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Human Embryoid Body Transcriptomes Reveal Maturation Differences Influenced by Size and Formation in Custom Microarrays

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Stem cell differentiation strategies and optimization for generating lineage-specific cells and tissues most frequently rely on a three-dimensional embryoid body (EB) intermediate. We previously applied nanotechnology tools of photolithography to generate custom microarrays that allow high throughput uniform formation of EBs of custom size for precise downstream analysis. Formation of EBs of 200 or 500 micron size revealed distinct morphological differences that are single or multicystic cores, respectively, independent of method of formation from single cells or two-dimensional (2D) clusters. Here we utilize photolithographic array generated EBs to obtain 3D cultures under a standardized platform for transcriptome analysis to compare EB size and the method of EB formation from single cells or mechanically passaged 2D clusters. Our analysis evaluates RNA expression in EBs formed from the human embryonic stem cell (hESC) line WA09 and from ethnically diverse human induced pluripotent stem cell lines (ED-iPSC) of African American and Hispanic Latino ethnicity recently derived in our laboratory. This is the first comprehensive study on EB transcriptomes including multiple size parameters, EB formation methodologies, and ethnicities. Our analysis indicates upregulation of genes involved in wound healing for mechanically passaged cells and of genes for embryonic tube formation in 500 micron multicystic EBs. We propose that EB maturation may be a longer process then previously realized. In addition, the type or extent of maturation possible may be influenced by EB size, with larger EBs capable of more extensive remodeling as revealed by multicystic morphology and initiation of early tube formation pathways while retaining pluripotency status. We anticipate that this information will be broadly useful to the stem cell and bioengineering communities in optimization of tissue engineering with pluripotent stem cells and understanding sources of variation.
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Article
Journal of
Nanoscience and Nanotechnology
Vol. 16, 8978–8988, 2016
www.aspbs.com/jnn
Human Embryoid Body Transcriptomes Reveal
Maturation Differences Influenced by Size and
Formation in Custom Microarrays
Martin L. Tomov1, Maria Tsompana2, Zachary T. Olmsted1, Michael Buck2, and Janet L. Paluh1
1State University of New York Polytechnic Institute, Colleges of Nanoscale Science and Engineering (SUNY PI CNSE),
Nanobioscience, Albany, NY, 12203, USA
2State University of New York Buffalo (SUNY Buffalo), Department of Biochemistry, Center of Excellence in Bioinformatics,
Buffalo, NY, 14203, USA
Stem cell differentiation strategies and optimization for generating lineage-specific cells and tissues
most frequently rely on a three-dimensional embryoid body (EB) intermediate.We previously applied
nanotechnology tools of photolithographyto generate custom microarrays that allow high throughput
uniform formation of EBs of custom size for precise downstream analysis. Formation of EBs of 200
or 500 micron size revealed distinct morphological differences that are single or multicystic cores,
respectively, independent of method of formation from single cells or two-dimensional (2D) clusters.
Here we utilize photolithographic array generated EBs to obtain 3D cultures under a standardized
platform for transcriptome analysis to compare EB size and the method of EB formation from sin-
gle cells or mechanically passaged 2D clusters. Our analysis evaluates RNA expression in EBs
formed from the human embryonic stem cell (hESC) line WA09 and from ethnically diverse human
induced pluripotent stem cell lines (ED-iPSC) of African American and Hispanic Latino ethnicity
recently derived in our laboratory. This is the first comprehensive study on EB transcriptomes includ-
ing multiple size parameters, EB formation methodologies, and ethnicities. Our analysis indicates
upregulation of genes involved in wound healing for mechanically passaged cells and of genes for
embryonic tube formation in 500 micron multicystic EBs. We propose that EB maturation may be a
longer process then previously realized. In addition, the type or extent of maturation possible may
be influenced by EB size, with larger EBs capable of more extensive remodeling as revealed by
multicystic morphology and initiation of early tube formation pathways while retaining pluripotency
status. We anticipate that this information will be broadly useful to the stem cell and bioengineering
communities in optimization of tissue engineering with pluripotent stem cells and understanding
sources of variation.
Keywords: Ethnicity, Induced Pluripotent Stem Cells, Embryonic Stem Cells, Nanotechnology,
Single Cells, Mechanical Passaging.
1. INTRODUCTION
Pluripotent stem cells offer unique and unparalleled oppor-
tunities in human tissue engineering through the ability
to direct differentiation along desired lineages to generate
specific cells, tissues or multicellular organoids. In addi-
tion to cell replacement therapies, biomedical advances
will benefit from stem cell studies that provide a new
understanding of normal processes of development and
repair or of malfunction of human tissues or organs as a
Author to whom correspondence should be addressed.
consequence of perturbation by aging, injury or disease.
The ability to manipulate human stem cells towards
biomedical applications in vivo,1or for on-chip therapeutic
devices2is challenging. It requires expanding our knowl-
edge of environmental, chemical, and physical influences
on stem cells in a niche as well as during in vitro manipu-
lations, and including studies with an increasing diversity
in stem cell lines in regard to age, sex and ethnicity.3
Transcriptome data along with bioinformatics analysis
provides a comprehensive means to cross-compare
pluripotent cell types such as hESC, hiPSC and human
8978 J. Nanosci. Nanotechnol. 2016, Vol. 16, No. 9 1533-4880/2016/16/8978/011 doi:10.1166/jnn.2016.12734
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Tom o v e t al. Human Embryoid Body Transcriptomes
embryonic carcinoma cells (hECCs)4–6 as well as multiple
independently generated pluripotent lines. Such analysis
compliments and extends in vitro multilineage differenti-
ation studies along with teratoma or more direct in vivo
functional studies. Open source comparative transcriptome
platform resources include Pluritest7and Cellnet.9Differ-
ences in transcriptome profiles of pluripotent stem cells
exist and are expected in part to reflect inherent varia-
tion in lines generated by different labs, applying differ-
ent reagents and protocols across the research community.
