Merakris Therapeutics' in vitro study on amniotic fluid exosomes and their potential in cutaneous wound healing - presented here is a novel multi-phase wound healing strategy that alters skin cell phenotypes using amniotic exosomes to support early phase healing, and then treating wounds with exosome-free biological material to promote late phase wound closure

1. Amniotic Fluid Exosomes Promote Wound Healing by Activating Epithelial to Mesenchymal Transition
W. Samuel Fagg, MS, PhD1,2; Naiyou Liu, PhD2; Jeffrey H. Fair, MD2; Mohammadali M. Shoja, MD2; T. Christopher Broderick1
1Merakris Therapeutics, LLC Research Triangle Park, NC; 2University of Texas Medical Branch, Transplant Division, Department of Surgery, Galveston, TX
§ Dermacyte® Amniotic Would Care Liquid (Dermacyte Liquid) is a sterile filtered amniotic fluid (AF)
allograft available in various volume aliquots and intended for subcutaneous injection to support
healing in chronic and acute wounds.
§ AF and extracellular vesicle (EV)/exosome-based therapies have gained significant attention for a
variety of potential regenerative medicine applications including, but not limited to, tissue repair and
immunomodulation by subcutaneous, intra-articular, and intravenous administration.
§ The molecular and cellular mechanisms of action through which beneficial effects of amniotic fluid
on target cell types are mediated are not fully understood.
§ Here, we test the hypothesis that amniotic fluid exosomes are required to promote wound healing
using in vitro scratch test assays with fibroblasts and myoblasts. Furthermore, we test the levels of
biomarkers for pathways relevant in wound healing to determine if different AF fractions induce
differential effects in target cell types active in the process.
INTRODUCTION
§ Amniotic fluid exosomes are necessary for rapid cell migration in fibroblasts, and are
necessary and sufficient for rapid myoblast migration.
§ Exosome-depleted amniotic fluid significantly inhibits migration of both fibroblasts and
myoblasts. This effect is concomitant with a conversion of normally mesenchymal-like
morphology of myoblasts into a more cobblestone-like, epithelioid morphology.
§ Activation of migration is mediated, at least in part, by promoting EMT in myoblasts
exposed to purified AF exosomes. Migration may be inhibited by exosome-depleted AF by
promoting MET and repressing EMT.
§ AF exosomes may contribute to activation of myoblasts and/or conversion to a
myofibroblast-like phenotype; a desirable ”early” effect in the wound healing process, but
potentially detrimental during late processes. Exosome-depleted AF has the opposite effect.
§ Exosome-depleted AF increases Stat3 expression, which may indicate active TGF-beta
signaling, or could be indicative or NFkb activation, and is under more investigation.
§ Taken together, these findings support the notion that purified AF exosomes and exosome-
depleted AF have different and sometimes opposing effects on target cells.
§ These different effects may be utilized to maximize efficacy by employing multi-phase wound
healing treatment approaches whereby AF exosomes activate early events required for
wound healing (i. e., EMT, myofibroblast activation, etc.), then total AF is used for intermediate
treatment to reduce cell activation, and finally exosome-depleted AF is administered to
promote re-epithelialization and keratinization.
CONCLUSIONS
RESULTS
Presented at 5th Annual Perinatal Stem Cell Society Congress | February 28- March 1, 2019 | Salt Lake City, UT | #1908
Disclosure: This study was sponsored by Merakris Therapeutics, LLC
§ Dermacyte Liquid was evaluated to quantitate exosomes from independent lots (N=3 per test)
using ZetaSizer PMX-120 (Malvern Instruments).
§ Exosomes were isolated from AF using using the ExoQuick TC Ultra kit (SBI) to produce an
exosome (EV+) fraction and exosome-depleted (EV-) fraction, and purity was confirmed by
western blot and ZetaSizer analysis.
