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A Long-Living Bioengineered Neural Tissue Platform to

Study Neurodegeneration
Nicolas Rouleau, William L. Cantley, Volha Liaudanskaya, Alexander Berk, Chuang Du,
William Rusk, Emily Peirent, Cole Koester, Thomas J. F. Nieland, and David L. Kaplan*

period, it is estimated that ≈85000 neu-

The prevalence of dementia and other neurodegenerative diseases continues rons are lost every day[3] without signifi-
to rise as age demographics in the population shift, inspiring the development cant replacement, contributing to normal,
of long-term tissue culture systems with which to study chronic brain disease. age-dependent decreases in brain weight,
cortical thickness, and neuronal den-
Here, it is investigated whether a 3D bioengineered neural tissue model derived
sity.[7,8] A 10% total reduction in neocor-
from human induced pluripotent stem cells (hiPSCs) can remain stable and tical neurons over a lifetime is typical,[3]
functional for multiple years in culture. Silk-based scaffolds are seeded with however, greater decrements in cerebral
neurons and glial cells derived from hiPSCs supplied by human donors who are volume are characteristic of neurodegen-
either healthy or have been diagnosed with Alzheimer’s disease. Cell retention erative diseases.[9] Those who are afflicted
by neurogenerative diseases such as
and markers of stress remain stable for over 2 years. Diseased samples display
dementias develop insidious cognitive,
decreased spontaneous electrical activity and a subset displays sporadic-like behavioral, and affective disorders earlier
indicators of increased pathological β-amyloid and tau markers characteristic in life—contributing to tremendous per-
of Alzheimer’s disease with concomitant increases in oxidative stress. It can be sonal hardship and societal burden.[10]
concluded that the long-term stability of the platform is suited to study chronic Our understanding of how neurodegen-
brain disease including neurodegeneration. erative diseases manifest at a mechanistic
level is unfortunately limited by experi-
mental bottlenecks including the non-
generalizability of animal models and the
1. Introduction absence of physiological relevance associated with traditional
cell culture systems which lack key features of the brain’s 3D
Human brains develop over an unusually protracted period microenvironment.[11] To address these limitations, investiga-
of time compared to other organs. Their rapid gestational tors are beginning to design and build 3D in vitro models that
expansions are contrasted by decades of maturation and sev- are assembled using appropriate cell types, biomimetic extracel-
eral more of gradual decline and deterioration.[1–6] With the lular matrix, and microenvironmental cues to ideally recapitulate
exception of significant early cell pruning during the perinatal normal physiology.[12] We designed and built a bioengineered
brain model which combined a porous scaffold composed of
Dr. N. Rouleau, Dr. W. L. Cantley, Dr. V. Liaudanskaya, A. Berk, Dr. C. Du, silk fibroin with an intercalated hydrogel to allow seeded neu-
W. Rusk, E. Peirent, C. Koester, Dr. T. J. F. Nieland, Dr. D. L. Kaplan rons and glia to form complex, functional 3D networks.[13–15]
Department of Biomedical Engineering The model’s gross structure reflects a separation of two distinct
School of Engineering regions, simulating gray and white matter respectively. Each
Tufts University
Science & Technology Center
component of the tissue construct was designed to be custom-
4 Colby Street, Medford, MA 02155, USA ized—from the pore size of the scaffold biomaterial,[16,17] to the
E-mail: david.kaplan@tufts.edu hydrogel composition,[18] and incorporated cell types. We hypoth-
Dr. N. Rouleau, Dr. W. L. Cantley, Dr. V. Liaudanskaya, Dr. C. Du, esized that if we could design a 3D human-derived neural culture
Dr. T. J. F. Nieland, Dr. D. L. Kaplan system that could be maintained for years or decades, we could
Initiative for Neural Science, Disease & Engineering begin to study chronic diseases of the nervous system under
Tufts University
Science & Engineering Complex physiological conditions in controlled settings. Without function-
200 College Avenue, Medford, MA 02155, USA ally viable long-term cultures, studies of neurodegeneration and
N. Rouleau, D. L. Kaplan senescence would continue to lack the physiological relevance
The Allen Discovery Center at Tufts University that could only be recapitulated by the passage of time.
Biology Department Building upon our existing tissue model system, we demon-
Tufts University
200 Boston Ave. Suite 4600, Medford, MA 02155, USA
strated that human induced pluripotent stem cells (hiPSCs) dif-
ferentiated to neurons and glia could be incorporated into the
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/mabi.202000004. scaffolds and remain functionally active for up to 9 months.[19]
As it was unclear whether the tissues would remain viable and
DOI: 10.1002/mabi.202000004 active indefinitely, we continued to generate batches of samples