Continued comparative analysis will allow variances and
similarities in gene expression to be defined along with
their relevance to specific biomedical applications.
Frequently used in pluripotent stem cell differentia-
tion strategies are embryoid body (EB) intermediates that
are naturally forming 3D stem cell aggregates that retain
pluripotent potential. Pluripotent stem cells grown in two-
dimensional culture have the capability to spontaneously
form 3D EBs when mechanically passaged and released
into non-adherent media conditions, as demonstrated for
mouse9–11 and human12 2D cell clusters. Biomedical scaled
up applications with human stem cells are alternatively
exploring use of dissociated to single cells followed by
seeding them in custom microarray templates to enhance
cell interactions and 3D assembly of iPSC13 or hESC14
EBs or by applying shear forces in a bioreactor.15 ESC-
EB shares some similarities with 3D embryonic-like
structures1617 such as the presence of a single internal
cyst-like core structure in naturally forming 200 micron-
sized EBs. However not all methods of EB formation gen-
erate a less dense core structure, such as in the use of high
shear in bioreactors to generate EBs that are then used for
differentiation. In addition, increased EB size can result
in a mulitcystic core15 as revealed by use of custom high
throughput photolithography-templated microarrays. Cur-
rent human EB transcriptome data18–20 have shown gene
activation involved with early differentiation and mRNA
levels in their analysis of naturally forming EB intermedi-
ates however there is no data on the effect of EB size, and
method of EB formation, on differentiation.
This study applies nanotechnology tools of photolithog-
raphy to generate uniformly-sized EBs for comparison
by transcriptome analysis. This is the first comprehensive
whole transcriptome analysis of EBs comparing EB size
(200 or 500 microns) as well as EB formation method
that is by single cells or 2D cell clusters. Our detailed
bioinformatics analysis includes comparative expression of
pluripotency genes as well as up-regulated gene ontol-
ogy (GO) pathways. In addition we compare EB tran-
scriptomes for the hESC line WA09 and newly derived
ethnically diverse induced pluripotent stem cell lines for
Hispanic-Latino and African American ethnicities.3We
observe slight but significant elevation of multiple pluripo-
tency genes in 500 micron EBs. Significant differences
in GO pathways were also present, reflecting influences
of EB formation and EB size and morphology. Our find-
ings provide new insights into EB maturation. They offer
a long-awaited explanation as to the unpredictable EB-
differentiation behavior observed between protocols and
laboratories, and should be taken into account by the stem
cell bioengineering community when designing stem cell
differentiation strategies.
2. RESULTS
2.1. Generation and Transcriptome Profiling of
Custom Sized EBs
To control EB size and formation we engineered the stem
cell microenvironment, by applying nanotechnology tools
of photolithography to produce custom templated microar-
rays. The designed Su-8 stencil was used as a template to
make a polydimethylsiloxane (PDMS) microarray platform
for generating uniform EBs of 200 to 500 microns from
hESC and ED-iPSC lines for analysis (Table I). Single
cells or 2D-clusters of cells were seeded into microar-
rays as previously described14 andallowedtoformEBs
over multiple days and grow to fill the well dimen-
sions before immunocytological (ICC) and transcriptome
analysis. Representative images of microarray formed EBs
from the hESC WA09 line as well as two ED-iPSC
lines and strategy for generating microarrays are shown
in Figure 1. Images reveal the typical single cyst internal
structure of 200 m EBs (Fig. 1(A)) versus the multicyst
internal structure of 500 m EBs (Fig. 1(B)) and include
brightfield and staining for OCT 4 and SSEA4 mark-
ers of pluripotency and the tight junction protein ZO1.
Microarray templating was used to ensure size and uni-
formity of EBs and compared to spontaneously formed
EBs in free-suspension (Fig. 1(C)). The process flow for
the lithography generated grids used in uniform EB for-
mation is illustrated in Figure 1(D) and loading of grids
in Figure 1(E). Differentiation potential of 200 versus
500 micron EBs is previously described.14
2.2. Transcriptome Variation is Present in 200 versus
500 Micron hESC WA09 EBs Independent of
Formation Method
To determine gene expression differences between EB
size and morphology, we used RNA-seq followed by
bioinformatics analysis. Overall, we observed a dramatic
Tab l e I . EB RNA samples used for microarray expression analysis.
200 m 200 m 500 m 500 m
EBformation EB2D EBsingle EB2D EBsingle
Method cluster cell cluster cell
Pluripotent hESC WA09 WA09 WA09 WA09
stem cells ED-iPSC F3.5.2
ED-iPSC H3.3.1
Notes:Abbreviations:ED=ethnically diverse; F =African American; H =
Hispanic-Latino; No samples for grey boxes. Two EBs analyzed for each sample.
