Wharton’s Jelly Mesenchymal Stem Cells-secreted IDO as Candidate

of Anti-inflammation Therapy

Wahyu Widowati

1,* a

, Teresa Liliana Wargasetia

, Fanny Rahardja

Rimonta F. Gunanegara

, Hanna Sari Widya Kusuma

, Seila Arumwardana

Cintani Dewi Wahyuni

, Cahyaning Riski Wijayanti

, Tri Handayani

and Rizal Rizal

2,3 j

Faculty of Medicine, Maranatha Christian University, Jl. Surya Sumantri No. 65, Bandung 40164, West Java, Indonesia

Biomolecular and Biomedical Research Center, Aretha Medika Utama, Jl Babakan Jeruk II No. 9, Bandung 40163,

West Java, Indonesia

Biomedical Engineering, Department of Electrical Engineering, Faculty of Engineering, Universitas Indonesia,

Depok 16426, West Java, Indonesia

hannasariw@amubbrc.co.id, seila.wardana91@gmail.com, cintanidewi@gmail.com, cahyaningwidodo@gmail.com,

mbaktrihandayani@gmail.com, rizal_biotek@yahoo.com

Keywords: Covid-19, Cytokine Storm, Indoleamine 2,3 Dioxygenase, Secretome, Wharton’s Jelly Mesenchymal Stem

Cells.

Abstract: Local inflammation in lung can induce by viral pneumonia which causes acute respiratory distress

syndrome (ARDS). ARDS also caused by COVID-19 SARS-COV-2 infection. hWJMSCs will release anti-

inflammatory signals such as indoleamine 2,3 dioxygenase (IDO) as tissue homeostasis between MSCs and

resident macrophages as anti-inflammatory signals. This led to the idea of investigating potential of

hWJMSCs-Secreted IDO as candidate of anti-inflammation therapy. The hWJMSCs have been isolated

from the human umbilical cord using an explant method and characterized using a flow cytometer to detect

the cell surface markers CD105, CD73, CD44, CD90, and negative lineage expression of hWJMSCs. The

hWJMSCs secretome was characterized by measuring the level of indoleamine 2,3-dioxygenase (IDO) in

various starving cells. The isolated umbilical cord hWJMSCs showed positive expression of CD105, CD73,

CD44, CD90, and negative lineage expression from 5, 10, and 15 passage. The hWJMSCs IDO secretion

level was 5.86 ng/mL for non-starving cells, 6.84 ng/mL for 24 h starving cells, 9.59 ng/mL for 48 h

starving cells, and 13.32 ng/mL for 72 h starving cells. The early, medium, and old passage of hWJMSCs

have the same characteristics. Longer starvation periods up to regulate the IDO level in hWJMSCs

secretome which indicate as anti-inflammation therapy.

https://orcid.org/0000-0002-5401-7794

https://orcid.org/0000-0002-9990-4741

https://orcid.org/0000-0003-2982-8437

https://orcid.org/0000-0003-3053-1120

https://orcid.org/0000-0002-7422-0036

https://orcid.org/0000-0003-0422-7379

https://orcid.org/0000-0002-7764-0482

https://orcid.org/0000-0002-3397-099X

https://orcid.org/0000-0001-9186-9841

https://orcid.org/0000-0003-2783-0672

Corresponding author

INTRODUCTION

Inflammation is a critical biological reaction to

damage that is linked to a variety of disorder such as

acute respiratory distress syndrome (ARDS). ARDS

is caused by viral pneumonia as the virus infects the

respiratory tract, it induces local inflammation, which

results in the production of pro- inflammatory

Widowati, W., Wargasetia, T., Rahardja, F., Gunanegara, R., Kusuma, H., Arumwardana, S., Wahyuni, C., Wijayanti, C., Handayani, T. and Rizal, R.

Wharton’s Jelly Mesenchymal Stem Cells-secreted IDO as Candidate of Anti-inﬂammation Therapy.

DOI: 10.5220/0010749700003113

In Proceedings of the 1st International Conference on Emerging Issues in Technology, Engineering and Science (ICE-TES 2021), pages 271-278

ISBN: 978-989-758-601-9

271

cytokines and chemokines. T cells and monocytes

from the blood are drawn to the infectious site as a

result (Tay et al., 2020; Canham et al., 2020).

