Rescue of Degenerating Neurons and Cells by Stem Cell Released Molecules: Using a Physiological Renormalization Strategy

Abstract

Evidence suggests that adult stem cell types and progenitor cells act collectively in a given tissue to maintain and heal organs, such as muscle, through a release of a multitude of molecules packaged into exosomes from the different cell types. Using this principle for the development of bioinspired therapeutics that induces homeostatic renormalization, here we show that the collection of molecules released from four cell types, including mesenchymal stem cells, fibroblast, neural stem cells, and astrocytes, rescues degenerating neurons and cells. Specifically, oxidative stress induced in a human recombinant TDP-43- or FUS‐tGFP U2OS cell line by exposure to sodium arsenite was shown to be significantly reduced by our collection of molecules using in vitro imaging of FUS and TDP-43 stress granules. Further, we also show that the collective secretome rescues cortical neurons from glutamate toxicity as evidenced by increased neurite outgrowth, reduced LDH release, and reduced caspase 3/7 activity. These data are the first in a series supporting the development of stem cell-based exosome systems therapeutics that uses a physiological renormalization strategy to treat neurodegenerative diseases.

Full text

ORIGINAL RESEARCH
Rescue of degenerating neurons and cells by stem cell
released molecules: using a physiological renormalization
strategy
Greg Maguire
1,2
, Lee Paler
1,2
, Linda Green
1
, Rosa Mella
3
, Maria Valcarcel
3
& Patricia Villace
3
1 BioRegenerative Sciences, Inc., San Diego, California
2 Auditory Sound Waves, LLC, San Diego, California
3 Innoprot, Derio-Bikkaia, Spain
Keywords
Neurodegeneration, neurons, secretome,
stem cells, stress granules.
Correspondence
Greg Maguire, BioRegenerative Sciences, Inc,
San Diego, CA.
Tel: 1-858-413-7372
Fax: 1-858-751-4714
E-mail:
gmaguire@bioregenerativesciences.com
Funding Information
No funding information provided.
Received: 15 February 2019; Revised: 26
March 2019; Accepted: 31 March 2019
doi: 10.14814/phy2.14072
Physiol Rep, 7 (9), 2019, e14072,
https://doi.org/10.14814/phy2.14072
Abstract
Evidence suggests that adult stem cell types and progenitor cells act collec-
tively in a given tissue to maintain and heal organs, such as muscle, through a
release of a multitude of molecules packaged into exosomes from the different
cell types. Using this principle for the development of bioinspired therapeutics
that induces homeostatic renormalization, here we show that the collection of
molecules released from four cell types, including mesenchymal stem cells,
fibroblast, neural stem cells, and astrocytes, rescues degenerating neurons and
cells. Specifically, oxidative stress induced in a human recombinant TDP-43-
or FUS-tGFP U2OS cell line by exposure to sodium arsenite was shown to be
significantly reduced by our collection of molecules using in vitro imaging of
FUS and TDP-43 stress granules. Furthermore, we also show that the collec-
tive secretome rescues cortical neurons from glutamate toxicity as evidenced
by increased neurite outgrowth, reduced LDH release, and reduced caspase 3/
7 activity. These data are the first in a series supporting the development of
stem cell-based exosome systems therapeutics that uses a physiological renor-
malization strategy to treat neurodegenerative diseases.
Introduction
Physiological renormalization of the immune system
instead of enhancement and direct attack is a new strategy
in the successful development of recent chemotherapeu-
tics for cancer (Sanmamed and Chen 2018), a strategy for
which the 2018 Nobel Prize in Physiology or Medicine
was awarded. In other words, strategies of immune nor-
malization therapy instead of enhancement of the
immune system or a therapeutic direct attack of the can-
cer cells, has renormalized T-cell physiology to perform
their normal attack of cancer cells through the B7-H1/
PD-1 pathway, bringing many new oncology “checkpoint
blockade” drugs to the market (Zappasodi et al. 2018).
Likewise, renormalization strategies for degenerative dis-
eases, including neurodegeneration, are a new means to
return the nervous system to homeostasis, including pro-
teostasis (Maguire 2018a,b), in diseases, such as ALS, that
have remained refractory to current therapeutic strategies
(Dorst et al. 2018; Maguire 2017). Physiological renor-
malization is a process that occurs naturally, for example,
in sleep-dependent synaptic downscaling, called synaptic
renormalization (Fink et al. 2013). Using a physiological
renormalization process to treat diseases may therefore be
a therapeutic development strategy that is more effica-
cious and safer than targeted, genomic approaches that
dominate today, and have been overhyped in the USA
and elsewhere (Woloshin et al. 2009; Prasad 2016).
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As in evolutionary studies, where physiology has
returned to the “centre stage” (Noble et al. 2014), thera-
peutic development must also bring physiology “centre
stage” (Moffat et al. 2014) given that genomics centric
therapeutic development strategies, having forgotten phys-
iology (Viney 2014), has failed to bring successful thera-
peutics to the market over the last two decades of
genome-centric research and development programs. In
this physiological renormalization strategy, the homeosta-
sis, including proteostasis, of the diseased nervous system
is restored through the application of exogenous stem cell
released molecules (secretome) in order to provide the
molecules, including heat shock proteins (HSP), needed
to mimic the functions of the cells and tissues and rebuild
the ECM and microenvironment, including the stem cell
niche.
Degenerative disorders and neurodegenerative disorders
in particular, are one of the most difficult challenges for
therapeutic development, medicine, and healthcare
because of their poorly understood epidemiology, etiol-
ogy, pathogenesis, and the heavy burden with which the
patients, healthcare system, and society must endure
(Dorsey et al. 2013). For example, exposure to multiple
toxins that act synergistically to cause neurodegeneration
add to the difficulty of understanding and predicting the
etiology of a given neurodegenerative disorder (Ku et al.