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(A) (B)
(C)
(D)
(E)
Figure 1. Three-dimensional formation of uniform stem cell hESC and hiPSC EBs in custom photo-lithography templated arrays. (A) Representative
images of 200 m EBs generated from the WA09 hESC line as well as from ED-iPSC lines stained for the pluripotency markers Oct4 and SSEA4
and for the tight junction marker ZO1. Brightfield images show the characteristic cyst found in EBs of that size. Images in the last column show
representative images of 200 m diameter EBs forming within a loaded 200 LTA-PDMS grid (top) and of 500 m diameter EBs recovered from a
500 LTA-PDMS grid (bottom). The insert image is of a 500 m diameter EB before removal from the LTA-PDMS grid. (B) ICC stained images for
the nuclear marker Hoechst show the multicystic nature of larger EBs. (C) Brightfield image shows representative images of free suspension EBs that
vary in size and shape. (D) Process flow starting with blank silicon wafer through the generation of LTA-PDMS grids for high throughput uniform EB
creation. (E) Generation of high throughput uniform EBs starting with either single cell suspension, or with 2D cell clusters. Scale bars in the figure
are 200 m.
difference in transcriptomes of 200 versus 500 micron
regardless of formation method. We found 1654 differ-
entially expressed genes at a false discovery rate (FDR)
<0.01 (less than 1% false positives) and 661 genes at
FDR <0.001 (less than 0.1% false positives). All fur-
ther analysis focused on the 661 differentially expressed
genes at a FDR of <0.001. The majority of the 661 genes
(476/661) were upregulated in the 500-micron compared
to the 200-micron EBs. To understand the gene expres-
sion relationship and consistence across all experimental
replicates we used hierarchical clustering analysis (Fig. 2)
and examined the gene class enrichments for up- or down-
regulated genes. Table II indicates the top genes up- or
down-regulated along with established roles. Functional
distinctions are further discussed in following sections.
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For the full list of genes refer to the GEO access number
GSE74792.
2.3. Pluripotency Gene Expression is Independent of
EB Size and Morphology or Formation Method
Previous reports have identified multiple genes contribut-
ing to pluripotency as well as the information on the rela-
tive levels of expression of pluripotency genes421–25 to each
other. We compared pluripotency gene expression levels
for our 200- or 500-micron EB hESC samples generated
using dissociated single cells (Table III) at the end point of
uniform EB formation for each method. We first focused
on genes that have been previously reported as important
for inducing and maintaining pluripotency in embryonic
and induced stem cell lines. This includes expression of
the Yamanaka pluripotency factors OCT4, SOX2, KLF4,
and c-MYC,26 plus the two additional factors NANOG and
LIN28A/B identified by James Thomson.27 In addition,
we evaluated several genes that have been more recently
implicated in pluripotency, that include the factors UTF1,
SALL4, NR5A2, TBX3, ESRRB, DPPA4,28–30 and REX-1
(zfp42).331 The FDR for all genes examined was <0.001.
Figure 2. Transcriptomic comparison of 200 and 500 micron EBs from WA09 hESC formed by two methods. RNA-seq, Heat Map and Jaccard
analysis. (A) 200 or 500 micron sized embryonic bodies (EBs) formed via 2D clusters or by templating of single cell (SC) suspensions from WA09
were compared by RNA-seq. The top 50 genes from 661 total differentially expressed genes at a FDR <0.001 were hierarchically clustered and the
top 50 genes were divided into (B) downregulated or (C) upregulated tables. Sequence counts at each gene was standardized for visualization by log2
(gene counts/gene mean). See Table II for top genes and the GEO access number GSE74792 for the full list of genes.
We observed small but insignificant up-regulation of KLF4
(1.34 fold), Sox2 (0.68), UTF1 (0.97), Lin28B (0.49), and
SALL4 (0.45) and downregulation of NR5A2 (0.92),
although again insignificant. Overall, we observe no sig-
nificant gene expression differences in pluripotency mark-
ers dependent on EB size and method of formation in our
pluripotent stem cell samples.
2.4. Expression of Early Embryonic Tube Forming
Genes in 500 Micron Multicystic EBs
We previously identified distinct morphological features
present in large 500 micron EBs versus 200 micron EBs
(Fig. 1(B); See also Ref. [14]) that is the presence of mul-
tiple cysts that are reminiscent of tubes or neural rosettes.
To provide information on these structures we performed
bioinformatics analysis on 200 versus 500 micron EBs.
GO analysis reveals up-regulation of genes associated with
embryonic tube formation (Table IV and Materials and
Methods). Specifically, genes involved in early tube for-
mation that are upregulated include DVL2, MIB1, TSC1,
TSC2, LAMA5, PTK7, PBX1, CELSR1, SCRIB, ZIC2,
HECTD1. The proteins encoded by genes for LAMA5,32
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Table II. Genes up- or down-regulated in 500 micron EBs.
