell/cm2) and treated with either rapamycin (10 nM) or control vehicle (DMSO) for up to 7 days. Senescence and autophagy were assessed by senescence-associated 𝛽-Galactosidase (SA 𝛽-Gal) staining and DAPI nuclear staining, respectively. Additionally, cell culture media was extracted at different time points to assess senescence-associated cytokines IL-6 and IL-8 via ELISA. Finally, total RNA was extracted for differential gene analysis via GeneChip Clariom S Human Arrays.

Results 
Rapamycin significantly decreased SA-B Gal staining at all time points, with 67.7% and 72.9% less area when compared to control at day 4 and 7, respectively. Senescent-associated cytokine IL-6 levels were significantly reduced when compared to control, with 614.3 pg/ml and 286.6 pg/ml decreases at day 2 and 4 of treatment, respectively. Interestingly, IL-8 expression had increased significantly at all time points, with a 979.9 pg/ml difference at day 4 of treatment. Autophagy associated vacuoles-stained area had a 3.94-fold increase at day 4 of treatment compared to vehicle. Microarray gene analysis identified a total of 429 genes with a 2-fold or greater change in expression following treatment. FoxO, cellular senescence, and cell cycle pathways are some of many effected with significantly altered expression of FBXO32, PSAT1, CXCL8, and MYC, acting as potential players in rapamycin’s anti-aging effects.

Conclusion 
Rapamycin has proven effective in prolonging pMSC lifespan. The speculation and understanding of genes enacting this function may aid in isolating this specific role for usage in prolonging sheet cultures to enhance bioengineering applications. Numerous pathways identified in this study may explain rapamycin’s anti-aging functionality and lead to the creation of therapeutics specifically targeting relevant genes. The presence of enhanced IL-8 may prove beneficial in preserving stem cell functionality, specifically for wound healing applications. These findings prove rapamycin is a candidate for improving stem cell preparation and support previous studies indicating IL-8 may enhance stem cell viability, results useful in advancing stem cell therapy usage for bone remodeling/bioengineering.


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Evaluation of Hydrodynamic Transfection for Brain Enzyme Replacement Therapy for Late-Infantile Neuronal Ceroid Lipofuscinosis

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