2016). Our exposome may account for 70–90% of
chronic disease (Rappaport and Smith 2010), including
the many cases of sporadic neurodegenerative diseases
where proteinopathies are greater than previously recog-
nized (Kovacs 2019). Neurodegenerative diseases (NDs)
such as glaucoma, sensorineural hearing loss, Alzheimer’s
disease, and amyotrophic lateral sclerosis among others
result from neuronal destruction in the central nervous
system. In NDs the volume of the brain is reduced and
the number of neurons and other cells decline over time.
These diseases reduce the function of patients and destroy
tissue and nerves of the brain because neurons in most
sections of the brain cannot reproduce themselves, except
for selective niches of the adult mammalian brain within
areas of the brain such as the hippocampal dentate gyrus
(Ming and Song 2005), amygdala (Jhaveri et al. 2017),
and the olfactory bulb, although even in the olfactory
bulb the regenerative capacity is not limitless (Child et al.
2018). The resident populations of neural stem and pre-
cursor cells that proliferate and differentiate into func-
tional neurons, drive neurogenesis in these regions
(Kempermann et al. 2018). Neural stem cells also release
molecules known to enhance survival of degenerating
neurons (Mendes-Pinheiro et al. 2018).
Many of the neurodegenerative disorders usually occur
at an older age, and are characterized by deficits in many
cell types and their surrounding tissue, including the
extracellular matrix (ECM) and its special condensed
form called perineuronal nets (PNN) that surrounds some
neurons, especially those with high spike rates (Cabungcal
et al. 2013). Stem cell function declines with age, and
therefore the ability of stem cells in the brain to make
and repair neurons and other tissues, including ECM, of
the brain will resultantly decline with age (Conboy et al.
2015).
Maintenance and healing of brain tissue relies on a
normal functioning ECM and microenvironment
(Maguire 2018a,b), including neural and mesenchymal
stem cells that provide maintenance factors, such as chap-
erones and heat shock proteins (Chiellini et al. 2008).
Neurons, once they have become differentiated, do not
produce these factors themselves (Oza et al. 2008), and
therefore must rely on neighboring stem cells as their
source of HSPs. The importance of HSP has recently been
demonstrated by Horwich’s laboratory where transgenic
expression of HSP110 extends the life of mice with a neu-
rodegenerative phenotype, a model that is much like
some aspects of ALS (Nagy et al. 2016). Furthermore,
other stem cells, such as mesenchymal stem cells, will
provide the molecules necessary to build the ECM and
microenvironment, and satellite cells in muscle, a type of
stem cell will help to regenerate the muscle tissue (Mur-
phy et al. 2011; Maguire 2017). Also, mesenchymal stem
cells can protect and rescue the extracellular matrix
(ECM), including perineuronal nets (PNN), a condensed
form of the ECM, and positively alter the course of neu-
rodegeneration in a mouse model of ALS (Forostyak et al.
2014). Other cells in the brain, including astrocytes,
releasing lipoxins, have been shown to be important in
rescuing CNS neurons during the degenerative process
(Livne-Bar et al. 2017).
Biology typically constrains intracellular molecules to a
particular domain by surrounding the molecules with
lipid membranes. However, another mechanism of con-
straint is present, cytoplasm phase separations (Walter
and Brooks 1995; Bowman et al. 2013) such as that
induced by the RNA-binding proteins that sequester
transcripts (Hentze et al. 2018). RNA-binding proteins
during oxidative stress events in the cell can bind pro-
teins that are unessential to cell viability, sequestering the
unessential proteins and allowing the cell to devote
energy and resources to the translation of necessary
“housekeeping” proteins. Recent studies have shown that
stress granules are assemblies of untranslating messenger
ribonucleoproteins (mRNPs) that form from mRNAs
stalled in translation initiation. Stress granule assembly
minimizes stress-related damage and promotes cell sur-
vival (Mahboubi and Stochaj 2017). Persistent or aber-
rant stress granule formation, such as FUS stress
granules, is associated with neurodegenerative disease and
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The Physiological Society and the American Physiological Society.
Rescue of Degenerating Cells by Stem Cell Secretome G. Maguire et al.

some cancers, and serves as an important biomarker for
neurodegenerative diseases (Protter and Parker 2016).
FUS is present in exosomes (Kamelgarn et al. 2016), sug-
gesting that the secretion of FUS might contribute to the
cell-to-cell spreading of FUS pathology in neurodegenera-
tive diseases such as ALS. Because FUS normally regu-
lates microRNA-based gene silencing, aberrant FUS
function can prevent microRNA gene silencing as shown
in a model for ALS (Zhang et al. 2018a,b), possibly
doing so in a spreading, cell-to-cell manner through exo-
some delivery. Likewise, TDP-43 stress granule formation
is a hallmark of a number of neurodegenerative diseases,
such as ALS (Gao et al. 2018).
In this series of studies, we reasoned that neurodegen-
erative diseases may best be treated with a collective of
molecules released from the key cell types in the nervous
system that have been shown to rescue neurons. Our
reasoning derives from evidence that adult stem cells
types and progenitor cells act collectively in a given tis-
sue to maintain and heal organs, such as muscle,
through a release of a multitude of molecules packaged
into exosomes from the different cell types (Murphy
et al. 2011; Fry et al. 2017). Our initial studies therefore
use an in vitro model of neurodegeneration, where neu-
rons or U2OS cells were insulted with arsenite or gluta-
mate, and the endpoints measured were stress granule
formation, or a number of mechanisms underlying neu-
rotoxicity. Our intervention was to use a proprietary and
patented mix of all the molecules in the secretome of
four cell types found in brain tissue to determine
whether this therapeutic candidate would prevent or les-
sen the formation of stress granules as an indicator that
the intervention prevented oxidative stress in the insulted
neurons. The results we present here show a significant
reduction in FUS and TDP-43 stress granule formation,
LDH release, and caspase 3/7 activation, along with an
increase in neurite outgrowth in the presence of our col-
lective secretome when the neurons were challenged with
glutamate. These data suggest that the molecules released
from stem cells, and other cell types, provide an impor-
tant new means to develop systems therapeutics
(Maguire 2014) that may provide key advantages over
standard small molecule-targeted approaches (Maguire
2014) and over the use of stem cells themselves (Maguire
2016a,b). Important to our development of a systems
therapeutic is allowing the stem cells to release their
molecules as opposed to using an extraction process
thereby allowing the molecules to fully form and fold
correctly, and to package into exosomes. The nanosphere
exosome will impart protective, targeting, and penetra-
tion qualities to the molecules that would not be present
in the molecule’s native state without packaging into
exosomes (Maguire 2016b).