Gene name Gene function Fold change log10 (p-value)
Genes downregulated in 500 micron EBs
PDXP Pyridoxine, vitamin B6 active form of vitamin B6 283 1456
FCRL4 Fc receptor-like 4 Fc receptor-like glycoprotein 249 1088
OR13C3 Olfactory receptor, fam 13, sub-fam C, mem. 3 initiates neuronal response to perceive smell 258 793
SLC15A4 Solute carrier family 15, member4 proton oligopeptide cotransporter 160 790
Genes upregulated in 500 micron EBs
MOGS Mannosyl-Oligosaccharide Glucosidase Mediates cell–cell adhesion 149 2038
NBAS Neuroblastoma amplified sequence involved in golgi-to-ER transport 122 1488
ISOC2 Isochorismatase domain containing 2 mitochondria localized protein binding 119 1380
FOXH1 Forkhead box H1 transcriptional activator of goosecoid (GSC) 144 1380
SH3KBP1 SH3-domain kinase binding protein 1 control of cell shape and migration 113 1366
SLC39A7 Solute carrier family 39, member 7 zinc transporter from the ER/golgi to the cytosol 146 1301
MIB1 Mindbomb E3 ubiquitin protein ligase 1 ubiquitinates notch protein ligands 089 1276
NUMA1 Nuclear mitotic apparatus protein 1 aligns mitotic spindle in asymmetric cell division 119 1249
SLC7A2 Solute carrier family 7, member 2 cationic amino acid transporter 142 1204
PIP5K1A Phosphatidylinositol-4-Phosphate 5-Kinase catalyzes phosphorylation of PtdIns4P 090 1190
ZBED4 Zinc finger, BED-type containing 4 zinc finger protein 095 1188
WDR82 WD repeat domain 82 involved in mitosis to interphase transition 079 1133
CAPNS1 Calpain, small subunit 1 remodeling of cytoskeleton during cell cycle 087 1116
NFATC1 Nuclear factor of activated T-cells gene expression in embryonic cardiac cells 159 1071
GTF2H4 General transcription factor IIH, polypeptide 4 involved in nucleotide excision repair 132 1040
USP9X Ubiquitin specific peptidase 9, X-linked chromosome alignment and segregation 219 1031
PLEC Plectin interlinks intermediate filaments and microtubules 121 1009
PTMS Parathymosin remodels chromatin structure 127 1002
YAP1 Yes-associated protein 1 regulates cell proliferation and apoptosis 136 1002
UNC13B Unc-13 homolog B synaptic vesicle maturation during exocytosis 118 982
TEF Thyrotrophic embryonic factor transcription factor for the TSHB promoter 167 982
ANKFY1 Ankyrin repeat FYVE domain containing 1 proposed effector of rab5 087 965
CTNNA2 Catenin, alpha 2 cell–cell adhesion and neural differentiation 134 914
CNOT1 CCR4-NOT transcription complex, subunit 1 maintenance of embryonic stem cell identity 102 897
CERS4 Ceramide synthase 4 involved in the production of sphingolipids 155 894
EMD Emerin formation of the nuclear actin cortical network 160 887
GRAMD4 GRAM domain containing 4 mediator of E2F1-induced apoptosis 131 878
IGSF3 Immunoglobulin superfamily, member 3 involved in cyst and duct formation 152 878
PTK7 Protein tyrosine kinase 7 (inactive) involved in Wnt/planar cell polarity signaling 159 878
LRP4 Low density lipoprotein receptor-rel. protein 4 involved in canonical Wnt pathway regulation 130 877
EFNA3 Ephrin-A3 migration, repulsion, and adhesion regualtor 137 868
MACROD1 MACRO domain containing 1 enhances ESR1-mediated transcription activity 149 868
EEF2 Eukaryotic translation elongation factor 2 ribosomal translocation in translation elongation 124 843
ORMDL3 Sphingolipid biosynthesis regulator 3 negative regulator of sphingolipid synthesis 119 834
ARHGEF11 Rho guanine nucleotide exchange factor 11 regulation of RhoA GTPase 083 817
NYNRIN NYN domain and retroviral integrase containing nucleic acid binding 162 817
CLASP1 Cytoplasmic linker associated protein 1 alignment of chromosomes to the mitotic spindle 118 813
NAV2 Neuron navigator 2 neuronal development, cell growth and migration 105 797
KLHL15 Kelch-like family member 15 protein ubiquitination and cytoskeletal organization 088 797
NMNAT2 Nicotinamide nucleotide adenylyltransferase 2 catalyze an essential step in NADP pathway 142 794
NUFIP2 Nuclear fragile X protein interacting protein 2 RNA binder 075 786
RACGAP1 Rac GTPase activating protein 1 controls cell growth and differentiation 120 782
HIPK1 Homeodomain interacting protein kinase 1 regulation and TNF-mediated cellular apoptosis 071 778
XPR1 Xenotropic and polytropic retrovirus receptor 1 G-protein coupled signal transduction 126 778
PITPNM1 Phosphatidylinositol transfer protein RHOA activity and cytoskeleton remodeling 086 765
SLC12A7 Solute carrier family 12, member 7 electroneutral potassium-chloride cotransporter 154 762
PTK7,33 and SCRIB3435 is a scaffold protein involved
in planar cell polarization processes prior to and dur-
ing early tube formation in both the neural tube and in
cardiogenesis. TSC1 and TSC236 and CELSR13738 are
genes that are associated primarily with cavitation driven
tube formation. MIB139–42 has been shown to be involved
in apoptosis driven tube formation. The general tube
formation regulator PBX1 is also upregulated.43 The pro-
teins encoded by DVL2,384445 ZIC2,46 and HECTD147–50
have been shown to be involved in later stages of tube
formation, specifically during tube closures.
Additional upregulated apoptosis mediating genes such
as AGTR2 and BMF may indicate a role for cell death
in remodeling of the internal multicyst formation in
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Table III. Comparison of pluripotency gene expression in 200 versus
500 micron hESC EBs.