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Figure 1.  Long-term 3D bioengineered neural tissues derived from hiPSCs from healthy donors remained viable and contained structured networks
of cells for over 2 years in culture. A) Silk fibroin was extracted from silk worm cocoons (Bombyx mori) to generate scaffolds, sculpted into toroidal
sponges (6 mm diameter, 2 mm height) with a silk-free central window region (cw), and seeded with neural progenitors sourced from human iPSCs
derived from a healthy donor. Once seeded, cells were embedded in a collagen (type-I) hydrogel and maintained in neural media to promote differen-
tiation and growth. Fluorescent imaging of live (calcein-AM, green) and dead (propidium iodide, red) cells within 3D CON samples that remained in
culture for 855 days (2.3 years) was performed to determine long-term viability. B) Both the silk-scaffold (blue) and hydrogel (black, right side of each
panel) remained populated with living cells with sparse dead cell staining (lumen separating both regions indicated by a dashed line). C) In some
cases, larger clusters (100–500 µm) of dead cells affixed to silk fibers (arrow) were observed embedded within living neural networks whose viability
was apparently unaffected. D) Fine-scale connections indicative of axons and dendrites were observed throughout the porous scaffolds. E) Cells were
not confined to any one compartment or region of the tissue construct; rather, live cell populations permeated the entirety of the sample, forming
both short- and long-range connections with neighboring structures (dashed line indicates the lumen of the hydrogel-only region). A scaled reference
scaffold is provided (ref.) with imaging regions (B–E), and the (cw) indicated for spatial orientation.

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(Figure  1A) at irregular intervals over time (Figure S1, Sup-
porting Information) using hiPSCs from donors who were
healthy controls (CON) or diagnosed with a sporadic form
of Alzheimer’s disease (ALZ). The model system would be
determined stable and suitable for neurodegeneration research
if, as we had predicted, networks would remain structurally and
functionally stable over years.
2. Results and Discussion
2.1. Structural Stability and Characterization
Twenty-six time points ranging from 3 months to 2.3 years
were assessed in search of time-dependent decrements asso-
ciated with the structure and function of the model system.
Both short-term and long-term cultures displayed similar
hydrogel quality (p  > 0.05, chi-square test, Figure S2, Sup-
porting Information), suggesting the materials used provided a
robust medium within which to maintain network formations.
To determine whether neural networks remained alive and
intact over long periods of time, a subset (n = 3) of the oldest
(2.3 years old) CON samples were stained for cell viability and
subsequently imaged, revealing diffuse populations of live
cells intermixed with sparse dead cells (Figure 1B–E). Cells
that had migrated into the collagen-based hydrogel window at
the center of the tissue construct remained alive and connected
to the broader, scaffold-bound network (Figure 1E). Immuno-
cytochemistry was then performed on CON samples (n  = 18)
that had remained in culture for periods between 94 and 855
days, revealing a reliable maintenance of network structure
containing both neural and glial cells (Figure 2A), with image
quantification revealing structural stability (Figure 2B–D) and
a trend toward increased glial marker expression in long-
term samples. ALZ samples (Total n = 29) were not stained or
imaged as their availability was limited relative to CON sam-
ples (Total n = 92); however, our previous report confirmed the
presence of similar structures in ALZ samples at 10 months.[19]
Instead, our limited ALZ samples were subjected to molecular
and functional assays and compared to healthy controls (CON).

