Running title: Directed differentiation via vesicle delivery
Extracellular vesicles derived from preosteoblasts influence embryonic stem cell differentiation
Rekha Nair1,2,3, Lívia Santos1,2,3, Siddhant Awasthi1,2, Thomas von Erlach1,2,3, Lesley W. Chow1,2,3, Sergio
Bertazzo1,3, Molly M. Stevens1,2,3*
1
Department of Materials, Imperial College London SW7 2AZ, UK
Department of Bioengineering, Imperial College London SW7 2AZ, UK
3
Institute of Biomedical Engineering, Imperial College London SW7 2AZ, UK
*Corresponding author
2
Author information:
Address: Royal School of Mines, South Kensington Campus, London SW7 2AZ
Fax: +44 (0)20 7594 6757
Phone: +44 (0)20 7594 6804
Email:
Rekha Nair: rekha.nair29@gmail.com
Lívia Santos: l.rocha-dos-santos@imperial.ac.uk
Siddhant Awasthi: siddhant.vit@gmail.com
Thomas von Erlach: t.von-erlach10@imperial.ac.uk
Lesley W. Chow: l.chow@imperial.ac.uk
Sergio Bertazzo: s.bertazzo@imperial.ac.uk
Corresponding author information:
Molly M. Stevens
Address: 208, Royal School of Mines, South Kensington Campus, London SW7 2AZ
Telephone: +44 (0)20 7594 6804
Fax: +44 (0)20 7594 6757
Email: m.stevens@imperial.ac.uk
Website: www.stevensgroup.org
1
ABSTRACT
Embryonic stem cells (ESCs) can differentiate into all cell types of the body and therefore hold tremendous
promise for cell-based regenerative medicine therapies. One significant challenge that must be addressed prior
to using ESCs in the clinic is to improve methods of efficiently and effectively directing the differentiation of
this heterogeneous cell population. The work presented here examines the potential of harnessing naturally
derived extracellular vesicles to deliver genetic material from mature cells to undifferentiated ESCs for the
purpose of manipulating stem cell fate. Vesicles were isolated from preosteoblast cells and were found to be
~170 nm in diameter and express the CD40 surface marker. Multiple interactions were visualized between
vesicles and ESCs using confocal microscopy, and no significant difference in cell viability was noted.
Incubation with vesicles caused significant changes in ESC gene expression, including persistence of
pluripotent gene levels as well as increased neurectoderm differentiation. Genetic cargo of the vesicles as well
as the cells from which they were derived were examined using a small microRNA (miRNA) gene array.
Interestingly, ~20% of the examined miRNAs were increased more than two-fold in the vesicles compared to
preosteoblast cells. Together, these results suggest that extracellular vesicles may be utilized as a novel method
of directing stem cell differentiation. Future work examining methods for controlled delivery of vesicles may
improve the clinical potential of these physiological liposomes for therapeutic applications.
Key words: Embryonic stem cells, directed differentiation, extracellular vesicles, microvesicles, exosomes,
microRNA
2
INTRODUCTION
Embryonic stem cells are derived from the inner cell mass of the pre-implantation blastocyst and can be
differentiated into any somatic cell of the body [1-3]. These pluripotent cells are well studied for their potential
for cell-based therapies in regenerative medicine and hold tremendous promise for applications where
endogenous cells are lost due to injury or disease. Despite great progress in the field, however, there is still a
relative lack of directed differentiation schemes that can reproducibly, effectively, and efficiently control stem
cell fate. These schemes are critical to develop if pluripotent stem cells, which are a naturally heterogeneous
cell population, are to be used for clinical therapies. Current methods of directing stem cell fate tend to rely on
soluble factors, culture conditions, or small molecules that are timed specifically to recapitulate developmental
processes [4,5]. These methods, however, tend to be inefficient in that each step of the process leads to
intermediate progenitor cell states until the final differentiated product is reached. Since differentiation
efficiency tends to decrease at each state, the resulting population is still relatively heterogeneous [4,6].