Pluripotency Log2FDR
gene fold change value p-value
Oct 4/POU5F1 +019 >0001 034
Myc/c-Myc 076 >0001 0025
KLF4 +134 >0001 000023
Sox2 +068 >0001 00024
Nanog 037 >0001 021
UTF1 +097 >0001 00043
LIN28A +043 >0001 0029
LIN28B +049 >0001 00027
SALL4 +045 >0001 000077
NR5A2 092 >0001 00041
TBX3 +069 >0001 012
ESRRB +058 >0001 016
DPPA4 011 >0001 051
Rex-1 (zfp42) +010 >0001 067
500 micron EBs. The list of genes includes functional
roles in cytoskeletal remodeling and cell adhesion, such as
E-cadherin and plakins plus various tight junction proteins.
Up-regulation of factors in signaling pathways for TGF-,
Tab l e I V. Tube formation genes in 200 versus 500 micron EBs.
Gene Fold
name Gene function change (p-value)
Genes upregulated in 500 micron EBs
DVL2 Dishevelled segment polarity protein
2 regulates Wnt signaling
pathways
+055 3.67E–07
MIB1 Mindbomb E3 Ubiquitin protein
ligase 1 ubiquitinates notch
protein ligands
+089 7.47E–17
TSC1 Tuberous sclerosis 1 negatively
regulator of mTORC1 pathway
+066 1.89E–07
TSC2 Tuberous sclerosis 2 negatively
regulator of mTORC1 pathway
+055 1.11E–05
LAMA5 Laminin, alpha 5 cell–cell
interactions in embryonic
development
+132 2.73E–06
PTK7 Protein tyrosine kinase 7 (inactive)
Wnt signaling mediated cell–cell
interactions
+159 2.79E–12
PBX1 Pre-B-cell leukemia homeobox 1
transcription factor involved in
cell patterning
+049 2.11E–05
CELSR1 Cadherin, EGF LAG g-type receptor
1 cell–cell signaling during neural
development
+104 1.77E–07
SCRIB Scribbled planar cell polarity protein
regulator of polarized cell-based
differentiation
+112 9.89E–06
ZIC2 Zic family member 2 transcription
factor in early CNS organogenesis
+123 1.05E–05
HECTD1 HECT Cont. E3 ubiquitin protein
ligase 1 required for neural tube
closure and others
+071 2.86E–05
Wnt, and EGF were also observed and in early organ
development are implicated in guiding early tube forma-
tion for multiple organ systems including trachea, kidneys,
and lungs. Genes that are involved in extra-cellular matrix
remodeling such as zinc finger proteins and other calcium
ion receptors and transporters are also upregulated. Genes
that may suggest a primed state also are significantly up
or down regulated and include USP9X and NYNRIN (up)
block protein degradation, EXOSC6 (down) targets RNA
for degradation, HES3 (up) inhibits Helix-loop-Helix pro-
teins, H1F0 and WDR82 (up) histone compacting genes,
BCORL1 (up) transcriptional repressor, and CCDC129
and CCDC96 (down) coiled-coil domain proteins. As well,
we observe upregulation in both ECM remodeling genes
and in early priming transcriptional factors implicated in
differentiation. These include for example, genes involved
in neurogenesis (SYT7, CNTFR, EFNB1, L1CAM), skele-
tal muscle (HRC, ACTN3), pituitary gland (TEF, GSX1),
pancreas (MAPK8IP1), germ cells (YBX2), and cardio-
genesis (SPNS2, PLEC, FOXH1).
2.5. Expression of Wound Responsive Genes in EBs
Generated by Mechanical Passaging
The formation of EBs is typically done by mechanical pas-
saging of colonies into 2D clusters or by use of single cells
in microarrays or bioreactors. Here we compared mechan-
ical passaging versus templating of single cells for 200
micron EBs (Fig. 3). We observed that compared to single
cell EB generation, mechanical passaging into 2D clusters
for EB formation activated genes within the GO wound
healing (Table V—GO:0009611Response to wounding)
that include PROCR, GATM, SERPINE1, DSP, PROS1,
and PLAU. Roles for these genes that may be relevant
include extracellular matrix remodeling, cytoprotection
(PROCR and PROS151–53), as well as ECM remodeling
and degradation (PLAU5455) and cell–cell tight junction
maintenance (DSP5657). SERPINE158 and GATM59 are
regulatory genes that are associated with monolayer repair
and cell migration. The timeline for EB formation from
2D clusters is 3–5 days for 200 micron EBs and 7–10 days
for 500 micron EBs, indicating that these genes if induced
by mechanical wounding persist in the newly formed EB
despite no noticeable variation in EB morphology versus
single cell formed EBs. For the full list of differentially
expressed genes see the GEO access number GSE74792.
3. MATERIALS AND METHODS
3.1. Maintenance of Human Pluripotent Stem Cell
ESC and ED-iPS Lines
The ethnically diverse (ED)-iPS cells3and hESC line
WA09 (WISC Bank, WiCell, Madison, Wisconsin) were
maintained in mTeSR2 complete media (Stem Cell Tech-
nologies, Vancouver, Canada) on StemAdhere (Stem Cell
Technologies, Vancouver, Canada) coated non-tissue cul-
ture treated dishes. Cells were enzymatically passaged
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Figure 3. Transcriptomic comparison of WA09 hESC 200 micron embryonic bodies derived from cell clusters (2D) or single cells (SC). Embryonic
bodies derived via 2D or single cell were compared by RNA-seq. (A) The 334 differentially expressed genes at a FDR <0.001 were hierarchically
clustered and the top 50 genes were divided into downregulated (B) or upregulated (C) tables. Sequence counts at each gene was standardized for
visualization by log2(gene counts/gene mean). See Tables III and IV for top genes and the GEO access number GSE74792 for the full list of
genes.
between days 5–7 using the Gentle Cell Dissociation
Agent (Stem Cell Technologies, Vancouver, Canada).