Figure 2.  Immunocytochemistry of long-term samples revealed a maintenance of neuronal and glial cell markers over time. A) Fluorescent imaging
revealed positive nuclear (DAPI), β-III tubulin (neuronal marker, TUJ1), and glial fibrillary protein (glial cell marker, GFAP) staining, revealing stable net-
work morphology over time. A 200 µm scale bar is provided for reference. Only CON samples were stained due to the limited availability of ALZ samples.
B) Nuclei remained of fixed size over the 2 year period (particle size, p  > 0.05). C) Neurons formed dense network-structures spanning hundreds of
micrometers to millimeters in length which were not affected by time in culture as indicated by stable mean grey values (MGV), a measure of fluorescent
intensity (p > 0.05). D) Glial structures remained stable over time (MGV, p > 0.05) with a potential trend toward more expression as a function of time.
Means are plotted with standard error of the mean (SEMs). Images were not quantified for the 530 day group due to limited sample size (n < 3).

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Figure 3.  DNA content, LDH activity, and ROS/RNS concentration within samples as a function of hiPSC donor source and time. A) DNA concentration
within both ALZ (red circles) and CON (blue triangles) groups did not correlate with time spent in culture; B) however, ALZ (red) samples displayed
increased DNA concentration relative to CON (blue) samples on average. C) Decreased cell toxicity over time as inferred by LDH activity (Z-scores)
was observed for both CON and ALZ samples. D) Markers of cell stress as indicated by ROS/RNS concentration did not change over time for either
group; E) however, CON samples displayed increased cell stress on average. F) DNA in ALZ samples (but not CON samples) was found to correlate
with ROS/RNS concentration. Means and standard error of the mean (SEM) are presented and significant differences are displayed within each panel
(N.S. = p > 0.05; *p < 0.05; ***p < 0.001). Red bars and circles indicate ALZ samples, Blue bars and triangles indicate CON samples.

2.2. DNA Quantification
To quantitatively assess the stability of cell retention within
both CON and ALZ samples over time, we measured the con-
centration of DNA within tissue constructs. Decrements of cell
content over time were not observed for either donor source
(Figure 3A; p > 0.05); however, ALZ samples did contain more
cells on average relative to CON samples (Figure 3B; t(78) =
4.22, p  < 0.001) regardless of time. Because the groups were
seeded with the same number of cells (5 × 105 cells per scaffold),
these results could indicate that there were initial, cell-donor-
dependent differences in early cell attachment or proliferation
rather than time-dependent decrements. When pooling the
groups together, a very weak relationship between DNA concen-
tration and time was identified (rho = −0.25, p < 0.05); however,
this amounted to a decrement of ≈100 ng mL−1 or 7% of total
DNA per year. Overall, it was concluded that cell retention was
stable over time. Subsequent measurements were normalized to
DNA concentration to account for and normalize to cell content.
F(1,77) = 11.37, p  < 0.001). Though both donor sources dis-
played decreased cell toxicity over time (Figure 3C), these
differences were less pronounced for ALZ samples (p < 0.05)
relative to CON samples (p  < 0.001). Reactive oxygen and
nitrogen species (ROS/RNS) activity remained stable over
time for both CON and ALZ samples (Figure 3D); however,
CON samples displayed increased markers of cell stress rela-
tive to ALZ samples (Figure 3E; t(78) = 8.14, p < 0.001). These
differences were apparent at all time points. Notably, there
was an inverse relationship between DNA concentration and
ROS/RNS activity for ALZ samples (rho =  −0.51, p  = 0.005)
that was not apparent for CON samples (Figure 3F). Together,
the results indicated that cell toxicity decreased over time
with differences between donor sources and that cell stress
was time-independent while moderately associated with cell
content in ALZ samples only.
2.4. Neurodegenerative Markers

Next, as we had not observed β-amyloid (Aβ) plaques within

2.3. Markers of Stress and Cell Toxicity
Molecular assays were then performed to determine if cellular
homeostasis was impacted by long-term culture conditions.
Cell toxicity and stress were assessed by measuring lactic
acid dehydrogenase (LDH) activity and the concentration of
ROS/RNS within the media associated with aged tissue con-
structs. A significant interaction between donor source and
time spent in culture was identified for cell toxicity (ANOVA,
the 10-month time frame of our previous study,[19] we sought to
evaluate the possibility that long-term culture conditions would
widen the observational window during which we might detect
Alzheimer’s markers such as an increased ratio of Aβ42 to
Aβ40[20,21] within media samples as detected by enzyme-linked
immunosorbent assays (ELISAs). Additionally, we examined the
ratio of phosphorylated Tau protein (p-tau) to total Tau (t-tau)
within the media—another clinically-relevant biomarker asso-
ciated with Alzheimer’s disease.[22,23] Statistically significant