Methods
Cell types and culture methodology
The secretome from four cell types was used to create our
therapeutic candidate: 1. Human skin-derived adipose mes-
enchymal stem cells (ADSCs) were acquired from Thermo-
Fisher Scientific (Cat. # R7788115), 2. Human skin-derived
fibroblasts (FBs) were acquired from ScienCell (Cat. #2300),
3. Human-derived astrocytes (Cell Applications, Cat # 882A-
05f), and 4. Human derived immortalized neural stem cells
(Millipore, Cat #SCC007). Standard protocols from the man-
ufacturers were utilized, and no antibiotics were used. Cell
types and cell culture methodology for the ADSCs and FBs is
described in detail in the patents issued to BioRegenerative
Sciences (US patent numbers 9545370; 9446075;
20140205563; 20130302273). The secretome from the four
cell types, containing both the exosome and the soluble frac-
tions, were combined in equal portions to form the therapeu-
tic candidate; that is, the secretome from each cell type
composed one-fourth of the total collective secretome. Only
the secretome from early passages (<10) of the cells was used.
In vitro testing
A series of three in vitro tests were performed in order to
assay the ability of a proprietary mix of secretome from
four different types of cells, including stem cells, to rescue
neurons from insult due to, (1) Glutamate, (2) Sodium
arsenite and the formation FUS stress granules, and (3)
Sodium Arsenite and the formation of TDP43 stress gran-
ules. All assays were performed in triplicate. In Figure 1
we show the basic protocol for running the cell culture
studies.
FUS study
Dose–responses for the SRM was performed using a cellu-
lar fluorescence redistribution assay after oxidative stress
induction in a human recombinant FUS-tGFP U2OS cell
line. The reduction, induced by the compounds, of the
FUS stress granules number within the cells after sodium
arsenite treatment was measured in triplicate. The FUS-
tGFP stress granules were quantified using the BD Path-
way HCS Reader and Attovision Compartmentalization
Software. The error bars represent the standard error of
the mean for the three replicate wells (Figs. 2 and 3).
TDP43 study
Dose–response functions for the stem cells released mole-
cules (SRM) were performed using a cellular fluorescence
redistribution assay after oxidative stress induction in
ª2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society.
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human recombinant TDP43-tGFP U2OS cell line. The
reduction, induced by the compounds, of the TDP-43
stress granules number within the cells after sodium
arsenite treatment was measured in triplicate. The
TDP43-tGFP stress granules were quantified using the BD
Pathway HCS Reader and Attovision Compartmentaliza-
tion Software. The error bars represent the standard devi-
ation among the three replicate wells (Table 1).
Glutamate neurotoxicity
Glutamate-induced excitotoxicity in rat cortical neuron
(RCN) primary cells was used to assay mitochondrial dam-
age, DNA damage, oxidative stress, neurite outgrowth,
membrane integrity measured by LDH release, and apopto-
sis as measured by Caspase 3 activation. The pattern of
neurite outgrowth of the neurons was analyzed by
immunocytochemistry against a Tubulin antibody,”TUJ.”
MK-801, a glutamate receptor (NMDA) antagonist was
used as a positive control. For determining plasma mem-
brane integrity, the supernatants were collected 24 h after
treatments and an LDH assay was performed following the
instructions of the kit manufacturer (Roche).
Our specific endpoints for glutamate toxicity were the
following:
(1) Apoptosis: Caspase-3 activation for Apoptosis
(2) Neurite Outgrowth: Tuj for Neurite Outgrowth
(3) Neuronal Viability: Cell Counting as viability
(Hoescht and WST-8 assay)
(4) Membrane Damage: LDH Release (Smith et al. 2011)
Protocol
Cortical neurons from embryonic 18 days rats were pla-
ted in poly-l-lysine coated 96-well plates with a number
of 30,000 cells per well. Cells were maintained in neu-
robasal medium supplemented with B-27 component
for 8 days at 37°C in a humidified 5% CO
2
atmo-
sphere. At day 8, cells were pretreated with seven
increasing concentrations of the conditioned media for
1 h in neurobasal medium supplemented with B-27
component, washed, and returned to drug-free medium
for up to 48 h before being subjected to a glutamate
excitotoxicity condition where cells were incubated with
100 lmol/L glutamate during 15 min in medium with-
out B-27 component. After glutamate exposure, med-
ium was replaced with neurobasal medium with
B27 factor, and then cells were incubated for an
additional 24 h. Some cells were treated as described
above with MK801 10 lmol/L as positive controls of
neuroprotection.
Statistics
A one-way ANOVA was applied in each experiment with
eight conditions, each condition reflecting a different con-
centration of the experimental secretome, from control to
100% concentration.
Results
For the three different studies (FUS, TDP43, and gluta-
mate neurotoxicity), each showed a significant reduction
in neuronal insult when the experimental arm was com-
pared to controls.
FUS
Seven concentrations of SRM were examined for their
inhibitory effects on FUS granules formation after
Protocol for Cell Culture
C
Experimental Control
Dose = percentage of added Exp. soluon,
Plus removal of same percentage medium
*Step one = removal of medium
*Step two = add experimental soluon
2. Add
Experimental
Soluon
1. Remove
Same
Amount
Of Medium
Dose = percentage of used medium,
Plus removal of same percentage medium
•Step one = removal of medium
•Step two = add used medium
2. Add
Used Medium
Soluon
1. Remove
Same
Amount
Of Medium
Cell
Culture
Well
Used medium is medium from past experiment for parcular cell type
Doses are : 0.1%, 1.0%, 5.0%, 10.0%, 20.0%, 50.0%, 100.0%
Figure 1. Basic protocol for cell culture in the experimental and control conditions.