Media was replaced one day after the first passage and
cells were grown overnight with addition of 10 l/mL slow
release hFGF2 (StemBeads FGF2; Stem Culture Incorpo-
rated, Rensselaer, NY) in fresh mTeSR2 media. Media
was then completely changed every two days, with fresh
hFGF2 beads added.
3.2. Formation of Uniformly Sized hESC and ED-iPS
EBs in Custom Microarrays
Generation of 200 or 500 micron uniform EBs was done
by templating in custom microarrays of polydimethyl-
siloxane (PDMS) as previously described.14 Essentially, to
load microarrays from single cells we chemically disso-
ciated 2D stem cell colonies using the Gentle Dissocia-
tion Agent (Stem Cell Technologies, Vancouver, Canada).
Alternatively we generated small 2D clusters, by mechan-
ical passaging for loading into microarrays. The loaded
microarrays were gently washed after loading with 2 mL
of fresh HBSS (Hank’s Balanced Salt Solution, Fisher
Scientific, Grand Island, NY) to remove excess cells not
loaded into the wells. After the wash, loaded microarrays
were incubated in mTeSR2 media containing 10 MRock
inhibitor (Sigma-Aldrich, St. Louis, MO) for two days.
The media was changed every two days, until EBs reached
the uniform 200 or 500 m diameter. No ROCK inhibitor
was added after the first two days. On average, EB for-
mation from 2D clusters required 3 to 5 days (200 m),
7to10days(500m). EB formation from single cells
required 5–7 days (200 m) or 10 to 14 days (500 m).
Once formed, the template EBs were readily removed by
liquid expulsion with a p1000 micropipette. Individual EBs
from each group to be analyzed were then collected man-
ually for processing by suction with a p200 micropipette
tip under a 10×microscope magnification.
3.3. RNA Isolation
RNA isolations for bioinformatics analysis of the hESC
and ED-iPS cell lines were done using the Ambion Pure-
Link Mini RNA isolation kit (Life Technologies). Eluted
total RNA from the stem cell EBs was stored at 80 C
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Tom o v e t al. Human Embryoid Body Transcriptomes
Tab l e V. Wound responsive genes in single cell versus 2D intermediates generated 200 micron EBs.
Gene name Gene function Fold change log10 (p-value)
Genes downregulated in single cell generated 200 micron EBs
TAGLN Transgelin actin cross-linking, contributes to senescence 246 2364
ANXA3 Annexin A3 regulates cell growth and signal transduction 224 2997
CXCL5 Chemokine (C-X-C Motif) ligand 5 involved in cell migration and invasion 204 2919
PROS1 Protein S (alpha) ECM remodeling protein 179 1134
SERPINE1 Serpin peptidase inhibitor, clade E serine protease inhibitor, ECM remodeling 152 647
ANXA1 Annexin A1 promotes membrane fusion and exocytosis 142 551
H1F0 H1 histone family, member 0 found in late differentiated non-dividing cells 136 897
PLAU Plasminogen activator, urokinase ECM modification, migration and proliferation 132 700
HSPB8 Heat shock 22 kDa protein 8 regulates cell proliferation and apoptosis 130 564
PRNP Prion protein neuronal development and synapse plasticity 125 897
MYO1C Myosin IC transcription factor for actin-based motors 124 933
CER1 Cerberus 1, DAN family BMP antagonist regulates nodal signaling during gastrulation 122 501
PROCR Protein C receptor, endothelial binds to and enhances activated protein C 120 563
EMP3 Epithelial membrane protein 3 cell proliferation and cell/cell interactions 115 327
APOBEC3B Apolipoprotein B MRNA editing enzyme implicated in growth or cell cycle control 115 359
CLDN3 Claudin 3 regulates tight junction formation 113 391
SCRN1 Secernin 1 calcium dependent secretase in mast cells 112 920
SERPINB6 Serpin peptidase inhibitor, clade B M6 regulates blood serine proteases in the brain 110 589
SOCS2 Suppressor of cytokine signaling 2 regulates cytokine transduction of GH/IGF1 108 526
BMP7 Bone morphogenetic protein 7 involved in embryonic TGFpathways 107 477
ACTA1 Actin, alpha 1, skeletal muscle involved in cell motility, structure and integrity 107 409
HPN Hepsin regulates cell growth and cell morphology 105 327
NMRK2 Nicotinamide riboside kinase 2 regulates laminin-based cell adhesion 104 519
TMSB4X Thymosin, beta 4, X chromosome cell proliferation, migration and differentiation 103 625
ADM Adrenomedullin regulates hormone secretion and angiogenesis 102 727
S100A10 S100 calcium binding protein A10 cell cycle progression and differentiation 102 622
ANXA2 Annexin A2 regulates cell growth and transduction 101 388
TAGLN2 Transgelin 2 putative marker of differentiated smooth muscle 101 590
HLA-DQB1 MHC, class II, DQ beta 1 plays a central role in the immune system 100 379
GATM L-Arginine:Glycine amidinotransferase embryonic muscle and CNS development 100 428
SERPINB1 Serpin peptidase inhibitor, clade B, M1 protects from inflammatory-based damage 093 326
DSP Desmoplakin junction protein linking to intermediate filaments 090 388