Another method of directing stem cell differentiation involves manipulation at the genetic level via ectopic
expression of genes of interest [7,8] or suppression using RNA interference [9-11] to influence the direction of
cell fate [12,13]. However, such genetic manipulation can be less than ideal for clinical applications since
exposure to these added genes or expression vectors may have long term negative consequences [13]. In recent
years, another method of altering recipient cell behavior at the transcript level has emerged that involves
horizontal gene transfer using particles naturally shed from cells. These extracellular vesicles have been
implicated in cell to cell communication in a variety of mammalian cell systems, including cancer cell lines
[14,15], stem cells [16-18], and primary cells [19,20].[21] Subclasses of these vesicles can be categorized based
on their biogenesis pathways and can sometimes be identified based on enrichment of specific surface markers
as well as particle size. For example, exosomes are formed via the endolysosomal pathway, are typically 40120 nm, and are enriched in tetraspanins, while microvesicles are formed through the outward budding of the
cell membrane, are 50-1,000 nm, and can be identified by expression of the CD40 ligand [21]. Both types of
vesicles have been shown to contain a variety of genetic information, including RNA, miRNA, and other non3
coding RNA [22]. A wide array of literature supports the presence of these vesicles in cancer cells [23,24], but
there has also been evidence to indicate their presence in a variety of other cell systems as well, including ES
cells [17,18,25], mesenchymal stem cells [26-31], and dendritic cells [32,33]. In several of these cases, research
has also shown that cells shedding these vesicles are able to transfer their own genetic material to distant cells
and that this genetic material can be incorporated and translated to protein in the recipient cell population
[22,34]. Such a transfer of genetic material holds tremendous promise for the devised manipulation of a targeted
cell population, and indeed, is already known to play roles in human physiology, including in stem cell
maintenance [17] and tissue repair [35].[21]
The aim of the work presented here was to determine whether vesicles derived from a non-embryonic source
could be used to manipulate cell fate in a pluripotent stem cell system. It was hypothesized that a preosteoblast
cell line would be of interest as a candidate for the vesicle source since its own potential for differentiation
suggests an active signaling base that could result in vesicle-bound paracrine cues, potentially leading to
quantifiable changes in the ES cells. In this study, vesicles were derived from a murine preosteoblast cell line
and characterized using several techniques, including nanoparticle tracking analysis, FACS, and immunogold
transmission electron microscopy. The ability of these cells to alter the behavior of differentiating ES cells was
then examined using confocal microscopy, viability studies, and quantitative PCR. Finally, the genetic content
transferred from the donor cell to its vesicles was examined in depth using a miRNA array. Results from this
work suggest that preosteoblasts can reproducibly produce vesicles containing genetic information that can
effectively alter ES cell fate. This novel means of manipulating stem cell differentiation may signify future
possibilities of altering cell behavior, thereby improving the potential of using heterogeneous pluripotent stem
cell populations for cell-based clinical therapies.
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MATERIALS AND METHODS
Cell culture
Mouse preosteoblasts derived from bone/calvaria (MC3T3-E1 subclone 4) were purchased from ATCC and
cultured using minimum essential medium (MEM) alpha without ascorbic acid (Invitrogen) supplemented with
10% (v/v) fetal bovine serum. Cells were trypsinized (0.05% (v/v) Trypsin EDTA) routinely once they reached
~90% confluence and were fed every 2-3 days. The preosteoblasts were grown to confluence and were switched
to serum free MEM alpha 48 hours prior to particle collection.
Undifferentiated mouse embryonic stem cells (D3 line, purchased from ATCC) were cultured feeder-free on
tissue culture treated polystyrene plates coated with 0.1% (w/v) gelatin in Dulbecco’s Modified Eagle’s
Medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum, 0.1 mM non-essential amino
acid solution, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, and 1000 U/mL leukemia inhibitory factor (LIF).
ESCs were fed every other day and passaged using 0.05% (v/v) Trypsin EDTA when cells reached 70%
confluence. For studies investigating ESC behavior and differentiation in the presence of preosteoblast particles,
cells were plated on 48-well plates at a density of ~5500 cells/cm2 and were cultured without LIF. Both the
MC3T3-E1 and D3 cell lines have been well studied and characterized by numerous research groups.
Researchers probing the behavior of mouse embryonic stem cells in particular have extensively examined the
D3 cell line.
Particle isolation
Conditioned media from preosteoblasts grown in serum-free conditions for 48 hours was collected and spun
down at 4000 rpm (3220 rcf) for 30 minutes at 4oC. The supernatant was then transferred to 25 mL sterile
Konical™ ultracentrifuge tubes (Beckman) and spun at 100,000 x g for two hours at 4oC in a Beckman Coulter
Optima L-100XP ultracentrifuge. Particles were concentrated at the bottom of the conical ultracentrifuge tubes
and could then be resuspended in phosphate-buffered saline (PBS) or ESC medium depending on the
application.