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The Physiological Society and the American Physiological Society.
Rescue of Degenerating Cells by Stem Cell Secretome G. Maguire et al.

cytotoxic stress induction by arsenite. The number of
stress granules containing FUS per cell was analyzed for
each compound.
SRM was positive in the inhibition of FUS granule for-
mation after arsenite treatment. SRM-induced stress gran-
ule inhibition in a dose-dependent manner with a
maximum of 30.5% at 100% of the concentration, and
the effect was no longer observable at a concentration of
20% or less.
TDP43
Seven concentrations of SRM were examined for their
inhibitory effects on TDP-43 granules formation after
cytotoxic stress induction by arsenite. The number of
stress granules containing TDP43 per cell was analyzed
for each compound.
SRM was positive in the inhibition of TDP43 granule
formation after arsenite treatment. SRM-induced stress
0
1
2
3
4
5
6
7
Arsenite Vehicle Riluzole 100 50 20 10 5 1 0.1
Average number granules FUS/cell
Dose -response in number of FUS granules
–20
0
20
Arsenite Vehicle Riluzole 100 50 20 10 5 1 0.1
40
60
80
100
120
140
Increment number FUS granule /cell
Dose-response in percentage of FUS granules
C
B
A
Figure 2. (A) Dose response relationship for experimental secretome at concentrations between 0.1% and 100%. Data points represent the
mean SD for each condition for a single experiment performed by triplicate. Results are expressed as the average granule number per cell.
The images were obtained with an objective of 20 9. 9 pictures of each well were taken. One-way ANOVA: Pvalue 52,637E-06. (B)
Percentage of the FUS granules increment quantification for experimental solution at the concentrations proposed by the Sponsor. Data points
represent the mean SD at each condition for a single experiment performed by triplicate. The images were obtained with an objective of
209. 9 pictures of each well were taken. The results were normalized according to sodium arsenite and vehicle, considering Sodium arsenite
and vehicle as 100% and 0%, respectively.(C) Representative images. The pictures are representative images corresponding to Vehicle (control),
treatment with Arsenite (Ars), treatment with Riluzole at 5 lmol/L concentration, experimental solution provide by BRS at 100% concentration.
ª2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society.
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G. Maguire et al.Rescue of Degenerating Cells by Stem Cell Secretome

granule inhibition in a dose-dependent manner with a
maximum of 60.2% at 100% of concentration, and the
effect was no longer observable at a concentration of 10%
or less.
Glutamate neurotoxicity
Neurite outgrowth was measured as an indicator for res-
cue from glutamate neurotoxicity. As shown below, we
measured three different parameters of neurite out-
growth to demonstrate that the experimental secretome
significantly rescued neurons from glutamate neurotoxi-
city. In Figure 4A is shown the average maximum length
for neurites for the different cell culture conditions.
Compared to controls where glutamate was shown to
significantly reduce neurite length, 5.0% of the experi-
mental secretome significantly enhanced the length of
neurites.
0
2
4
6
8
10
12
Ars Vehicle Rilu 100 50 20 10 5 1 0.1
Average number TDP43 granule/cell
Dose-response in number of TDP-43 granules
–20
0
20
40
60
80
100
120
140
Ars Vehicle Rilu 100 50 20 10 5 1 0.1
% Increment average number
TDP43/cell
Dose-response in percentage of TDP-43 granules
C
B
A
Figure 3. (A) Dose response relationship for experimental solution at the concentrations between 0.1% and 100%. Data points represent the
mean SD for each condition for a single experiment performed by triplicate. Results are expressed as the average granule number per cell.
The images were obtained with an objective of 209. 9 pictures of each well were taken. One-way ANOVA: Pvalue 1,84,356E-05. (B)
Percentage of the TDP43 granules increment quantification for experimental solution provide by BRS at the concentrations proposed by the
Sponsor. Data points represent the mean SD at each condition for a single experiment performed by triplicate. The images were obtained
with an objective of 209. 9 pictures of each well were taken. The results were normalized according to sodium arsenite and vehicle,
considering Sodium arsenite and vehicle as 100% and 0%, respectively. (C) Representative images. The pictures are representative images
corresponding to Vehicle (control), treatment with Arsenite (Ars), treatment with Riluzole at 5 lmol/L concentration, experimental solution at
100% concentration.
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Rescue of Degenerating Cells by Stem Cell Secretome G. Maguire et al.

Another parameter for measuring neurite outgrowth is
the number of neurite roots extending from the neuron’s
soma. In Figure 4B is shown the mean number of roots
counted under the different experimental conditions.
Compared to the control condition where the neurons
were exposed to glutamate, the experimental secretome at
a concentration of 10% significantly increased the number
of neurite roots.
The number of branches on the roots of the neurites
was another parameter we measured as an assay for
neurotoxicity. In Figure 4C we show that glutamate sig-
nificantly reduces the number of branches, and that the
experimental secretome significantly increases the num-
ber of branches compared to the control glutamate con-
dition. The rescue of number of branches was maximal
at a concentration of 5.0% experimental secretome.
Higher concentrations of the experimental secretome
were not as effective in rescuing neurite outgrowth,
likely because the diluted culture medium did not sup-
ply all of the requisite factors needed for optimal neurite
growth.
Glutamate neurotoxicity –caspase
activation
Caspase activation levels were measured in primary rat cor-
tical pyramidal neurons as an indicator for rescue from glu-
tamate neurotoxicity. Caspases are a family of
endoproteases important for maintaining homeostasis
through regulating cell death and inflammation. In primary
cortical cells, glutamate-induced cell death occurs through
upregulation of caspase-3 and its activation of a caspase-
dependent pathway involving mitochondrial signaling
(Zhang and Bhavnani 2005). We therefore measured cas-
pase levels in cortical neurons challenged by glutamate to
determine whether our collective conditioned media could
inhibit caspase-3 and -7 levels. At concentrations of 10%
and higher, our collective secretome significantly reduced
the levels of caspase 3/7 compared to those levels induced
by overexposure to glutamate, and even reduced the cas-
pase 3/7 levels below that of the control condition where
no glutamate was applied.