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Figure 4.  Neurodegenerative markers of Alzheimer’s disease and increased reactive oxygen species concentrations were expressed within a subset of
ALZ samples. A) Ab42 to Ab40 ratios for CON and ALZ samples were equivalent (p > 0.05); however, B), a sporadic-like (Sp) phenotype was observed
in a subset of ALZ samples that were cultured for over 1 year. C) The ratio of p-tau to t-tau was greater for ALZ samples relative to CON samples (p <
0.05). D) The same samples (ID18, 21, and 32) expressed an Sp phenotype relative to other ALZ samples. E) Sp-ALZ samples (n = 3) displayed grater
reactive oxygen species (ROS/RNS) concentrations relative to the remaining ALZ samples.

effects of time and donor source (Figure  4A) on normalized
Aβ isoforms were not observed; however, we noticed that the
three highest Aβ42 to Aβ40 ratios were associated with the ALZ
group (IDs 18, 21, and 32) and each had spent more than 1 year
in culture (Sp circle, Figure 4B). In other words, only among
older ALZ samples did we begin to observe sporadic-like cases
expressing a pathological β-amyloid hallmark of Alzheimer’s
disease; however, the major source of variance was a decrease
in Aβ40 relative to Aβ42 for the Sp-ALZ subset (Table S1, Sup-
porting Information; p  < 0.05). Due to the limited subgroup
of sporadic-like samples, a linear relationship between Sp-like
phenotypes and time could not be discerned. Normalized Aβ40
was observed to decrease over time for both ALZ (p < 0.05) and
CON (p  = 0.05) conditions. A similar effect was not observed
for Aβ42 (p > 0.05).
Examining measures of Tau, it was evident that CON sam-
ples displayed more normalized t-tau (p < 0.01) and p-tau (p <
0.05) relative to ALZ samples. However, ALZ samples dis-
played increased p-tau to t-tau ratio relative to CON samples
(Figure 4C; t(65) = 2.96, p  < 0.005). The major contributor
to ratio differences was a decrease in t-tau relative to p-tau
(Table S1, Supporting Information). Plotting p-tau to t-tau
ratio over time spent in culture revealed the same Sp-ALZ
subgroup that was identified when examining the ratio of
Aβ42 to Aβ40 (Figure 4D). We then compared samples with
sporadic-like phenotypes (Sp-ALZ; n  = 3) of increased neuro-
degenerative markers and all other ALZ samples (n  = 11) to
determine whether the two groups could be distinguished
on the basis of other measures. Indeed, we found that media
associated with Sp-ALZ samples displayed a 68% increased
concentration of normalized ROS/RNS relative to other ALZ
samples (Figure 4E; t(12) = 2.71, p < 0.05), suggesting the two
samples belonged to distinct populations. Differences of DNA
concentration, LDH, or local field potentials (LFPs) were not
evident (p  > 0.05). These data suggested that neurodegenera-
tive markers of Alzheimer’s disease did not vary linearly over
time; however, donor source may have potentially impacted the
number of sporadic-like cases which emerged after 1 year in
culture where extreme values were only apparent for a subset
of the ALZ group (Sp-ALZ) which also displayed increased cell
stress. This latter relationship between ROS/RNS and neurode-
generative markers may be particularly relevant since the role
of oxidative stress in Alzheimer’s disease has been highlighted
in the literature.[24]
2.5. Electrophysiological Characterization
Finally, to determine whether network activity was impacted
by time spent in culture, we measured spontaneous local