§ Both the EV+ and EV- fractions were added to serum-free media in the presence of myoblasts or
fibroblasts, two cell types actively involved in wound healing in humans. Scratch test assays were
performed to determine migration capacity of cells in the presence of total AF, EV+, or EV- AF.
§ RNA was extracted from cells 24h after incubation with each respective total AF or AF-derived
fraction to to measure the abundance of relevant biomarkers for migration, inflammation, and cell
signaling by RT-qPCR.
METHODS
Figure 3. How do AF exosomes contribute to myoblast migration?
Figure 2. How do AF exosomes contribute to fibroblast migration?
Figure 1: AF Exosome Measurement and Purification
CD63
Albumin
CD9
antibody:
sample: amniotic fluid
total
exoCrude
exoPure
exo(-)AF
MW (kDa)
68
68
24
FIG xx
A B
C D
Figure 1: measurement of exosome quantity and size, then purification and validation of AF exosome purity. A. Histogram from ZetaView report of one representative donated
sample of Dermacyte Liquid from Merakris Therapeutics showing size distribution (nm, X-axis) and count (Y-axis) of extracellular vesicles (mean value of extracellular vesicles in
Dermacyte is ~ 5e10/ml). B. Histogram from ZetaView report of one representative donated sample of Dermacyte Liquid showing size distribution (nm, X-axis) and vesicle volume
(mm3 e-6, Y-axis). C. Freeze-frame view of extracellular vesicles from ZetaView report of the same representative donated sample of Dermacyte Liquid shown in A & B. D. Western
blots from AF measuring CD63 (exosome marker), albumin (abundant protein in AF absent from exosomes), and CD9 (exosome marker) in total AF, crude purification of exosomes
(exoCrude), purified exosome eluate (exoPure), and exosome-depleted AF (exo(-)AF); MW in kDa shown on right.
time (h)
relativepercentarea
Fibroblast scratch test assay: AF +/- exosomes
uncSFM+AF
uncSFM+AFexosomes
uncSFM+exo-AF
0 6 12 18 24
0.0
12.5
25.0
50
60
70
80
90
100
110
uncSFM+AFTime (h)
0
12
18
24
FIG xx
A
B
uncSFM+
AFexos
uncSFM+
exo(-)AF
**
* *
uncSFM+AFTime (h)
0
12
18
24
FIG xx
A
B
uncSFM+
AFexos
uncSFM+
exo(-)AF
0 4 8 12 16 20 24
0
25
50
75
100
time (h)
Myoblast scratch test assay: AF +/- exosomes
uncSFM+AF
uncSFM+AFexos
uncSFM+exo(-)AF
relativepercentarea
**
***
*** **** ****
• AF exosomes are sufficient to
promote early/rapid fibroblast
migration
• AF exosomes are necessary to
activate fibroblast migration over
the 24h timeframe
• AF exosomes are necessary and
sufficient to promote myoblast
migration.
• Exosome-depleted AF potently
inhibits myoblast migration and
induces a cobblestone-like cell
morphology.
SFM
+A
F
SFM
+A
Fexo
SFM
+exo(-)A
F
0
50
100
150
200
media
ddCt
N-Cadherin/E-Cadherin mRNA abundance
*P < 0.05, **P < 0.01; by student’s t-test
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; by student’s t-test
SFM
+A
F
SFM
+A
Fexo
SFM
+exo(-)A
F
0
1
2
3
4
media
ddCt
Stat3 mRNA abundance
**
***
SFM
+A
F
SFM
+A
Fexo
SFM
+exo(-)A
F
0
50
100
150
200
media
ddCt
Vimentin mRNA abundance
*ns
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant (P > 0.05); by one-way ANOVA
Figure 4. Do exosome-enriched or -depleted AF fractions modulate
migration- and signaling-associated gene expression signatures?