Excessive release of proteases and reactive oxygen

species (ROS) is caused by the uncontrolled invasion

of inflammatory cells into the lungs (Abraham &

Krasnodembskaya; 2020). Cytokine Storm Syndrome

(CSS) or Cytokine Release Syndrome (CRS) is a

condition in which the body produces excessive

amounts of cytokines (Ye et al., 2020; Azmi et al.,

2020). SARS-CoV-2 binds to the angiotensin-

converting enzyme 2 (ACE2) receptor on the surface

of human cells for cell entry (Azmi et al., 2020),

Increased serum cytokines such as Interleukin-1 (IL-

1), IL-6, and tumor necrosis factor (TNF)-, peripheral

lymphopenia, elevated ferritin level, lactate

dehydrogenase (LDH), d-dimer, C-reactive protein

(CRP), and coagulation factors are all part of the

host's immune response to COVID-19 (Zhou et al.,

2020; Chen et al., 2019; Acosta, 2020). Infection

with SARS-CoV-2 causes an increase in IL-1

Receptor Antagonist (IL- 1RA), IL-2, IL-6, IL-7, IL-

9, IL-10, Interferon Gamma (IFN), Interferon

Gamma Inducible Protein-10 (IP-10), Monocyte

Chemoattractant Protein-1 (MCP1), Granulocyte-

Colony Stimulating Factor (GCSF), Basic Fibroblast

Growth Factor (FGF), Platelet-Derived Growth

Factor (PDGF), and Inflammatory Protein 1- Alpha

(MIP1-α), MIP-1β (Durand et al., 2020, Huang et al

2020; Canham et al., 2020). There are also high

levels of chemokines (CCL2, CCL3, CCL5, CXCL8,

CXCL9 and CXCL10) (Huang et al., 2019; Williams

& Chambers, 2014; Cetin & Topcul; 2020), edema in

the alveoli, reduced efficiency of gas exchange,

ARDS, and acute cardiac injury (ACI), which can

result in hypoxemia, secondary infection (Huang et al

2020; Canham et al., 2020), and death (Huang et al.,

2020).ARDS caused by COVID-19 is due to

respiratory failure (53%), respiratory failure coupled

with cardiac failure (33%), myocardial damage and

circulatory impairment (7%), or death by unknown

cause (Gibson et al., 2020). CSS refers to a group of

disorders that result in a violent immune system

assault on the host body, such as systemic

inflammation, multiorgan failure, and

hyperferritinemia, and, if left untreated, death.

(Behrens EM, Koretzky, 2017; Cetin & Topcul;

2020).

Because of the COVID-19's serious respiratory

effects as a result of CSS, infection prevention,

surveillance, and supportive care, such as

supplementary oxygen and mechanical ventilation,

are now needed in the clinical management of

critically ill patients (Baruah & Bose, 2020; Golchin

et al., 2020). Since there is currently no specific cure

for COVID-19 it is critical to develop new treatment

approaches that are more innovative, safe, and

promising in the treatment of ARDS. Cell-based

therapy, especially stem cell therapy, is currently

viewed as a promising therapy for curing incurable

diseases (Golchin & Farahany, 2019; Golchin et al.,

2020). Adult stem cells derived from mesenchymal

stem cells (MSCs) is a promising source for cell

therapy and tissue engineering (Widowati et al.,

2015). MSCs are a more superior care than the

others, and they've gotten a lot of coverage because

of: i). source potential, easily accessible, and can be

isolated from a variety of tissues including bone

marrow (BM), adipose tissues (AT) (such as

infrapatellar fat pad, abdominal fat, and buccal fat

pad), neonatal birth-associated tissues such as

placenta (PL), amniotic fluid (AF), Wharton jelly

(WJ), umbilical cord (UC), and cord blood (CB),

dental pulp, menstrual blood, buccal fat pad, fetal

liver; ii). high proliferation rate; iii). multipotent

stem cells with high proliferation rate; iv). simple

culture and harvesting procedures; v). easy ex vivo

expansion to clinical volume; vi). can be processed

for repeated therapeutic use; vii). trophic paracrine

secretion, producing a large amount of therapeutic

growth factor and cytokines; and viii). autologous

and allogenic clinical therapy (Golchin et al., 2018;

Golchin et al., 2020). MSCs have low major

histocompatibility complex (MHC) type 1

expression and no MHC type 2 expression, making

them non-immunogenic and suitable for allogeneic

therapy (Berglund et al., 2017; Canham et al., 2020).

MSCs have ability to restore the balancing

immunological response at inflammation sites and in

the surrounding environment by communicating with

different immune system components. MSCs have

the ability to interact with adaptive immune and

innate immune systems by sensing the inflammatory

state and detecting the presence of microbes through

stimulation of Toll-like receptors (TLRs) on their

surface. In the presence of an inflammatory

microenvironment, such as high levels of Interferon-γ

(INF-γ) and Tumor Necrosis Factor-α (TNF-α) or

TLR3 receptor stimulation by viral RNA, MSCs will

release anti-inflammatory signals such as

indoleamine 2,3 dioxygenase (IDO), Tumor Growth

Factor- β (TGF-β), and prostaglandin E-2 (PGE-2),

as tissue homeostasis between MSCs and resident

macrophages as anti-inflammatory signals that cause

the emergence of both regulatory T and dendritic

cells (Bernardo & Fibbe, 2013; Glenn & Whartenby,

2014; Canham et al., 2020). When MSCs interact

directly with immune cells, they perform paracrine

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

272

modulation as a result of the immune response by

releasing cytokines such as IDO, IL-10, TGF- β, and

IL-1 receptor antagonist (IL- 1RA), and nitric oxide

(NO) (van Buul et al., 2012). Several studies have

shown that tryptophan catabolism occurs

predominantly at areas of tissue inflammation and

that IDO expression might well be involved in

inhibiting the inflammatory reaction and therefore

decreasing tissue injury (Wolf et al, 2004; Nikolaus,

et al., 2017). IDO is a rate-limiting enzyme of

tryptophan catabolism along the kynurenine (Kyn)

pathway. The immunosuppressive mechanism of

IDO is mediated by depletion of tryptophan,

accumulation of kynurenines (Lee et al., 2016).