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Figure 5.  Assessments of spontaneous electrical activity revealed long-term samples remained. A) Extracellular recordings of local field potentials
(LFPs) were obtained from CON and ALZ samples by positioning an electrode alongside silk fibers along the lumen of the central window. A refer-
ence electrode was placed into the extracellular solution surrounding the tissue construct. B) No time-dependent changes in spontaneous activity
were noted. C) ALZ and Sp-ALZ samples displayed similarly decreased activity relative to CON samples. D,E) Representative traces are provided for
LFP recordings from CON and ALZ samples. Error bars indicate standard error of the mean (SEM) and significant differences are indicated (N.S. =
p > 0.05; *p < 0.05).

field potentials (LFPs) from single electrodes inserted into the
superficial layers of the hydrogel next to silk fibers (Figure 5A).
Each sample was measured once for a particular timepoint and
subsequently processed for use in other assays (i.e., DNA quan-
tification or immunostaining). LFP spike potentials, which are
indicative of cellular electrical activations, were quantified and
compared across conditions. Effects of time were not detected
(Figure 5B), with 3 month and 2.3 year old tissues displaying
similar levels of spontaneous activity (p  > 0.05). However, it
was evident that despite there being more cells in ALZ sam-
ples, spontaneous LFP spikes per minute were decreased in
ALZ samples relative to CON samples (Figure 5C,D; t(77) =
2.34, p < 0.05). Sp-ALZ samples displayed similarly decreased
activity relative to CON samples (p < 0.05); however, no signifi-
cant difference could be discerned when comparing ALZ and
Sp-ALZ groups (p  > 0.05). This finding suggested that tissue
constructs seeded with hiPSC-derived neurons from a donor
diagnosed with Alzheimer’s disease were functionally sup-
pressed relative to healthy controls, displaying less electrical
activity on average.
3. Conclusion
In summary, our data show that the current neural tissue
model derived from hiPSCs can remain structurally and func-
tionally stable in culture for over 2 years. Based upon the
observed rate of decrement of cell retention (≈100 ng mL−1 per
year) and assuming linearity, we project that our long-term cul-
ture system could be extended for up to a decade; however, it
may be unnecessary and is likely impractical to maintain the
cultures for more than 5 years. Indeed, multi-year experiments
could be designed with many points of observation over time
for robust within-subject designs.
In addition to the extended viability of the tissues, the model
system displayed stable markers of cell stress and neurodegen-
eration over time, indicating that the cellular microenvironment
was conducive to homeostatic function. The original design of
the scaffold and hydrogel materials were made porous to facili-
tate nutrient perfusion across the tissue substrate. The current
findings validate the use of complex topology to maximize sur-
face area while remaining permeable to the culture media. One