5
Transmission electron microscopy (TEM)
Particles were fixed in paraformaldehyde directly after isolation and incubated on a TEM grid (copper grid with
300 mesh, carbon coated; Electron Microscopy Sciences) for 10 minutes. For samples to be immunostained,
excess sample solution was gently wicked off using filter paper, and the grid containing the particles was
blocked in a 1% (v/v) bovine serum albumin (BSA)/PBS solution for 10 minutes. The sample was then
incubated with anti-CD40 primary antibody (diluted 1:100 in BSA/PBS; Abcam) for one hour and washed five
times (one minute each, in 1% (v/v) BSA/PBS). Particles were then incubated with immunogold solution
(diluted 1:1 in PBS; 7.5-10 nm particles synthesized according to [36,37]) for one hour, washed five times (one
minute each, in PBS), fixed in 4% (v/v) gluteraldehyde for five minutes, and finally washed again five times
(one minute each, in water). Samples were imaged at 80 kV using a JEOL 2010 TEM.
Nanoparticle tracking analysis (NTA)
Particles were resuspended in 10 mL PBS immediately after isolation. After ensuring even distribution of the
particles within PBS through repeated pipetting, 1 mL of the solution was drawn into a syringe and inserted into
the NanoSight LM10. Particle movement was tracked for 60 seconds where particle size distribution was
monitored using Nanoparticle Tracking Analysis software. Within one batch of isolated particles, this process
was repeated an additional two times, and all readings were averaged to obtain n = 1. NTA readings were taken
from four separate batches of isolated particles (n = 4).
Flow activated cell sorting (FACS)
Isolated particles were suspended in 50 uL PBS/0.1% (w/v) BSA. Samples were then labeled with CD40 (5 L;
FITC hamster anti-mouse CD40, BD Pharmingen™) or isotype control (FITC hamster IgM, 1 isotype control,
BD Pharmingen™) and incubated for 20 minutes in the dark. Particle populations were analyzed in a three
laser, 11 color Becton Dickinson Fortessa instrument equipped with DiVa software; ten thousand events were
collected for each sample.
6
Confocal microscopy
ESCs were plated at a density of ~9000 cells/cm2 onto u-Slide 8-well ibiTreat chamber slides (Ibidi) and were
allowed to adhere overnight at 37oC and 5% CO2. Immediately following isolation, particles were suspended in
a solution containing CellMask™ Orange (1:1000 dilution in PBS; Invitrogen) in order to label their
membranes. Undifferentiated ESCs that had been allowed to attach overnight were switched to differentiation
media (ESC medium without LIF) containing the stained vesicles and were then returned to growth conditions
(37oC and 5% CO2). After six and 24 hours, wells were rinsed with PBS to remove traces of medium and nonadherent particles, fixed for 30 minutes at room temperature in 4% (v/v) paraformaldehyde and blocked for 1h
with 4% (w/v) BSA. Wells from each time point were then incubated for three minutes with 300 uL 3 mM
DAPI solution (1:1000 in water), rinsed in PBS, incubated with 300 uL 488 Phalloidin (1:350 in PBS;
Invitrogen) for 20 minutes, and finally rinsed three times in PBS prior to coverslipping. The samples were
analyzed with a Leica SP5 inverted confocal microscope with 63x (1.4n/a) oil immersion objective under
standard imaging conditions.
Viability (alamarBlue® and LIVE/DEAD®)
ESCs were plated onto 24-well plates at a density of ~5500 cells/cm2 and allowed to adhere overnight at 37oC
and 5% CO2. Immediately after isolation, particles were suspended in ESC medium without LIF and distributed
to wells; each experimental group was given vesicles derived from 6 mL of conditioned MC3T3 medium. Cells
were then returned to growth conditions (37oC and 5% CO2). At 24 and 48 hours, cells were rinsed in PBS and
replenished with a solution of ESC medium without LIF containing a fresh batch of isolated vesicles. After four
and six days, wells were rinsed with PBS and incubated in 10% (v/v) alamarBlue® (Invitrogen) solution for 2.5
hours at 37oC and 5% CO2). Fluorescent readings of the wells containing cells alone and cells incubated with
particles were taken in a Spectramax M5 plate reader (Molecular Devices) (excitation 570 nm, emission 585
nm) to determine the relative levels of metabolic activity of the two populations.
7
In parallel, separate wells were incubated with LIVE/DEAD® solution (Invitrogen; 3 uL 1 mg/mL calcein + 3
uL ethidium homodimer-1 in 1.5 mL PBS) for 20 minutes at 37oC and 5% CO2. Cells were then rinsed in PBS
and imaged using a 1X51 inverted microscope (Olympus) and DP70-BSW software (Olympus). Cells
fluorescing green are considered live since the calcein (excitation 495 nm, emission 515 nm) is well retained in
live cells while those fluorescing red are considered dead since the ethidium homodimer-1 (excitation 528 nm,
emission 617 nm) was able to penetrate the membrane.