Several soluble factors either produced

constitutively by MSCs or as a result of cross-talk

with target immune cells have been attributed to

immunomodulatory property of MSCs, including

PGE2, IDO, NO, IL-10, and hepatocyte growth

factor (HGF) (Meesuk et al., 2016). MSCs exhibit as

anti- inﬂammatory several factors, including IDO

and TNF- stimulated gene 6 (TSG-6). IDO controls

the TSG-6 mediated anti-inﬂammatory therapeutic

potent of MSCs (Wang et al., 2018). TSG-6-

knockdown (TSG- 6-KD) MSCs have less

therapeutic effect on lipopolysaccharide (LPS)-

induced Acute Lung Injury (ALI) model mice

compared to MSCs control. This data demonstrates

that IDO expression by MSCs has capability to

alleviates ALI by regulating the TSG-6. Expression

(Wang et al., 2018).

The microenvironment of cells, such as food

deprivation and low oxygen stress, has an effect on

their characteristics (Ferro et al., 2019). Many

studies show that starving MSCs in vitro to mimic in

vivo post- transplantation improves MSC survival

and therapeutic efficacy (Ferro et al., 2019). Human

MSCs are protected from a rapid transition from in

vitro culture to a harsh environment in vivo by using

fetal bovine serum (FBS) and glucose deprivation

before transplantation (Moya et al., 2015; Ferro et

al., 2019). COVID-19 therapy using stem cells,

especially WJMSCs, has several advantages,

including a high capability for regeneration and

differentiation, as well as the ability to rapidly

expand (Garzon et al., 2020). WJMSCs are a non-

controversial stem cell source (Yang et al., 2012;

Bongso et al., 2013). WJMSCs are more useful and

straightforward in terms of donor entry, expansion,

proliferative ability, and banking; they can also be

used in clinical and experimental therapy (Tamura et

al., 2011). Between the amniotic epithelium and the

umbilical vessels is the WJ, which is embryonic

mucous connective tissue. Adult tissue- derived

MSCs have a lower proliferation rate and self-

renewal capability than WJ derived MSCs or

WJMSCs (Marino et al., 2019; Widowati et al.,

2014). This study looked at the immunophenotyping

of human WJMSCs (hWJMSCs) at different

passages, including positive CD105, CD73, CD44,

CD90, and negative lineage expression, as well as

the IDO secretion of hWJMSCs.

MATERIAL AND METHOD

2.1 hWJMSCs Isolation

Human umbilical cords (UC) were collected from

normal delivery women aged 25 to 40 who signed an

informed consent document that was accepted by the

Institutional Ethics Committee of Maranatha

Christian University, Bandung, Indonesia, and

Immanuel Hospital Bandung, Bandung, Indonesia

(Widowati et al., 2017). Phosphate Buffer Saline

(PBS) (Biowest, X0515500) was used to wash UC's

blood, which was then supplemented with antibiotics

and transported to the laboratory using transport

medium (Widowati et al., 2019a; Widowati et al.,

2019b).

The vessel was extracted from the UC after being

transferred and washed in PBS (1x). Wharton jelly

tissue explants were dissected into 1-2 mm

pieces

and plated on 6 well plates in Minimum Essential

Medium (MEM- α) (Biowest, L0475-500)

supplemented with 10% fetal bovine serum (FBS)

(Biowest, S1810-500), 1% ABAM, 0.1%

Gentamicin (Gibco, 15750060), 1% Amphotericin B

(Amp B) (Biowest, L0009-100), and 1%

Nanomycopulitin (Biowest, LX16-100). After 3

weeks of incubation at 37

C in a humidified

atmosphere with 5% CO2, adherent cells and tissue

fragments were detached using Trypsin-EDTA

solution (Biowest, L0931-500) and washed with

basal medium. The cells were harvested and re-

plated at a density 8 x 10

cells/cm

when cells

reached 80-90% confluence (Widowati et al., 2014;

Widowati et al., 2017; Widowati et al., 2019a;

Widowati et al., 2019b).