AK3 Adenylate kinase 3 maintains homeostasis of cellular nucleotides 079 340
AMT Aminomethyltransferase critical component of glycine cleavage system 075 343
FGFBP3 Fibroblast growth factor binding protein 3 involved in heparin binding and function 075 388
Genes upregulated in single cell generated 200 micron EBs
HIST1H1A Histone cluster 1, H1a regulator of individual gene transcription 271 2579
NLRP2 NLR family, Pyrin domain containing 2 suppresses TNF/CD40-induced NFKB1 activity 169 1799
ZNF883 Zinc finger protein 883 involved in transcriptional regulation 157 769
SYT11 Synaptotagmin XI involved in vesicular trafficking and exocytosis 154 95
TYW3 TRNA-YW synthesizing protein 3 homolog stabilizes codon-anticodon interactions 13621
LMO3 LIM domain only 3 (Rhombotin-Like 2) involved in chromosomal translocations 126 529
SYT4 Synaptotagmin IV involved in vesicular trafficking and exocytosis 123 380
CRYZ Crystallin, zeta (quinone reductase) binds NADP and enhances mRNA stability in BCL2 121 578
DOCK2 Dedicator of cytokinesis 2 involved in cytoskeletal rearrangement 118 360
HIST1H1B Histone cluster 1, H1b regulator of individual gene transcription 117 482
ZNF619 Zinc finger protein 619 involved in transcriptional regulation 115 388
TMEM132B Transmembrane protein 132B integral membrane component 108 563
P2RY1 Purinergic receptor P2Y receptor for extracellular ATP and ADP 108 343
BTBD17 BTB (POZ) domain containing 17 transcription factor in early differentiation 100 360
ZFP42 ZFP42 zinc finger protein (Rex-1) acquisition and maintaining of ES pluripotency 088 590
until ready to ship for analysis. For RNA-seq analysis,
total RNA was processed for sequencing using the TruSeq
RNA Sample Preparation Kit (Illumina). Samples were
four-plexed and 50 bp single-end sequenced on an Illumina
HiSeq2500 producing 30 to 50 million sequence reads to
generate bioinformatics data.
3.4. RNA-Seq Data Processing
Raw sequencing reads were mapped to the Homo sapi-
ens genome (hg19 build) using Tophat—version 2.0.7,60
count files generated with HTSeq-count.61 Significantly
enriched genes (FDR <0.001) were then determine
using DESeq2.62 Enriched genes where then tested
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Human Embryoid Body Transcriptomes Tom ov e t a l .
(A)
(B)
(C)
Figure 4. 500 micron EBs retain pluripotency while also expressing
early embryonic tube formation pathway genes. Representative 3D ren-
derings of monocystic 200 and polycystic 500 micron EBs are shown
in (A), arrows highlight the internal cystic structures. Simulated scale bar
is 200 microns. (B) 3D renderings of early embryonic tube formation path-
ways that appear active in the 500 micron EBs, based on observed gene
expression (listed genes). (C) ICC staining of a polarized cyst in a 500
micron EB showing levels of nuclear pluripotency marker Sox2, Hoechst
nuclear stain, and ZO1 tight junction marker. Scale bar is 50 microns.
for annotation enrichment with DAVID63 and clustered
using unsupervised hierarchical clustering with com-
plete linkage and Euclidean distance. We identified
the following GO terms to be of interest during our
analysis: GO: 0060562 epithelial tube morphogenesis;
GO: 0035295 tube development; GO: 0021915 neural
tube development; GO: 0001841 neural tube formation;
GO: 0001843 neural tube closure; GO: 0060606 tube
closure; GO: 0001838 embryonic epithelial tube forma-
tion. The GEO access number is GSE74792.
4. DISCUSSION
Nanotechnology and microtechnology provides critical
tools for customized in vitro analysis of stem cells.64–67
Here we applied photolithography to generate custom
microarrays of defined size to control EB formation and
size uniformity for comparative transcriptome analysis. Our
findings indicate that manipulation of stem cells for EB
formation, as well as EB size, each significantly alter tran-
scriptome profiles which should be considered when dif-
ferentiation methodologies utilize EBs (Figs. 4(A and B)).
Thus EBs formed by different strategies may not pro-
vide comparable results, consistent with concerns for more
uniformity in protocols in the stem cell field.68–70 The
expression of pluripotency genes was consistent and inde-
pendent of method of formation or EB size (Fig. 4(C)).
Gene ontology (GO) analysis revealed an upregulation of
genes for wound healing when mechanical passaging is
used to form EBs from 2D clusters, not present when sin-
gle cell dissociation is used. Comparison of multicystic 500
micron EBs versus monocystic 200 micron EBs indicates
upregulation of GO pathways for early embryonic tube for-
mation and organogenesis, including but not limited to neu-
ral tube formation. This analysis fills important gaps in our
understanding of the EB stem cell intermediate and has
significance for understanding potential variation and effi-
ciencies in differentiation protocols employing EBs.