RNA and DNA extraction and quantification
ESCs were plated onto 24-well plates at a density of ~5500 cells/cm2 and allowed to adhere overnight at 37oC
and 5% CO2. Immediately after isolation, particles were suspended in ESC medium without LIF and distributed
to wells; each experimental group was given vesicles derived from 6 mL of conditioned MC3T3 medium. Cells
were then returned to growth conditions (37oC and 5% CO2). At 24 and 48 hours, cells were rinsed in PBS and
replenished with a solution of ESC medium without LIF containing a fresh batch of isolated vesicles. After 0
(the day of initial particle distribution), 3, and 7 days, wells containing treated and untreated cells were rinsed
with PBS, trypsinized (0.05% Trypsin EDTA), and spun down (300 x g, 5 minutes). The resulting pellet was
rinsed in PBS and resuspended in 350 uL RNeasy lysis buffer (Qiagen). RNA was then extracted according to
the manufacture’s protocol. Briefly, the lysed cells were homogenized using a Qiashredder and incubated with
an equal volume of 70% ethanol. Samples were then transferred to the RNeasy spin column and rinsed with
Buffer RWI and Buffer RPE prior to final elution in water. RNA was then quantified using a Nanodrop 2000c
(Thermo Scientific).
Isolated particles were examined for the presence of DNA and RNA (see protocol above). For DNA analysis,
particles were dissolved in 6M guanidine hydrochloride immediately after isolation. DNA was then quantified
using the Quant-iT™ PicoGreen® assay (Invitrogen) according to manufacture instructions. Briefly, samples as
well as calf thymus DNA standards were incubated with PicoGreen® buffer TE and solution containing
PicoGreen® dsDNA reagent, a fluorescent nucleic acid stain, and read on a Spectramax M5 plate reader
8
(excitation: 485 nm, emission: 528 nm). A linear equation was generated from DNA standards and used to
determine DNA concentrations in the samples.
Quantitative real time polymerase chain reaction (qRT-PCR)
To explore alterations at the gene expression level of ESCs incubated with the particles, cDNA was first
synthesized from isolated RNA using the QuantiTect Reverse Transcription kit (Qiagen) according to
manufacture’s protocol. Briefly, RNA diluted to 40 ng/uL using RNase-free water was incubated with 2 uL
gDNA Wipeout Buffer for 2 minutes at 42oC. Samples were then incubated with Reverse Transcription (RT)
Master Mix (at a ratio of 1 volume Reverse Transcriptase: 4 volumes RT Buffer: 1 volume RT Primer Mix) at
42oC for 15 minutes, followed by 3 minutes at 95oC. Freshly synthesize cDNA samples were then combined
with QuantiTect SYBR Green and PCR primers and tested under the following cycle conditions: hold at 95oC
for 15 minutes, cycle (40 cycles) at 94oC for 15 seconds, 58oC for 30 seconds, and 72oC for 30 seconds. A melt
curve was then run between 70-95oC. For annealing temperatures and primer details, see Supplementary Table
1. Gene expression of oct4, nanog, sox2, sox17, gata6, foxa2, nestin, sox1, and fgf5 were calculated relative to
undifferentiated ESC samples using the Pfaffl method (reference), with glyceraldehyde-3-phosphate
dehydrogenase (gapdh) used as the housekeeping gene.
miRNA array and analysis
To further explore genetic content contained within the vesicles, a miScript™ miRNA PCR array (Qiagen) was
run on both particles and the cells from which they were derived. Samples were isolated through
ultracentrifugation (particles) or trypsinization (cells) and analyzed according to manufacture’s protocols.
Briefly, total RNA (including small RNAs) was isolated using the miRNeasy Micro Kit (Qiagen) wherein
samples were lysed using QIAzol Lysis Reagent and homogenized via vortexing. Samples were then incubated
at room temperature for 5 minutes, and chloroform was added to induce phase separation. Ethanol was then
added to the aqueous phase, and total RNA including small RNAs were bound, washed, and eluted in 14 uL
RNase-free water. To synthesize cDNA, 250 ng RNA from each sample was incubated with miScript™ 5x
9
HiSpec buffer, 10x Nucleics Mix, and Reverse Transcriptase Mix for 60 minutes at 37oC, then 5 minutes at
95oC. To run the array, cDNA samples were combined with 2x QuantiTect SYBR Green PCR Master Mix, 10x
miScript™ Universal Primer, and RNase-free water and dispensed equally (10 uL per well) into Rotor-Disc®
100 Mouse Cell Differentiation & Development arrays. Arrays were then sealed using a Rotor-disc heat sealer
(Qiagen), placed into the RotorgeneQ PCR machine (Qiagen), and run using the following settings: initial
activation step of 95oC for 15 minutes, 3-step cycling (40 cycles) of 94oC for 15 seconds, 55oC for 30 seconds,
and 70oC for 30 seconds (fluorescence data collection was performed at this step).