Glutamate treatment resulted in a twofold increase of
caspase 3/7 activation in cortical neurons when measured
24 h after exposure at 100 lmol/L concentration, and
positive control MK801 prevented glutamate-induced
apoptosis by 65% (Fig. 5). Glutamate-induced caspase 3/7
activation was also prevented by application of the highest
concentrations of S4RM-N (10, 20, 50, and 100%).
Therefore, S4RM-N secretome supplementation signifi-
cantly reduced caspase 3/7 activation and improved the
survival of cortical neurons.
LDH-based cytotoxicity
To assess the effects of glutamate on plasma membrane
integrity, LDH quantification in supernatants of treated
cells was employed. Glutamate induced a 270% increase
of LDH in cultured cortical neurons, while the positive
control using MK801 normalized the plasma membrane
Table 1. Summarizing the magnitude of the rescue effects of the experimental Secretome.
Compound 0.1% bio 1% bio 5% bio 10% bio 20% bio 50% bio 100% bio
Caspase 0 0 0 2 2 2 2
Max length 0 2 3 2 1 1 2
Root/neuron 3 3 3 3 3 2 3
LDH 2 2 2 2 2 1 0
Total 5 7 8 9 8 6 7
Drug classification Moderate Moderate High High High Moderate Moderate
Compound 0.1% bio 1% bio 5% bio 10% bio 20% bio 50% bio 100% bio
Drug classification Moderate Moderate High High High Moderate Moderate
MK801 10 lmol/L
Caspase 3
Max length 3
Root/neuron 3
LDH 2
Total 11
Drug classification High
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0
5
10
15
20
25
30
35
40
Arbitrary Units
Average maximum length
0
0.5
1
1.5
2
2.5
3
Root count
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Extremity count
A
B
C
Figure 4. (A) Maximum length of neurite outgrowth as a function of glutamate toxicity and rescue from toxicity with experimental secretome
at concentrations between 0.1% and 100%. (B) Mean neurite root count as a function of glutamate toxicity and rescue from toxicity with
experimental secretome at concentrations between 0.1% and 100%. (C) Mean neurite branch (extremity) count as a function of glutamate
toxicity and rescue from toxicity with experimental secretome at concentrations between 0.1% and 100%.
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Rescue of Degenerating Cells by Stem Cell Secretome G. Maguire et al.

integrity by 45% (Fig. 6). Glutamate induced cell death
was also prevented by application of all concentrations
tested with the exception of 100% concentration. There-
fore, the test therapeutic (S4RM-N secretome) signifi-
cantly reduced LDH release and improved survival of the
cortical neurons. Different grades of neuroprotection were
observed with each compound at the different concentra-
tions tested (Fig. 6).
We summarize the protective effects of S4RM-N secre-
tome in the glutamatergic neurotoxicity studies as follows.
For the therapeutic secretome (S4RM-N) studied under
the various parameters, a variation of at least 20% in flu-
orescence intensity or in the corresponding morphological
parameter in relation to untreated cultures was estab-
lished. In order to compare the degree of neuroprotec-
tion, the level of change for each parameter at 24 h was
studied at each concentration. Four different scores of
neuroprotection were established according to the level of
variation when compared with control cells: 0 (no neuro-
protection or variation lower than 20%), 1 (variation 20–
40%), 2 (variation 40–60%), and 3 (variation >60%).
The sum of each individual score resulted in the total
level of neuroprotection for each compound and was
defined as its degree of neuroprotection. From this calcu-
lation, a neuroprotective scale was established: high (>8),
moderate (5–7), low (1–4), and no neuroprotection (0).
Figure 5. (A) Validation of caspace 3/7 activity in control
conditions, glutamate exposure, and the antagonism of glutamate
by MK801. This study validated that glutamate induces an increase
in caspase 3/7 activity in cortical neurons and that MK801, a
glutamatergic NMDA receptor antagonist, blocked the effects of
glutamate on the induction of caspase 3/7 activity. (B) Caspase 3/7
activity in primary cortical neurons exposed to glutamate in
combination with the S2RM collective secretome. The control
shows the baseline level of caspase activity without exposure to
glutamate or the S2Rm. The glutamate bar is for exposure
to glutamate without S2RM. The bio bars indicate exposure to
glutamate plus the addition of the various percentages of the
S4RM-N secretome at concentrations from 0.1% to 100%. Each
condition was run in triplicate. These data show that concentrations
of the S2RM collective secretome at 10% and higher significantly
reduced the induction of caspase 3/7 activity in cortical neurons.
Figure 6. (A) LDH secretion determination using positive and
negative control. Neurons were pre-treated with MK801 10 lMin
complete medium for 1h and then returned to drug-free complete
medium for 48h. Cultures were then exposed to glutamate 100 lM
for 15 min and 24 h later LDH assay was performed. (B) LDH
secretion in cells treated under glutamate excitotoxicity condition.
Neurons were pre-treated with increasing concentrations of the
compound in complete medium for 1h and then returned to drug-
free complete medium for 48h. Cultures were then exposed to
glutamate 100 lMfor 15 min and 24 h later LDH assay was
performed. Data points represent the mean SD for each
condition. The results of the compounds were normalized
according to the control cells. “Bio” indicates the concentration of
the experimental secretome added to the culture dish.
ª2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society.