2.2 Markers Detection of hWJMSCs

using Fluorescence Activated Cell

Sorting

The hWJMSCs surface marker was observed in

Passage 5 (P5), 10, and 15 cell cultures. The cells

culture that had reached 80-90% confluence were

Wharton’s Jelly Mesenchymal Stem Cells-secreted IDO as Candidate of Anti-inﬂammation Therapy

273

harvested and analyzed for surface marker using

flow cytometry (MACSQuant Analyzer 10, Miltenyi

Biotec). The cells were stained with specific

antibodies (CD90 FITC, CD73 APC, CD105 PerCP-

Cy5, CD44 PE, negative lineage:

CD34/CD45/CD11b/CD19/HLA-DR PE) according

to manufacturer’s protocol (BD stem flow

kit,

562245). The surface marker of hWJMSCs were

conducted in triplicate for each passage (Widowati

et al., 2014; Widowati et al., 2019a; Widowati et al.,

2019b).

2.3 Preparation of Conditioned

Medium From hWJMSCs

The medium was collected and centrifuged at 3000

rpm for 4 minutes at room temperature, and the

supernatant was ﬁltered by a 0.22-mm MillexeGV

Filter Unit with Durapore (Millipore Corporation,

SLGV 033 RS) and used as hWJMSCs secretome

(Widowati et al., 2015).

2.4 IDO level of hWJMSCs Secretome

The hWJMSCs P5 cell culture was used for

experiments. The cells were seeded 8x10

cells/cm

in complete medium. After the cells reached 80-90%

confluence, the cells were grown in starving medium

(MEM-α no- phenol red, 1% ABAM, 0.1%

Gentamicin, 1% Amphotericin B and 1%

Nanomycopulitin) for 24, 48 and 72 hours.

The level of IDO in the cell-free supernatant of

hWJMSCs was measured using human IDO

(Indoleamine-2,3- Dioxygenase) ELISA Kit

(Elabsci, E-EL-H2162). Regarding the manual 50

µL of stop solution was applied to each well, and the

absorbance was read at 450 nm microplate reader

(Multiskan Go, Thermos Fisher Scientific)

(Widowati et al., 2017).

RESULTS AND DISCUSSION

3.1 The Effect of Different Passages on

hWJMSC Markers

The result of evaluating the effect of different

passages (P5, P10, P15) on hWJMSC surface

markers. The hWJMSCs were positive for CD90,

CD44, CD105, CD73 and negative for CD11b,

CD19, CD34, CD45, and HLA-II. The effect of

different passages on the surface marker of

hWJMSCs are given in Table 1. Positive and

Table 1: Effects of different passages on the percentage of

hWJMSCs with positive and negative surface marker

lineages.

Passage

CD44

(%)

CD73

(%)

CD90

(%)

CD10 5

(%)

negative-

lineage

(%)

P5 99.44±0.01 96.97±0.24 99.44±0.03 99.56±0.03 0.23±0.02

P10 98.77±0.26 97.68±0.09 98.79±0.13 98.86±0.27 0.71±0.05

P15 99.75±0.06 99.36±0.20 99.71±0.03 99.78±0.05 0.43±0.04

Data are expressed as mean ± standard deviation from 3

replications; CD90, CD44, CD105, CD73 are positive

lineage- markers; CD14, CD19, CD34, CD45, HLA-II are

negative lineage- markers.

negative surface marker expression of hWJMSCs

P5, P10, and P15 were not significantly different

(p>0.05). MSCs should have CD44, CD73, CD90,

and CD105 positive lineage markers and CD11b,

CD19, CD34, CD45, and HLA-DR negative lineage

markers (Figure 1). Propyl etidium (PE), fluorescein

isothiocyanate (FITC), and peridinin chlorophyll

protein-5 (PerCP-Cy5) staining were used to detect

the surface markers of hWJMMSCs. The MSC

characteristics were visible in different passages,

including early (P5), medium (P10), and old passage

(P15) (Table 1).

This finding is also in line with another previous

study that human adipose tissue-derived MSCs

(hATMSCs) exhibited positive lineage markers

(CD44, CD73, CD90, CD105) and negative lineage

markers (CD11b, CD19, CD34, CD45, HLA-DR)

from passage 4 to 15 (Widowati et al., 2014;

Widowati et al., 2019a; Widowati et al., 2019b). The

passaging from P4-P15 affects the cells proliferation

but not affects the cells morphology and cells

characteristic (Widowati et al., 2019b). Our previous

research that passage 3 and passage 8 of hWJMSCs

isolated by explant and enzymatic method exhibited

un-significantly differences between P3 and P8,

between explant and enzymatic isolation (Widowati

et al., 2019a). Our previous research exhibited that

passage P4 and P8 of hWJMSCs cultured in

normoxic and hypoxic condition showed hWJMSCs

un-significantly differences between P4 and P8,

between normoxic and hypoxic condition (Widowati

et al., 2014). The flow cytometric analysis showed

that oxygen level, isolation method and passage did

not affect the MSC’s character. The hWJMSCs from

P5, P10, P15 showed a very little expression (0.23-

0.71%) of negative lineage (CD11b, CD19, CD34,

CD45, HLA-DR).