Using the miScript miRNA PCR Array Data Analysis Web Portal provided by SABiosciences, a normalization
gene was selected among the examined miRNAs since the housekeeping genes typically used for quantitation
were dissimilar between the cells and vesicles. Software analysis assessed the most tightly regulated gene
amongst those examined and selected miR-1a-3p as the normalization gene. Fold changes in gene expression
were subsequently analyzed using the Ct method of quantitation, whereby vesicles were compared relative to
the cells from which they were shed after individual samples were normalized to internal miR-1a-3p levels. The
Data Analysis Web Portal was also utilized to generate a two-dimensional hierarchical clustering of the data
using average linkage clustering. Clustering results were represented visually by a heat map, with green
indicating decreased expression and red indicating increased expression. The degree of color intensity
corresponds to the magnitude of fold change (either an increase or a decrease). A scatter plot examining
log10(2^Delta Ct) of the vesicles v. cells was also generated to highlight fold regulation, with the red dots and
text indicating an up-regulation in gene expression in vesicles compared to cells and green indicating a decrease
in fold regulation.
Statistics
10
Significance testing was conducted using SYSTAT software. For individual genes in PCR analysis, expression
fold change comparisons within and across time points were evaluated using analysis of variance (ANOVA)
with subsequent post-hoc Tukey analysis to determine significance (p < 0.05).
11
RESULTS
Vesicle characteristics
Particles isolated from mouse preosteoblasts were characterized to investigate morphology, size, and surface
markers (Figure 1). Isolated particles were visualized using immunogold labeling of CD40 and transmission
electron microscopy (TEM). Positive labeling indicating the presence of CD40 on the surface of the particles
was observed throughout. Particles were irregularly shaped, and TEM-based diameter analysis of the images
indicated particle sizes of approximately 200 nm. Additional images of stained and unstained vesicles are
provided in Supplementary Figure 1. Further, nanoparticle tracking analysis (NTA) was used to more accurately
assess particle size. The average size from four different batches of isolated particles was determined to be
174.2 ± 22 nm, confirming data observed from TEM analyses. All NTA assessments displayed similar size
distributions (data from a representative batch is displayed in Figure 1), with only one distinct population seen.
Particle sizing determined by both TEM and NTA suggest that the particles isolated from the preosteoblasts fall
within the size range of microvesicles and not exosomes [21]. Surface markers are often used to further
distinguish between these types of particles, with the presence of CD40 again identifying a microvesicle
population. As noted above, immunogold labeling of the particles revealed the presence of CD40. In addition,
FACS analysis also identified a strong CD40-positive population within the isolated particles. Forward v. side
scatter (FSC-W v. SSC-W) as well as count assessments indicated a distinct positive shift in fluorescent signal
compared to isotype controls.
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Cellular uptake efficiency of vesicles
The interaction and uptake efficiency of preosteoblast-derived particles incubated with undifferentiated
embryonic stem cells was analyzed qualitatively by high resolution confocal scanning microscopy at multiple
time points (Figure 2). The vesicle membrane was fluorescently labeled with a cell membrane dye prior to
incubation with adherent stem cells to allow for cellular tracking. Within six hours, a large number of vesicles
(red) were detected within all analyzed ESC clusters, indicating high cellular uptake efficiency of the
microvesicles. The particles were widely dispersed within the ESC cluster and were primarily co-localized
within the nuclei, the site of action of the vesicle cargo, which indicates endosomal escape and cargo delivery of
the vesicles within the cell. By 24 hours, some evidence of the particles still remained, though fewer were
visible through confocal microscopy, possibly due to lysosomal degradation of the fluorescently labeled vesicle
membrane within the cells.
The viability of differentiating ESCs was not affected by the presence of the particles. alamarBlue® readings of
cells incubated with preosteoblast-derived vesicles did not change significantly compared to untreated cells at
either day 4 or day 6 after incubation. Further, cells at each time point that were incubated with LIVE/DEAD®
stain primarily stained green (live) with very few visible red (dead) cells, supporting the alamarBlue® results
and indicating that the vesicles did not negatively affect ESC viability. These results were consistent across
vesicles derived from separate batches of MC3T3s (data not shown).