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implication of successfully maintaining low levels of cell stress
is the increased likelihood that future long-term experiments
will proceed without confounding factors of time-dependent
changes to cell physiology related to oxidative stress.
Differences between donor sources were evident. Most
notable among them, we observed decreased electrical activity in
ALZ samples which, in vivo, is characteristic of dementia;[25,26]
however, these effects were not time-dependent. Similarly, con-
centrations of LDH were markedly different between cell donor
sources; however, these too were not time-dependent and fol-
lowed similar trends (i.e., as time increased, LDH decreased).
One potential explanation is that an initial period of toxicity was
followed by an eventual steady-state after sufficient maturation.
That we observed sporadic cases of older ALZ samples
expressing increased pathological β-amyloid and Tau markers
relative to CON samples was interesting and potentially indica-
tive of the experimental time-frame that is necessary to observe
degenerative processes in vitro that are not explicitly induced by
genetic manipulation. Most saliently, the concomitant increases
of pathological β-amyloid, Tau, and oxidative stress markers
in a subset of ALZ samples reveals the potential to study spo-
radic emergence of disease phenomena in the laboratory. We
interpret these results with caution, as conclusions cannot be
drawn from a single donor. However, we maintain that as cul-
ture time increases for tissues composed of cells from donors
with a diagnosed history of sporadic Alzheimer’s disease, the
likelihood that subsets of samples will express classic markers
of Alzheimer’s disease should increase.
To experimentally address neurodegeneration, investiga-
tors require tools that transcend existing model systems. While
animal models offer complete, well-defined, patterned tissues,
it can be challenging to isolate the underlying mechanisms
amid a multitude of confounding factors inherent to the func-
tion of a complex organism with the well-documented differ-
ences between rodent and human brain networks and their
functions.[27–29] Likewise, the ease of interrogation associated
with in vitro or 2D, cell-based model systems is contrasted by
poor predictive validity due in part to an absence of a suitable
microenvironment. To increase physiological relevance and
the propensity to translate results from bench to bedside, 3D
model systems were quickly adopted by the scientific commu-
nity. Neural organoids in particular, which express the nascent
morphology of the developing brain, have become increasingly
valuable as model systems. Their complex morphology—which
includes ventricles, cortices, discretized domains, choroid plex-
uses, meninges, and retina—offer a unique substrate with which
to model disease.[27,30] However, their lack of a circulatory system
limits their growth to ≈4 mm and generates necrotic cores—
extensive regions of cell death within the center of the organoids.
The model system we have characterized here is not develop-
mentally restricted and represents a more flexible platform
which is suitable to conduct long-term research on neurodegen-
erative processes. Though we did not incorporate a laminated
cortical structure into the present system, the 3D model can
be constructed using concentric toroidal silk-based scaffold to
more closely approximate other gross features of the six-layered
neocortex.[14] Indeed, fewer rings could be used to instead simu-
late the three-layered structure of the entorhinal cortex and hip-
pocampus (i.e., archicortex), areas which are well-known to be
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affected in Alzheimer’s Disease.[31] However, with any modifica-
tion to the structural design of the tissues there is the possibility
that cell stress and longevity could be affected in unexpected
ways that differ from the results presented here. While it may
be difficult to predict how changes might impact tissue function,
preserving adequate nutrient transport and cell density are likely
important constants which should not be compromised.
In the present study, an existing bank of tissues that had
been maintained in culture for years was sub-sampled for the
purposes of demonstrating model longevity and its potential
relevance to neurodegeneration research. One clear limitation
of this approach was a low sample size without the practical
possibility of generating more tissues. As such, prioritization
of certain experiments over others was necessary where we
sought to demonstrate structural and functional longevity over
all else. For example, ELISAs were performed using cell media
rather than cell lysates to multiplex each sample. Whereas we
had previously reported that hiPSC source (donor) influenced
gene expression,[19] we did not pursue a similar characteriza-
tion here; an increased sample size would have permitted a
thorough genetic characterization over time within each hiPSC
source group. Implementations of any long-term culture
system, including the present model, would benefit from the
foresight of a specific, hypothesis-driven experimental design
which would dictate the number of generated samples a priori.
Future work should aim to harness the long-term capabili-
ties of the present model system to explore questions related
to Alzheimer’s Disease as well as other neurodegenerative dis-
eases (e.g., Parkinson’s Disease, Multiple Sclerosis, etc.) using
embedded electrodes for continuous, long-term recordings of
electrophysiological activity.[32,33] The long-term model system
presented here is highly tunable and can be modified to con-
tain combinations of cell donor types, selectively mutated or
otherwise, and exposed to environmental conditions which
are hypothesized to drive or exacerbate neurodegeneration
including chronic vascular insufficiency or toxic exposures.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
The authors would like to thank Dr. Mattia Bonzanni, Dr. Josh Erndt-
Marino, and Dr. Nirosha J. Murugan for their suggestions. This work
was supported by the NIH (R01NS092847, P41EB002520, NIHS10
OD021624.
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
N.R., W.L.C., and D.L.K. conceptualized the study; N.R. performed
all data analysis and visualization; N.R., W.L.C., V.L., A.B., C.D., W.R.,
© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
E.P., and C.K. performed the investigation; N.R. curated the data;
N.R., W.L.C., V.L., T.N., and D.L.K. developed methodology, performed
validation, and wrote the original draft; D.L.K. and T.N. acquired funding,
provided resources, and performed project administration; D.L.K. and
T.N. supervised the study; N.R., W.L.C., V.L., T.N., and D.L.K. reviewed
and edited the final manuscript.
Keywords
Alzheimer’s disease, bioengineering, biomaterials, induced pluripotent
stem cells, neurodegeneration
Received: January 7, 2020
Revised: January 13, 2020
Published online:
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