Figure 3: Brightfield microscopy (20X objective) showing
representative images of C2C12 myoblasts during scratch test
wound healing assay at time (hours) 0, 12, 18, and 24
incubated with unconditioned serum-free media + 10%
amniotic fluid (uncSFM+AF), uncSFM with an equal amount of
exosomes derived from amniotic fluid as that in uncSFM+10%
AF (uncSFM+AFexos), or uncSFM plus 10% exosome-
depleted amniotic fluid (uncSFM+exo(-)AF). Dotted lines
outline area not occupied by cells; scale bar denotes 50 µm.
Quantitation of scratch area (in percent area relative to total
scratch at time zero) in conditions described in A. Area was
calculated using ImageJ software and six independent
replicates for each condition and time point were measured.
Each data point shows the mean area percent relative to that
at time zero, +/- standard deviation (*P < 0.05, **P < 0.01, ***P
< 0.001, ****P < 0.0001 by student’s t-test, relative to
uncSFM+AF or uncSFM+AFexos, by student’s t-test).
Figure 4: Following 24h incubation in respective media type for scratch test assays, RNA was extracted from C2C12 myoblasts, reverse transcribed, then mRNA
abundances were measured by qPCR to determine level of each biomarker mRNA relative to hydroxymethylbilane synthase (housekeeping gene). Top left panel
shows the relative abundance of ratio of N-Cadherin (N-Cad) and E-Cadherin (E-Cad; N-Cad/E-Cad), top right panel shows the relative abundance of vimentin,
bottom left shows relative abundance of smooth-muscle actin (Acta2), bottom right shows relative abundance of of Signal transduction and activator of transcription
3 (Stat3). Ordinary one-way ANOVA was used to measure statistically significant differences, with ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P <
0.0001 denoting results.
Figure 2: A. Brightfield microscopy (20X objective) showing
representative images of C2C12 myoblasts during scratch test
wound healing assay at time (hours) 0, 12, 18, and 24
incubated with unconditioned serum-free media + 10%
amniotic fluid (uncSFM+AF), uncSFM with an equal amount of
exosomes derived from amniotic fluid as that in uncSFM+10%
AF (uncSFM+AFexos), or uncSFM plus 10% exosome-
depleted amniotic fluid (uncSFM+exo(-)AF). Dotted lines
outline area not occupied by cells; scale bar denotes 50 µm.
B. Quantitation of scratch area (in percent area relative to total
scratch at time zero) in conditions described in A. Area was
calculated using ImageJ software and six independent
replicates for each condition and time point were measured.
Each data point shows the mean area percent relative to that
at time zero, +/- standard deviation (*P < 0.05 or **P < 0.01 by
student’s t-test, relative to uncSFM+ 10% exosome-depleted
AF, by student’s t-test).
Merakris Therapeutics, LLC | 800 Park Offices Drive | Suite 3305 | Research Triangle Park NC 27709 | (919) 921-8105 | info@merakris.com | www.merakris.com
0 6 12 18 24
0.0
12.5
25.0
50
60
70
80
90
100
110
time (h)
relativepercentarea
Fibroblast scratch test assay: AF +/- exosomes
uncSFM+AF
uncSFM+AFexosomes
uncSFM+exo-AF
0 4 8 12 16 20 24
0
25
50
75
100
time (h)
Myoblast scratch test assay: AF +/- exosomes
uncSFM+AF
uncSFM+AFexos
uncSFM+exo(-)AF
relativepercentarea
B
B
**
* *
** *** *** **** ****
• AF exosomes
promote EMT;
exosome-depleted
AF inhibits
EMT/promotes
MET
• AF exosomes
promote, and
exosome-depleted AF
inhibits smooth-
muscle actin (Acta2)
expression, a marker
of myofibroblast
activation
• Exosome-depleted
AF promotes Stat3
expression
SFM
+A
F
SFM
+A
Fexo
SFM
+exo(-)A
F
0
100
200
300
media
ddCt
Acta2 mRNA abundance
*** ****
*