2019 | Vol. 7 | Iss. 9 | e14072
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G. Maguire et al.Rescue of Degenerating Cells by Stem Cell Secretome

In the present study glutamate toxicity was linked to
an increase in caspase 3/7 activation, LDH secretion, and
decreased neurite outgrowth. The preventive effects of
S4RM-N against glutamate toxicity are associated with
restoration of caspase 3/7 activity, stabilization of neurite
outgrowth, and decrease in LDH secretion. All concentra-
tions tested of S4RM-N scored an optimum degree of
neuroprotection level and was shown to be an efficient
strategy for the treatment of glutamate toxicity. Neuro-
protection from glutamate toxicity was most efficacious at
the concentrations of 5, 10, and 20% compared to the
other concentrations.
Discussion
Our data show that molecules released from a collective
of four cell types known to be important to neuronal
function and regeneration can rescue isolated neurons
from glutamate insult, and rescue U2OS cells from arsen-
ite insult as measured in vitro. Specifically, the secretome
from neural stem cells, mesenchymal stem cells, astro-
cytes, and fibroblasts was able to mitigate FUS- and TDP-
43 stress granule formation in U2OS cells, and a number
of key mechanisms underlying glutamate neurotoxicity in
CNS neurons, including : 1. Mitochondrial function, 2.
Neurite outgrowth, 3. Membrane integrity, 4. Neuronal
viability, and 5. Apoptosis.
Our methodology for therapeutic development depends
on targeting pathways at multiple levels of the system,
including protein and genomic levels. Considering the
protein-level pathways, many natural molecular, cellular,
and tissue functions are initiated and maintained by pro-
tein-level circuits. As an example, caspase mediated pro-
grammed cell death, apoptosis, is orchestrated by a circuit
of proteases that activate one another by cleavage (Budi-
hardjo et al. 1999). Modifiable protein circuits offer a
number of advantages over genetic circuits, including fas-
ter operation, direct coupling to endogenous pathways,
single-transcript delivery, and function without genomic
integration (Gao et al. 2018). Indeed, protein-level thera-
peutics may be very important to neurodegenerative dis-
eases because the disease state may often occur at the
protein level, not the genomic level (Maguire 2017). In
this regard, Horwich’s laboratory at Yale showed that in
an animal model of ALS, although genomic correlates
have been found to the disease, transcripts were found to
be normal, suggesting that the disease state occurs at the
level of protein translation or posttranslation despite the
association of some genetic defects (Bandyopadhyay et al.
2013).
Inhibiting stress granule number, as is shown in this
study, has been shown to diminish nucleocytoplasmic
transport defects as well as neurodegeneration
in C9ORF72-mediated ALS/FTD model (Zhang et al.
2018a,b). The formation of these stress granules is context
dependent, and the content of the stress granules reflects
that context (Markmiller et al. 2018). This means the
assemblies of proteins that condense in tight clusters to
interact with RNA when cells respond to cellular stresses
are different depending on the type of stress experienced
by the cell. People with neurodegenerative disease may
have an aberrant formation of stress granules (Markmiller
et al. 2018) depending on factors from the patient’s expo-
some, phenotype, genotype, or structural cross-seeding
mechanisms in the self assembly of proteins (Nizynski
et al. 2018). When stressors are removed, many stress
granules disassemble, but a significant proportion relies
on autophagy for their elimination (Buchan et al. 2013).
The mechanisms by which our secretome reduced the
number of stress granules may relate to the prevention of
stress granule formation or to their enhanced removal by
disassembly or autophagy. Because RNA has recently been
shown to have chaperone activity and help to fold pro-
teins (Docter et al. 2016), aberrant stress granule forma-
tion that may bind RNA chaperones could be a factor in
causing protein misfolding and their self-templating and
prion-like spreading in neurodegenerative diseases (Goed-
ert et al. 2010). This prion-like, protein-only somatic
inheritance may contribute to many diseases, including
cancer phenotypes given that p53, a protein with the task
of suppressing tumor formation in the body, may show a
typical prion-like behavior when mutated (Bom et al.
2012).
Mitochondrial dysfunction is common to most neu-
rodegenerative diseases, including ALS, Alzheimer’s,
Parkinson’s (Smith et al. 2017), glaucoma (Lee et al.
2012), and possibly sensorineural hearing loss (Kaheel
et al. 2018). Our study measured TDP-43, known to bind
and regulate the processing of transcripts encoding mito-
chondrial proteins (Izumikawa et al. 2017), within stress
granules and found that our experimental secretome
reduced the amount TDP-43 in the granules compared to
control cells.
Glutamate is the most prominent excitatory transmitter
in the nervous system, and under normal conditions the
concentration in interstitial tissue is well regulated by glu-
tamate transporters that serve to transport glutamate
from the extracellular to the intracellular compartments.
However, under conditions that mimic neurotoxicity, the
transporter can reverse (Szatkowski et al. 1990; Grewer
et al. 2008) leading to increased glutamate accumulation
in the extracellular compartment of the CNS (Maguire
et al. 1998). An increased concentration of extracellular
glutamate in the nervous system and the ensuing toxicity
are thought to partially underlie a number of neurode-
generative diseases (Lewerenz and Pamela Maher 2015).
2019 | Vol. 7 | Iss. 9 | e14072
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ª2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society.
Rescue of Degenerating Cells by Stem Cell Secretome G. Maguire et al.

In our studies we have shown a direct effect of the experi-
mental secretome on neurons to block glutamate toxicity.
The direct effects of the secretome can be through many
mechanisms, including the induction of neuroglobin
(Baez-Jurado et al. 2018) to reduce ROS production, sub-
sequently upregulate membranous Atp1b1, suppressing
the glutathionylation of Atp1b1, and eventually preserving
the activity of proteins such as NKA (Wen et al. 2018),
involved in pumping ions across the membrane and
assembly of protein complexes (Cui and Xie 2017).
Another possible mechanism for the rescue of cells by
our collective secretome is through protection of the
stressed cell’s proteins is by heat shock proteins (HSPs)
contained in the secretome of stem cells (Chiellini et al.
2008; Teixeira et al. 2015; Nie et al. 2018), and known to
be contained in exosomes. Neurons are particular vulner-
able to stress because they do not make their own HSPs
(Oza et al. 2008). As a result, stressed neurons not in
contact with surrounding stem cells and the HSPs that
are supplied by the surrounding stem cells, are at risk.