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

274

3.2 Effect Starvation on hWJMSCs-

secretome IDO Levels

The secretome (conditioned medium) and P5 of

hWJMSCs that had been starved (fee FBS) for 24,

48, and 72 hours were harvested, and the IDO levels

of the hWJMSCs-secretome was calculated. The

IDO level was measured using enzyme-linked

immunosorbent assay (ELISA) kit assay. The IDO

levels of hWJMSCs- secretome are shown Figure 1.

The results indicate that hWJMSCs secrete IDO at

concentrations ranging from 5.86 ng/mL to 13.12

ng/mL or 39.39 ng/mg protein to 82.05 ng/mg (Figure

1). This data was supported with previous study that

MSCs release TGF-β, IL-10, IL-1RA, NO, IDO (van

Buul et al., 2012). MSCs change inflammation from

releasing pro- inflammatory cytokines including IL-1,

IL-6, IL-12, IL- 17, MCP-1, MIP-2, CXCL-1, CXCL-

2, TNF-α, IFN-γ, proteases like MMP-2, MMP-9 and

MMP-12 to an anti- inflammatory status with

releasing anti-inflammatory TGF-β, CCL18, IL-4, IL-

10, PGE2, IDO, NO, inﬂammation resolving lipoxin

A4 (LXA4) which enable reduce inﬂammation and

improve tissue repair (Zheng et al., 2015; Mao et al.,

2015). MSCs control excessive inﬂammation,

improve the microenvironment for tissue repairing

LPS-induced ALI model mice but MSCs of IDO

knockdown (IDO- KD) didn’t increase the

inflammation compared to control group, indicating

that IDO is important in mediating the inflammation

therapeutic role of MSCs (Wang et al., 2018).

IFN-γ, IL-12, and IL-18 are powerful inducers of

IDO expression. However, IDO acts as a negative

feedback loop that can inhibit pro-inflammatory

activation (IFN-γ, IL-12, and IL-18). Thus Wolf et al

(2004) hypothesized that IDO has an anti-

inflammatory role characterized by Th1

overexpression (Wolf et al., 2004; Nikolaus et al.,

2017). This study was supported by previous

research, it has shown that indoleamine 2,3-

dioxygenase (IDO) plays a critical role in the

immunomodulatory ability of human MSCs. This

enzyme catalyzes the first and rate-limiting step of

tryptophan catabolism along the kynurenine

pathway, and IDO and several of its downstream

Figure 1: Dot plot of immunophenotype representative hWJ-MSCs from P5, P10, P15.

Wharton’s Jelly Mesenchymal Stem Cells-secreted IDO as Candidate of Anti-inﬂammation Therapy

275

Figure 2: Effect starving time on hWJMSCs IDO levels (A) IDO level (ng/mL); (B) IDO level (ng/mg protein). The data

was presented as mean + standard deviation. Different letters (a,b) show a significant different between different starving

time for IDO level in ng/mL (Figure A). Different letters (a,b,c) show a significant different between different starving time

for IDO level in ng/mg protein (Figure B) based on Dunnett T3 post hoc test (p<0.05).

metabolites, such as kynurenine (KYN) and 3-

hydroxyanthranilic acid, not only inhibit effector T-

cell proliferation but also induce regulatory T-cell

differentiation (Treg). Notably, IDO has been found

to influence inflammation-associated gene

expression, either directly as a signaling factor or

indirectly through the production of bioactive

intermediates such as kynurenic acid via the

kynurenine pathway. MSC has a metabolite of IDO

that controls the TSG-6-mediated anti-inflammatory

therapeutic effects (Wang et al., 2018). The other

study reported that MSCs can improve inflammation

and repair tissue from chronic inflammation

(Rubtsov et al., 2017).

Figure 1 shows that longer deprivation increased

IDO levels, with the longest deprivation resulting in

the highest level of IDO. This data was confirmed

by a previous study, which found that deprivation

did not cause an obvious apoptotic response in

immortalized human MSC (ihMSCs) until~120 h of

deprivation (Nuschke et al., 2016). In response to

starvation stress, cells cause adaptive responses such

as angiogenesis, which promote tissue

reorganization and repair, as well as up-regulation of

multiple cytokines and chemokines, including IL-6

and IL-8 (Püschel et al., 2020).

Starvation for 3 days (see Figure 2) on umbilical

cord MSCs (UCMSCs) increase L-Kynurenine

(correlated IDO activity) 6 µM compared to untreated

UCMSCs 2 µM. IFN-γ, IFN-β, TGF- β increase IDO

activity 27 µM, 10 µM, 3 µM (de Witte et al., 2017).

IDO level increase in human adipose stem cells

(hASCs) in the presence of activated peripheral blood

mononuclear cells (PBMCs) (Rubtsov et al., 2017).

The amnion-derived MSCs (AM- MSCs) and BM-

MSCs induced by Phytohemagglutinin (PHA) and

IFN-γ exhibit that the IDO gene expression increase

compared negative control (AMMSCs, BMMSCs)

(Meesuk et al., 2016).