Alteration of recipient cell behavior
In addition to examining the viability of recipient cells, alterations in the population’s gene expression were
investigated (Figure 3). ESCs grown under differentiation conditions (no LIF) were either left untreated or were
incubated with vesicles, and RNA was collected after three or seven days. Levels of gene expression of
pluripotent (oct4, nanog, sox2) and differentiation (mes/endoderm: sox17, gata6, foxa2; ectoderm: nestin, sox1,
fgf5) markers were analyzed using RT-PCR and expressed as fold change values compared to undifferentiated
ESCs (day 0). ESCs incubated with the preosteoblast-derived vesicles expressed higher levels of all pluripotent
13
markers at almost all time points examined. At day 3, oct4 levels were significantly higher in vesicle-treated
cells (p < 0.01), as were nanog levels (p < 0.05) at day 7. In all cases, the fold change was less than 1, with the
exception of oct4 (1.39 ± 0.3) and sox2 (1.07 ± 0.5) at day 3 in the vesicle-treated population. Taken together,
these results suggest that both populations exhibited some extent of differentiation by day 7, but the presence of
the vesicles in the ESC culture delayed the onset of differentiation.
Analysis of lineage markers also indicated a modification in the differentiation trajectory of the treated cell
population. This panel of markers was specifically chosen to represent each of the three germ lineages, with an
up- or down-regulation in any group suggesting that the ES cells were moving toward or away from those
lineages, respectively. No significant differences were noted in the mesendoderm markers, although the vesicletreated cells exhibited higher fold changes at all time points, with the exception of gata6 at day 7 (Figure 3).
However, neurectoderm markers nestin and sox1 displayed significant increases in expression at day 7 in the
vesicle-treated cells compared to untreated cells, with nestin fold changes increasing from 1.34 ± 0.8 to 13.39 ±
4.8 and sox1 shifting from 0.18 ± 0.1 to 11.18 ± 6.6 (p ≤ 0.001 in both cases). The gene expression level of fgf5
similarly increased at day 7 from 3.34 ± 2.6 to 41.47 ± 20.3, though the change was not statistically significant.
These results were consistent across vesicles derived from separate batches of preosteoblasts (data not shown)
and suggest that the presence of the vesicles resulted in a shift towards the neurectoderm lineage in the
differentiating ESCs.
Identification of cargo contained within particles
In order to probe why the presence of the vesicles in differentiating ESC cultures resulted in gene expression
changes compared to untreated cells, the content of the particles was examined. For initial assessments on the
cargo contained within the preosteoblast-derived particles, total DNA, RNA, and miRNA was isolated from
multiple batches. On average, the particles isolated from a monolayer of preosteoblasts grown to confluence on
a T225 flask contained 7.06 ± 1.6 ng DNA and 1.17 ± 0.3 mg RNA. To further explore the relevance of this
14
content, a miRNA array was utilized to identify which miRNA molecules were most concentrated in the
vesicles compared to the preosteoblast population from which they were derived (Figure 4).
In total, 84 different miRNA genes affiliated with development and differentiation were analyzed in cell and
isolated vesicle populations (n = 3 in both cases). Heat map analysis clustered individual cell and vesicle groups
together, as expected, with the latter demonstrating overall higher expression levels of the miRNAs examined
(indicated in red) (Figure 4A). Cluster analysis suggested that miRNA contained within the cell population was
generally expressed at lower levels (green) than their vesicle counterparts, indicating that cells undergo a
process that concentrates genetic material within the particles. This trend is corroborated by examining specific
Ct values from each group: 44% of the isolated vesicles exhibited miRNA Ct values under 20 (indicating
relatively high gene expression), while 37% fell under a Ct of 20 in the cell group. Log scatter plots of the
vesicle versus cell populations highlight these differences between the groups (Figure 4B). Points falling along
the diagonal line crossing the center of the graph indicate miRNA expression levels that are equivalent between
groups. As corroborated by the heat map cluster analysis, most points (81%; 68/84 genes) fall above this
diagonal (fold change > 1), suggesting a general increasing expression trend in vesicle miRNAs compared to
the cells from which they were derived. The two outer diagonal lines delineate a two-fold expression increase or
decrease in vesicle as compared to cells. Approximately 20% (17/84 genes) of the analyzed miRNA genes in
isolated vesicles exhibited a greater than two-fold increase compared to cells (red dots). In contrast, only 6% of
genes (5/84) showed greater than two-fold decreased expression (green dots). Of the miRNAs exhibiting an
increase in fold regulation in vesicles compared to cells, five displayed a ten-fold increase or greater: miR-1225p (12.1 fold), miR-451a (312.3 fold), miR-183-5p (17.5 fold), miR-144-3p (67.5 fold), and miR-142-5p (14.4
fold). In contrast, only one miRNA (miR-541-5p) was down-regulated more than 10-fold. Figure 4C highlights
these differences, showing the differing percentages of total up- and down-regulated genes. In addition, the
breakdown of up- or down-regulation is displayed, indicating the percentage of genes within those that are upregulated (red bar) that exhibit a 2-10 fold increase versus a greater than 10 fold increase. Similarly, the
percentages of genes within those that are down-regulated (green bar) are differentiated based on either a 2-10
15
fold down-regulation or a greater than 10 fold down-regulation (for a list of which genes fall into these
categories, see Supplementary Table 2).