Tube formation has been shown to be a prerequisite for
proper development in multiple organs and there are mul-
tiple factors that direct this process. The morphological
processes of early tube formation share common features
that include molecular events to establish apical membrane
polarity. Genes that govern this process are upregulated
in large 500 micron EBs compared to the smaller 200
micron EBs (Fig. 4(B)). Critical genes that are involved in
tube formation include tight junction proteins and calcium
ion receptors and transporters, which are involved in ECM
remodeling to generate conditions that promote tube for-
mation. Additionally, other genes involved in ECM remod-
eling have been shown to prevent the apical surfaces of the
forming tube from sticking together. Within the cell, multi-
ple genes that code for cytosolic transport proteins, which
are involved in early tube formation cues are also consis-
tently upregulated. These factors regulate the concentra-
tion of small soluble molecules in the ECM, which help
generate the tight junction between the cells within the
forming tube walls that may affect the size and shape of
the tube being formed. In addition, genes that are involved
8986 J. Nanosci. Nanotechnol. 16, 8978–8988,2016
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Tom o v e t al. Human Embryoid Body Transcriptomes
in programmed cell death are also upregulated in the mul-
ticystic 500-micron EBs. These genes assist formation of
cavities, such as pre-amniotic cavities during embryonic
development in vertebrates. Transcriptome analysis further
indicates the expression of genes related to remodeling of
ECM, cytoskeleton, cell–cell interactions, and signaling,
consistent with the observed multicystic internal structure.
The 500-micron EBs may be primed for formation of these
more complex tissues and organoids.71 Select genes that
code for proteins found at early stages of multilineage
differentiation are in some cases upregulated while later
expressed proteins remain downregulated in 500-micron
EBs. Together the combination of upregulated and down-
regulated genes in larger 500-micron EBs is intriguing and
may in some cases provide unique advantages for differ-
entiation when tube formation is needed.71 In this regard
it is interesting that stem cell differentiation for cardiogen-
esis has been proposed to occur with higher frequency by
use of larger EBs in the range of 500 microns, but smaller,
in the range of 200 microns, EBs were shown to be more
enriched for beating cardiomyocytes.72–74
Our findings reveal new insights into size-dependent
variability in EBs as well as retention of signals arising
from manipulation of stem cells for EB formation. Ele-
vated expression of genes involved in wound healing when
mechanical passaging is used is in retrospect not surpris-
ing and consistent with in vitro wounding models typically
used in culture5875 However the retention of these signals
up to 5 days during the formation of EBs in microarray
templates indicates that EB maturation may be a longer
process then realized. In addition, the type or extent of
maturation possible is also reflected in EB size, with larger
EBs capable of more extensive remodeling as revealed by
multicystic morphology and initiation of early tube forma-
tion pathways while retaining pluripotency status.
Acknowledgments: The authors thank Jose Cibelli,
Michigan State University and LARCEL, Spain, for criti-
cal feedback. The work was a collaborative effort between
SUNY Polytechnic Institute and SUNY Buffalo and sup-
ported by internal funds and NYSTEM Award C026186.
Author contributions: Janet L. Paluh designed and super-
vised experiments, analysis, and manuscript and figure
preparations. Michael Buck performed and supervised
RNAseq bioinformatics; Maria Tsompana performed bioin-
formatics and RNAseq sample processing. Martin L.
Tomov performed experiments for lithography, stem cell
growth, maintenance, EB generation, microscopy and RNA
harvesting, and assisted manuscript writing and figures.
Zachary T. Olmsted performed experiments for stem cell
growth, maintenance, and assisted manuscript figures.
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8988 J. Nanosci. Nanotechnol. 16, 8978–8988,2016
... The ED-iPSC lines analyzed in this study fit this need and include replicate lines of Asian or of Hispanic-Latino designation that were derived from fibroblasts and analyzed under a single platform. In addition, we initiate differentiation protocols from lithography templated uniform EBs to increase accuracy of our comparative analysis 13,22,23 . In our initial validation of these ED-iPSC cell lines we confirmed normal karyotype, verified pluripotency biomarkers by qRT-PCR, and confirmed teratoma formation as well as in vitro tri-lineage commitment, summarized here in Table 1. ...
... Stochastic epigenomic differences fall into two primary clusters, each capable of pluripotency as gauged by teratoma and qRT-PCR 13 as well as our new data on multi-lineage differentiation into pyramidal neurons, CD44+ /GFAP+ astrocytes, RPE cells, pancreatic progenitors, smooth muscle and contractile cardiomyocytes. We use previously established protocols for differentiation and initiate differentiation from custom templated uniformly sized EB intermediates for consistency in comparative analysis 22,23 . At the epigenetic level, ED-iPSC line reprogramming was evaluated by ChIP-seq for histone modifications H3K4me1 and H3K27ac. ...
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... Importantly, to maintain cell viability and functionality, the scaffolds that are employed in tissue engineering must satisfy several key biophysical and biochemical requirements, both during and post manufacturing, such as cell viability, proper niche recapitulation, spatial fidelity, and biodegradability [7][8][9]. These parameters, together with a reliable method to generate functional vascular networks within the engineered constructs, are critical for generating high-fidelity tissue analogs [10]. ...
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... The ability to incorporate different cell types within the same assembly has also been shown to improve maturation and development of the tissue arrays [125]. Recent advances in the field have enabled a range of patterning techniques that make it possible to produce biocompatible surfaces, without the often-toxic chemicals that are common in traditional lithography process flows [126,127]. Multiple modes of patterning, such as reactive ion etching, light-based crosslinkers, enzymatic, or charge-based adhesion, have been used to generate hard and soft stamped surfaces without significant cell toxicity, allowing for advances into the translational and drug discovery areas [51,115,117]. ...
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