The AD-MSCs secrete IDO 52.82 IU/mL, IFN-γ

induction on AD-MSCs increase IDO level 81.25-

94.79 IU/mL, higher IFN-γ increase IDO level

(Laksmitawati et al., 2011). hWJMSCs secretes IDO

as an anti- inflammatory (Zheng et al., 2015; Mao et

al., 2015), indicating that hWJMSCs secretion is a

promising therapy candidate for enhancing cytokine

storm in COVID-19.

CONCLUSIONS

The hWJMSCs have distinct MSCs until passage 15

that differ in a non-significant way in both positive

and negative lineage surface markers. The IDO is

secreted by hWJMSCs, and longer deprivation

increases IDO levels. However, the longer starvation

periods up to regulate the IDO level in hWJMSCs

secretome are one of method as an alternative

Covid- 19 therapy.

ACKNOWLEDGMENTS

This study was funded by the Ministry of Research,

Technology and Higher Education of the Republic

of Indonesia and research grant 2021 (Penelitian

Terapan Unggulan Perguruan Tinggi). This research

was also supported by Aretha Medika Utama

Biomolecular and Biomedical Research Center,

Bandung, Indonesia. We also acknowledge the

technical support of Ervi Afifah from Aretha Medika

Utama-Biomolecular and Biomedical Research

Center, Bandung, Indonesia.

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

276

REFERENCES

Abraham, A., Krasnodembskaya, A., 2020. ‘Mesenchymal

stem cell‐derived extracellular vesicles for the

treatment of acute respiratory distress syndrome’. Stem

Cells Transl. Med. 9(1), 28-38.

Acosta, M.A.T., Singer, B.D. 2020. ‘Pathogenesis of

COVID-19- induced ARDS: implications for an

ageing population’. Eur. Respir. J. 56(3), 2002049.

Azmi, N.U., Puteri, M.U., Lukmanto, D. 2020. ‘Cytokine

storm in COVID-19: an overview, mechanism,

treatment strategies, and stem cell therapy

perspective’. Pharm. Sci. Res. 7(4), 1- 11.

Baruah, V., & Bose, S. 2020. ‘Immunoinformatics‐aided

identification of T cell and B cell epitopes in the

surface glycoprotein of 2019‐nCoV’. Journal of

medical virology, 92(5), 495-500.

Behrens, E.M., Koretzky, G.A. 2017. ‘Review: Cytokine

storm syndrome: Looking toward the precision

medicine era’. Arthritis Rheumatol. 69(6), 1135-1143.

Berglund, A.K., Fisher, M.B., Cameron, K.A., Poole, E.J.,

Schnabel, L.V. 2017. ‘Transforming growth factor-β2

downregulates major histocompatibility complex

(MHC) I and MHC II surface expression on equine

bone marrow- derived mesenchymal stem cells

without altering other phenotypic cell surface

markers’. Front. Vet. Sci. 4, 84.

Bernardo, M.E., Fibbe, W.E. (2013). ‘Mesenchymal

stromal cells: sensors and switchers of inflammation’.

Cell Stem Cell. 13(4), 392-402.

Bongso, A., Fong, C.Y. 2013. ‘The therapeutic potential,

challenges and future clinical directions of stem cells

from the Wharton’s jelly of the human umbilical

cord’. Stem Cell Rev. Rep. 9(2), 226-240.

Canham, M.A., Campbell, J.D., Mountford, J.C. 2020.

T’he use of mesenchymal stromal cells in the

treatment of coronavirus disease 2019’. J. Transl.

Med. 18(1), 1-15.

Çetı̇ n, I., Topçul, M. 2020. ‘Can mesenchymal stem cells

be used to treat COVID-19-induced pneumonia?’.

Biomed. Rep. 13(6), 1-1.

Chen, G., Wu, D.I., Guo, W., Cao, Y., Huang, D., Wang,

H. 2020. ‘Clinical and immunological features of

severe and moderate coronavirus disease 2019’. J.

Clin. Invest. 130(5), 2620-2629.

De Witte, S.F.H., Merino, A.M., Franquesa, M., Strini, T.,

van Zoggel, J.A.A., Korevaar, S.S. 2017. ‘Cytokine

treatment optimises the immunotherapeutic effects of

umbilical cord- derived MSC for treatment of

inflammatory liver disease’. Stem Cell Res. Ther.

8(140), 1- 12.

Durand, N., Mallea, J., Zubair, A.C. (2020). ‘Insights into

the use of mesenchymal stem cells in COVID-19

mediated acute respiratory failure’. NPJ Regen. Med.

5(1), 1-9.

Engin, A.B., Engin, E.D., Engin, A. 2020. ‘The effect of

environmental pollution on immune evasion

checkpoints of SARS-CoV- 2’. Environ. Toxicol.

Pharmacol. 81(2020), 103520.

Ferro, F., Spelat, R., Shaw, G., Duffy, N., Islam, M. N.,

O'Shea, P.M., et al. 2019. ‘Survival/adaptation of bone

marrow‐derived mesenchymal stem cells after long‐

term starvation through selective processes’. Stem

Cells. 37(6), 813-827.