16
DISCUSSION
Particles naturally shed from a variety of cell types have been previously categorized most commonly as
apoptotic bodies, microvesicles, exosomes, as well others [21]. These “physiological liposomes” have been
shown in several cell systems to contain genetic material originating from the host cell and in many instances to
contain a combination of RNA and miRNA [21]. The extracellular vesicles presented in this study were
harbored within the preosteoblast microenvironment and were isolated using ultracentrifugation techniques.
They were found to be ~170 nm in diameter, and immunogold and FACS analyses indicated the presence of
CD40 in the population, suggesting that at least a portion of the isolated particles may fall into the
“microvesicle” category established by recent literature [21]. Preosteoblast vesicles appeared to impact the
maintenance of pluripotency as well as promoted neurectoderm differentiation in treated ESCs. Both mRNA
and miRNA were found in the preosteoblast vesicles, and the definitive alteration in gene expression in the
recipient ESCs suggests that the vesicles may have responded to this cargo. While the role of mRNA in
influencing gene expression is well understood, the impact of miRNA on stem cell pluripotency differentiation
has only recently been investigated and continues to evolve [38-44].
As outlined previously, approximately 20% (17/84) of the miRNAs examined exhibited at least a two-fold
increase in the vesicles compared to the cells from which they were derived. Previous work in other cell
systems has similarly shown an increase in genetic material in vesicles relative to the cells [15,22,45]. Twelve
of the seventeen up-regulated miRNAs in the preosteoblast system are affiliated with the mesoderm lineage,
which may be explained by its derivation from a mesoderm-derived cell source. Of importance to note is that
miRNAs may play roles in multiple lineages, so those associated with the mesoderm may also be important in
other developmental processes. Five of the miRNAs that were up-regulated at least two-fold (miR-182-5p (2.1
fold), miR-183-5p (17.5), miR-320-3p (2.1), miR-205-5p (2.5), miR-133b-3p (8.8)) have been implicated in
pluripotency, which may explain why ESCs incubated with these vesicles exhibited higher levels of pluripotent
genes. In addition, miR-141-3p, which promotes self-renewal in ESCs,[46] was up-regulated 8.2-fold while all
examined miRNAs in the pro-differentiation let-7 family [41,47] exhibited levels around 1 (0.9 – 1.5), with no
17
significant increase relative to preosteoblast cells. Interestingly, three of the miRNAs up-regulated at least twofold (miR-182-5p, miR-183-5p, miR-205-5p) have also been reported to influence development of the
ectoderm, specifically in relation to neurological and inner ear development, as well as epidermal differentiation
[48]. Given the up-regulation of these miRNAs in the isolated vesicles, it is possible that one or all of them
played a role in the neurectoderm differentiation observed in the ESCs treated with vesicles. In addition to miR183-5p, only two other miRNAs exhibited at least a 15-fold increase in the vesicles: miR-451a (312.3 fold) and
miR-144-3p (67.5 fold), both of which have been implicated in erythropoiesis and megakaryopoiesis [49].
Although research is still relatively young regarding the roles of miRNAs and their corresponding effects in
pluripotent cells, the work presented here has established the importance of understanding what is contained
within naturally shed vesicles since the genetic material enclosed within them may have significant effects on
any recipient cell type. As an example, in other cases where documented protective effects are induced by shed
particles, it is possible that miRNA cargo contained within the particles and transferred to the recipient cell play
critical roles [30,31]. As demonstrated here, one potential application of this transferrable genetic material is as
a means of directing the differentiation of stem cells. It is possible that the stem cell niche in vivo is influenced
by particles shed from cells in that environment, which could direct the fate of localized stem cells. The
technology of a biomaterials construct in which vesicles are incorporated could be one method of delivering
packaged miRNAs to heterogeneous pluripotent populations as a means of controlled modulation at the gene
level. In addition to this potential critical application as a means of controlling differentiation, the transfer of
genetic material between cells has tremendous potential in the development of therapies. It is currently not well
known how cells select the mRNA or miRNA that will be contained within the vesicles, although evidence
suggests a non-random packaging [45], but controlling or understanding that process will be critical for
manipulating vesicle transfer for therapeutic purposes. As an example, consider miR-133b-3p, which was upregulated almost nine-fold in the preosteoblast vesicles. This miRNA belongs to the cluster mmiR-133, which
has been described in multiple reports for their potential in the treatment of heart disease [50,51] and promoting
angiogenesis [52,53]. In addition, miR-133b has been shown to inhibit the growth of non-small-cell lung cancer
18
[54] and invasion of prostate cancer cells [55]. If properly harnessed, it is possible that this miRNA, along with
related molecules, may be able to integrate into critical niches to enhance or provide therapeutic efforts towards
the treatment of specific diseases. It is, however, first critical to ensure that the select group of miRNAs are
consistently up-regulated and transferred into the vesicles. Future work coupling controlled delivery methods
along with vesicle biology may also serve to direct the targeting and uptake of the vesicles to the desired
recipient cell population.