However, neurons have been shown to be rescued using
exosomes (Jarmalavi
ci
ut_
e et al. 2015) released from sup-
port cells that contain chaperone proteins (Sreekumar
et al. 2010). Our study is similar, having used a collective
secretome, known to contain exosomes (Maguire et al.
2013) and thought to contain HSPs (Maguire 2017).
In studies where indirect effects of the secretome can
be measured, unlike our isolated primary neurons in the
present study, we would expect additional efficacy because
the ECM and the microenvironment (ECM-M) surround-
ing the neurons is built and repaired from the molecules
in the secretome, including matrix proteins, and the
ECM-M may be critical to regulating glutamate toxicity
and hyperexcitability (Chen et al. 2008; Frischknecht et al.
2009; Vedunova et al. 2013). The importance of a well
maintained ECM-M is fundamental, even at the level of
regulating stem cell function, where a disrupted stem cell
niche may profoundly alter the stem cell’s gene expression
profile (Van Velthoven et al. 2017). Indeed, adult stem
cells sense their environment and develop epigenetic mem-
ories of the event that persist for long periods (Naik et al.
2018). The rebuilt ECM-M may also facilitate the forma-
tion and function of tunneling nanotubes (Osteikoetxea-
Moln
ar et al. 2016), allowing the stem cells to supply
molecules and even organelles, such as mitochondria, to
their surrounding neurons in need of rescue (Wang and
Gerdes 2015). Furthermore, because the immune system,
including T-cell regulation plays a large role in neurode-
generation, including glaucoma (Chen et al. 2018a,b) and
multiple sclerosis (Haase et al. 2018), the immune modula-
tory capacities of MSCs are suggested by the inhibition of
T- and B-cell proliferation (Duffy et al. 2011; Franquesa
et al. 2012), inhibition of the production of
H
2
O
2
from neutrophils (Raffaghello et al. 2008), and T
and NK cytotoxicity (Spaggiari et al. 2008), as well as sup-
pression of the differentiation and maturation of mono-
cytes into dendritic cells (Ivanova-Todorova, 2009).
The immune system, acting though T cells, has now
been shown to interact with resident HSPs in the resident
neural tissue undergoing neurodegeneration (Chen et al.
2018a,b). T cells, conditioned by bacteria in the gut, can
invade neural tissue once thought to be immunoprivi-
leged. In the case of a glaucoma model, elevated intraocu-
lar pressure upregulates membrane bound and
extracellular HSPs in the surrounding neural tissue, and
allows T cells to enter the neural tissue compartment
because of a compromised blood-retinal-barrier (Flammer
et al. 2002). Because HSPs are so well conserved from
bacteria to mammals, T cells conditioned by HSPs in gut
bacteria will attack the mammalian HSP because of
molecular mimicry between, for example, bacterial HSP65
and human HSP65 (Rajaiah and Moudgil 2017). Once
the HSPs in the neural tissue are compromised by the
invading T cells, neurons in that tissue may degenerate in
a spreading fashion (Lackie et al. 2017). Thus infusion of
normal, exogenous HSPs instead of indogenous, mal-
formed HSPs, into degenerating neural tissue may be an
important therapeutic strategy in general, and for ALS
and glaucoma specifically (Maguire 2017).
Without consideration of glutamatergic mechanisms,
we would expect the efficacy of our experimental secre-
tome to be greater within intact tissue because our mix-
ture includes molecules that should work well by indirect
mechanisms, that is, not directly on the compromised
cells themselves, rather working on other cell types to
activate a number of pathways leading to a number of
therapeutic events such as the rebuilding of stem cell
niches and the microenvironment/ECM of the affected
tissues (Maguire 2017, 2018a,b). In a state of dynamic
reciprocity (Bissell and Aggeler 1987), the ECM acting
through a number of mechanisms, including mechanical
forces at chromatin (Maniotis et al. 1997), may activate
stem cells to genetically reprogram themselves to rebuild
tissue (Ransom et al. 2018). Essentially a reversion to a
more primordial, developmental state in adult stem cells
underlies new tissue growth. Other approaches to amelio-
rating neurodegeneration involve implantation of stem
cells, including neural stem cells. Recent studies show that
transplanted neural stem cells derived from iPSCs are able
to engraft into injured spinal cords and form synapses
(Kumamaru et al. 2018). Engraftment of stem cells,
whether they are autologous or from a different patient,
may have the unintended consequence of inducing aging
of the tissue as measured by a p16 biomarker (Wood et al.
2016). While neural stem cell transplantation is an impor-
tant approach for neuroregeneration, there are many other
ª2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society.
2019 | Vol. 7 | Iss. 9 | e14072
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G. Maguire et al.Rescue of Degenerating Cells by Stem Cell Secretome

cells and tissues involved in the repair process in addition
to the neural stem cells. As such, the regenerative thera-
peutic candidate needs to be considered as acting within
the system, and address repair of the other cells types. In
neurodegenerative indications such as trauma to the spinal
cord, cellular transplants will likely be required in order to
reconnect the two distal ends of the truncated tissue, that
is, spinal cord. However, in other neurodegenerative indi-
cations, such as neurodegenerative diseases, for example
ALS and Alzheimer’s, physical truncation of tissue, includ-
ing nerves, will be less severe or nonexistent. In these
cases, especially with early detection strategies, enabling
repair of the neurons and other tissues in the nervous sys-
tem may be best facilitated with the molecules that stem
cells release (Maguire 2016a,b, 2017). In this manner,
repair of the system, not just one cell type, can be best
addressed, facilitating repair of all the neural tissue.