Garzon, I., Chato-Astrain, J., Campos, F., Fernandez-

Valades, R., Sanchez-Montesinos, I., Campos, A., et

al. 2020. ‘Expanded differentiation capability of

human Wharton's jelly stem cells toward pluripotency:

a systematic review’. Tissue Eng. Part B: Rev. 26(4),

301- 312.

Gibson, P.G., Qin, L., Puah, S. 2020. ‘COVID-19 ARDS:

clinical features and differences to ‘usual’ pre COVID

ARDS’. Med. J. Australia. 213(2), 54-56.

Glenn, JD., Whartenby, K.A. 2014. ‘Mesenchymal stem

cells: Emerging mechanisms of immunomodulation

and therapy’. World J. Stem Cells. 6(5), 526–539.

Golchin, A., Seyedjafari, E., Ardeshirylajimi, A. 2020.

‘Mesenchymal stem cell therapy for COVID-19:

present or future’. Stem Cell Rev. Rep. 16(3), 427-433.

Golchin, A., Farahany, T.Z. 2019. ‘Biological products:

cellular therapy and FDA approved products’. Stem

Cell Rev. Rep. 15(2), 166- 175.

Golchin, A., Farahany, T.Z., Khojasteh, A., Soleimanifar,

F., Ardeshirylajimi, A. 2019. ‘The clinical trials of

mesenchymal stem cell therapy in skin diseases: an

update and concise review’. Curr. Stem Cell Res.

Ther. 14(1), 22- 33.

Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., et

al. 2020. ‘Clinical features of patients infected with

2019 novel coronavirus in Wuhan, China’. Lancet.

395(10223), 497-506.

Laksmitawati, D.E., Sardjono, C.T., Pawitan, J.A. Sadikin,

M., Sandra, F. 2010. ‘Secretion of Indoleamine 2,3-

dioxygenase, an immunomodulatory substance, by

adipose- derived mesenchymal stem cell’. IJCC. 1(2),

92- 98.

Lee, S.M., Park, H.Y., Suh, Y-S., Yoon, E.H., Kim, J.,

Jang, W.H. 2017. ‘Inhibition of acute lethal pulmonary

inflammation by the IDO–AhR pathway’. Proc. Natl.

Acad. Sci. USA. 144(29), E5881–E5890.

Mao, Y.X., Xu, J.F., Seeley, EJ., Tang, X. D., Xu, L.L.,

Zhu, Y.G., et al. 2015. ‘Adipose tissue‐ derived

mesenchymal stem cells attenuate pulmonary infection

caused by Pseudomonas aeruginosa via inhibiting

overproduction of prostaglandin E2’. Stem Cells.

33(7), 2331-2342.

Marino, L., Castaldi, M.A., Rosamilio, R., Ragni, E.,

Vitolo, R., Fulgione, C., et al. 2019. ‘Mesenchymal

stem cells from the Wharton’s jelly of the human

umbilical cord: biological properties and therapeutic

potential’. Int. J. Stem Cells. 12(2), 218-226.

Moya, A., Larochette, N., Paquet, J., Deschepper, M.,

Bensidhoum, M., Izzo, V., et al. 2017. ‘Quiescence

preconditioned human multipotent stromal cells adopt

a metabolic profile favorable for enhanced survival

under ischemia’. Stem Cells. 35(1), 181-196.

Meesuk, L., Tantrawatpan, C., Kheolamai, P.,

Manochantr, S. 2016. ‘The immunosuppressive

Wharton’s Jelly Mesenchymal Stem Cells-secreted IDO as Candidate of Anti-inﬂammation Therapy

277

capacity of human mesenchymal stromal cells’.

Biochem. Biophy. Rep. 8(2016), 34–40.

Nikolaus, S., Schulte, B., Al-Massad, N., Thieme, F.,

Schulte, DM., Bethge, J., et al. 2017. Increased

Tryptophan Metabolism Is Associated With Activity

of Inflammatory Bowel Diseases. Gastroenterology,

153(6): 1504-1516.

Nuschke, A., Rodrigues, M., Wells, A.W., Sylakowski, K.,

Wells, A. 2016. ‘Mesenchymal stem cells/multipotent

stromal cells (MSCs) are glycolytic and thus glucoseis

a limiting factor of in vitro models of MSC

starvation’. Stem Cell Res. Ther. 7(1), 1-9.

Püschel, F., Favaro, F., Redondo-Pedraza, J., Lucendo, E.,

Iurlaro, R., Marchetti, S., et al. 2020. ‘Starvation and

antimetabolic therapy promote cytokine release and

recruitment of immune cells’. Proc. Natl. Acad. Sci.

USA. 117(18), 9932-9941.

Rubtsov, Y., Goryunov, K., Romanov, A., Suzdaltseva,

Y., Sharonov, G., Tkachuk, V. 2017. ‘Molecular

Mechanisms of Immunomodulation Properties of

Mesenchymal Stromal Cells: A New Insight into the

Role of ICAM-1’. Stem Cells Int. 2017(6516854), 1-

15.