The horizontal transfer of genetic material via extracellular vesicle delivery may be a potent mediator for
directing the differentiation of pluripotent populations. The work presented here identifies particles shed from
preosteoblasts that resemble microvesicles and contain both mRNA and miRNA. The presence of these vesicles
in differentiating ESC cultures significantly influenced stem cell fate, prolonging expression levels of
pluripotent markers while increasing later markers of neurectoderm differentiation. Future work investigating
the control of miRNA packaging as well as targeted delivery of the vesicles may significantly enhance the
potential of this technology in cellular reprogramming and directed differentiation.
19
ACKNOWLEDGMENTS
The authors would like to acknowledge Kharissa Nitiputri for her contributions to the immunogold labeling of
the extracellular vesicles. RN was supported by a Whitaker Foundation International Scholarship, and LS was
supported by a Marie Curie Actions Intra-European Fellowship.
20
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DISCLOSURE STATEMENT
No competing financial interests exist.
26
FIGURE LEGENDS
Figure 1. Vesicle characterization. Vesicles isolated from preosteoblast cultures stained positive for CD40
using immunogold transmission electron microscopy (immunoTEM; top left). Nanoparticle tracking analysis
indicated that particles were on average 174.2 ± 22 nm (top right). Flow cytometric analysis identified a CD40positive population (bottom panel), confirming immunoTEM indications of the surface marker.
Figure 2. Cell-vesicle interactions. Extracellular vesicles were stained with CellMask™ Orange and visualized
on clusters of actin (phalloidin/green)- and nuclei (dapi/blue)-stained embryonic stem cells after 6 (top left) and
24 (top right) hours. The presence of vesicles in ESC culture did not influence viability, as indicated by
alamarBlue® (bottom left) and LIVE/DEAD® (bottom right) assays. Scale bars in LIVE/DEAD® images are
200 m and are both from day 6.
Figure 3. ESC gene expression changes. The influence of extracellular vesicles on differentiation ESCs was
monitored using RT-PCR. Pluripotent genes oct4 and nanog as well as neurectoderm differentiation markers
nestin and sox-1 were significantly increased in ESCs treated with vesicles.
Figure 4. Vesicle cargo characterization. MicroRNA packaged within the preosteoblast-derived vesicles were
analyzed using gene expression arrays for mouse cell differentiation and development. (A) Heat map and (B)
scatterplot results indicate the increase in specific miRNAs in vesicles compared to the preosteoblast cells from
which they were derived. (C) The bar graph displays the breakdown of differential up- and down-regulation of
all miRNAs examined.
27
Figure 1
Vesicle characterization. Vesicles isolated from preosteoblast cultures stained positive for CD40 using
immunogold transmission electron microscopy (immunoTEM; top left). Nanoparticle tracking analysis
indicated that particles were on average 174.2±22 nm (top right). Flow cytometric analysis identified a CD40positive population (bottom panel), confirming immunoTEM indications of the surface marker.
28
Figure 2
Cell-vesicle interactions. Extracellular vesicles were stained with CellMask™ Orange and visualized on clusters
of actin (phalloidin/green)- and nuclei (DAPI/blue)-stained embryonic stem cells (ESCS) after 6 h (top left) and
24 h (top right). The presence of vesicles in ESC culture did not influence viability, as indicated by
alamarBlue® (bottom left) and LIVE/DEAD® (bottom right) assays. Scale bars in LIVE/DEAD images are
200 μm and are both from day 6. Live cells are indicated by green fluorescence while dead cells (insets) are
indicated by red.
29
Figure 3
ESC gene expression changes. The influence of extracellular vesicles on differentiation ESCs was monitored
using real-time–polymerase chain reaction. Pluripotent genes oct4 and nanog as well as neurectoderm
differentiation markers nestin and sox-1 were significantly increased in ESCs treated with vesicles.
30
Figure 4
31
32
Vesicle cargo characterization. microRNA (miRNA) packaged within the preosteoblast-derived vesicles were
analyzed using gene expression arrays for mouse cell differentiation and development. (A) Heat map and (B)
scatterplot results indicate the increase in specific miRNAs in vesicles compared with the preosteoblast cells
from which they were derived. (C) The bar graph displays the breakdown of differential up- and downregulation of all miRNAs examined.
33
34