Stem cells are known to release many of the members
of the insulin-like growth factor-binding protein (IGFBP)
family (Park et al. 2010). Yamahara et al. (2018) have
recently shown that exogenously applied IGF-1 alone can
modestly mitigate the effects of traumatic mechanical
insult during cochlear implant in a guinea pig model. A
slight decrease in thresholds for auditory brainstem
responses was measured at low-frequency stimulation
when IGF-1 was administered compared to controls in
which saline was used. Reductionist approaches using one
growth factor or a few growth factors, such as NGF (Aloe
et al. 2015) in an attempt to prevent or mitigate neural
damage has been widely used for years with poor results,
including poor results in the treatment of ALS (Petrou
et al. 2016). Rather, a wide variety of factors, secreted
from multiple cell types, including lipids secereted by glia
(Livne-Bar et al. 2017), microRNA (El Fatimy et al.
2018), and proteins (Gumy et al. 2010), likely play a role
in forming a collective secretome that can more com-
pletely rescue neurons and neural tissue during various
conditions such as ALS (Maguire 2017). The approach we
use therefore is called homeostatic renormalization, where
not only proteostasis is achieved with administration of
the therapeutic but also renormalization of homeostasis
for lipids (Livne-Bar et al. 2017) and microRNA (El
Fatimy et al. 2018; Rizzuti et al. 2018) for example.
Future studies will need to tease apart the factors in our
collective secretome responsible for rescue, including the
factors contained within the exosomes and within the
secretome solution, and those factors that may not be
involved in rescue (Maguire 2018a,b).
Organizing principles for stem cell function
Reductionists strategies still dominate the field of thera-
peutics, including stem cell therapeutics, where, for
example, “Determining the most appropriate cell type
and tissue source is crucial for successful clinical transla-
tion of cell-based therapies” is still the thinking (Chaubey
et al. 2018). Instead, we should first recognize that there
are many distinct adult stem cell phenotypes, each pheno-
type optimized for the particular tissue in which it oper-
ates, with more than one stem cell phenotype operating
within a given tissue. Bone marrow stem cells are distinct
from neural stem cells for example, and each cell type will
release a pool of 100s of molecules that act collectively in
a synergistic manner to promote homeostasis, such as
proteostasis and cellulostasis (cell survival), for example
(Nie et al. 2018).
Different cell types, including adult stem cells, are
known to secrete unique pools of proteins and other
molecular constituents (Statna and van Eyk 2012; Berardis
et al. 2014). We suggest that one or the organizing princi-
ples in stem cell function is that the more differentiated
the stem cell, the more specialized will be the SRM from
the stem cell (Maguire 2018a,b). This means that embry-
onic stem cells will release a more general set of molecules
than will adult stem cells. This also means that stem cells
in a given tissue, even within subregions of a given tissue,
will release tissue-specific molecules. A bone marrow stem
cell, for example, will not release the same set of mole-
cules as does a neural stem cell. Each stem cell, progenitor
cell, or other cell type within a given region of tissue will
release a given set of molecules to maintain or renormal-
ize homeostasis in that given tissue.
A hierarchical organization of stem cells and progeni-
tors cells has been demonstrated in some tissues. Stem
cells within a tissue, intrinsic stem cells, act on intrinsic
progenitor cells. Such a hierarchy has been demonstrated
in the skin where intrinsic stem cells, called ker-
atinocytes (Fuchs 2018), are able to control fibroblasts
(Ghaffari et al. 2009) and other cells through the release
of exosomes (Cicero et al. 2015). A similar hierarchical
organization has been demonstrated in muscle that facil-
itates muscle regeneration (Fry et al. 2017). Also, bone
marrow stem cells (BMSCs) that circulate through the
body within the blood may be considered intrinsic
under certain conditions, such as wounding. Normally
these bone marrow stem cells are not found in tissues
such as the skin or brain, but upon demand, during
injury will be recruited to the damaged tissue, including
the brain during neurodegenerative events (Kierdorf et al.
2013). The purpose for recruiting the BMSCs maybe that
the intrinsic stem cells do not secrete GDF-11 (Lee et al.
2010), whereas the BMSCs do secrete GDF-11 (Lai et al.
2010). This is important because GDF11 will inhibit prolif-
eration and drive differentiation of stem cells (Williams
et al. 2013). Essentially this is a maturation effect that is
required to form new somatic cells to replace the injured
2019 | Vol. 7 | Iss. 9 | e14072
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ª2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of
The Physiological Society and the American Physiological Society.
Rescue of Degenerating Cells by Stem Cell Secretome G. Maguire et al.

cells. Careful recruitment of BMSCs only for injury is
important so that constant differentiation and lack of pro-
liferation does not exhaust the intrinsic stem cell supply
and therefore prematurely age the tissue.
In conclusion, our studies show that the collective con-
ditioned media from four cell types intrinsic to neural tis-
sue directly rescue neurons and cells from stress and
glutamate-induced neurotoxicity. We also expect that
paraneurons, such as auditory hair cells (G
el
eoc and Holt
2014) and retinal photoreceptors that, under some condi-
tions in humans, may be able to regenerate using our
strategy (Horton et al. 2015). We also expect, and have
experiments underway to determine the effects of this col-
lective conditioned media on neurons and other cells
through indirect mechanisms, such as the rebuilding of
the ECM/microenvironment, basement membranes, and
the stem cell niches within the neural tissue. Although our
initial study here used the collective secretome from four
cell types demonstrated to be relevant to the nervous sys-
tem and to neural repair, future studies will need to
explore two key areas: (1) tease apart whether all compo-
nents are necessary for the observed effects, or whether a
subset of the four components used are sufficient to
observe the therapeutic effect, and (2) we have also begun
mass spectrometry studies to understand the molecular
components our collective secretome, and metabolite and
lipid analysis will also be necessary to fully characterize the
content, including microRNA species known to be impor-
tant to homeostasis of the nervous system (Pegtel et al.
2014). Understanding these two areas may lead to an
approach where a reductionist set (system) of molecules,
the minimum molecule set (MMS), that is not overly
reductionist so as to be ineffective, but instead uses the
least number of necessary molecules that are sufficient to
realize a safe and efficacious MMS therapeutic (Maguire
2014), can be used to treat neurodegenerative diseases.
Conflict of Interest
None declared.
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