Tamura, M., Kawabata, A., Ohta, N., Uppalapati, L., G

Becker, K., Troyer, D. 2011. Wharton's jelly stem

cells as agents for cancer therapy. Open Tissue Eng

Regen Med J. 4(1), 39-47.

Tay, M.Z., Poh, C.M., Rénia, L., MacAry, P.A., Ng, L.F.

2020. ‘The trinity of COVID-19: immunity,

inflammation and intervention’. Nat Rev Immunol.

20(6), 363- 374.

Van Buul, G.M., Villafuertes, E., Bos, P.K., Waarsing,

J.H., Kops, N., Narcisi, R., et al. 2012. ‘Mesenchymal

stem cells secrete factors that inhibit inflammatory

processes in short-term osteoarthritic synovium and

cartilage explant culture’. Osteoarthr. Cartil. 20(10),

1186-1196.

Wang, G., Cao, K., Liu, K., Xue, Y., Roberts, A.I., Li, F.,

et al. 2018. ‘Kynurenic acid, an IDO metabolite,

controls TSG-6- mediated immunosuppression of

human mesenchymal stem cells’. Cell Death Different.

25(7), 1209–1223.

Widowati, W., Wijaya, L., Bachtiar, I., Gunanegara, R.F.,

Sugeng, S.U., Irawan, Y.A., et al. 2014. ‘Effect of

oxygen tension on proliferation and characteristics of

Wharton's jelly-derived mesenchymal stem cells’.

Biomarkers Genomic Med. 6(1), 43- 48.

Widowati, W., Wijaya, L., Murti, H., Widyastuti, H.,

Agustina, D., Laksmitawati, D. R., et al. 2015.

‘Conditioned medium from normoxia (WJMSCs-

norCM) and hypoxia-treated WJMSCs (WJMSCs-

hypoCM) in inhibiting cancer cell proliferation’.

Biomark. Genom. Med. 7(1), 8-17.

Widowati, W., Gunanegara, R.F., Rizal, R., Widodo,

W.S., Amalia, A., Wibowo, S.H.B., et al. 2019.

‘Comparative analysis of Wharton’s jelly

mesenchymal stem cell (WJ- MSCs) isolated using

explant and enzymatic methods’. J. Phys. Conf. Ser.

1374(1), 012024.

Widowati, W., Noverina, R., Ayuningtyas, W.,

Kurniawan, D., Kusuma, H.S.W., Arumwardana, S., et

al. 2019. ‘Proliferation, characterization and

differentiation potency of adipose tissue- derived

mesenchymal stem cells (AT-MSCs) cultured in fresh

frozen and non-fresh frozen plasma’. Int. J. Mol. Cell

Med. 8(4), 283-293.

Widowati, W., Widyastuti, H., Murti, H., Laksmitawati,

D.R., Kusuma, H.S.W., Rizal, R., et al. 2017.

‘Interleukins and VEGF secretome of human

wharton's Jelly mesenchymal stem cells-conditioned

medium (hWJMSCs-CM) in different passages and

oxygen tensions’. Biosci. Res. 14(4), 776-787.

Williams, A.E., Chambers, R.C. 2014. ‘The mercurial

nature of neutrophils: still an enigma in ARDS?’. Am

J. Physiol. Lung Cell Mol. Physiol. 306(3), L217-

L230.

Wolf, AM., Wolf, D., Rumpold, H., Moschen, AR., Kaser,

A., Obrist, P., et al. 2004. Overexpression of

indoleamine 2,3- dioxygenase in human inflammatory

bowel disease. J. Clin. Immunol. 113, 47-55.

Xu, Z., Shi, L., Wang, Y., Zhang, J., Huang, L., Zhang, C.,

et al. 2020. ‘Pathological findings of COVID-19

associated with acute respiratory distress syndrome’.

Lancet Respir. Med. 8(4), 420-422.

Ye, Z., Zhang, Y., Wang, Y., Huang, Z., Song, B. 2020.

‘Chest CT manifestations of new coronavirus disease

2019 (COVID-19): a pictorial review’. Eur. Radiol.

30(8), 4381-4389.

Zheng, G., Ge, M., Qiu, G., Shu, Q., Xu, J. 2015.

‘Mesenchymal stromal cells affect disease outcomes

via macrophage polarization’. Stem Cells Int.

2015(989473), 1-11.

Zhou, F., Yu, T., Du, R., Fan, G., Liu, Y., Liu, Z., et al.

2020. ‘Clinical course and risk factors for mortality of

adult inpatients with COVID- 19 in Wuhan, China: a

retrospective cohort study’. Lancet. 395(10229), 1054-

1062.

Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J.,

et al. 2020. ‘A novel coronavirus from patients with

pneumonia in China, 2019’. N. Engl. J. Med.

Massachusetts Med. Soc. 382(8), 727-733.

ICE-TES 2021 - International Conference on Emerging Issues in Technology, Engineering, and Science

278