The Effectiveness of Mesenchymal Stem Cell and Colostrum Bovine

Combination in Post Hepatectomy Liver Failure with Liver Fibrosis

Animal Model

Dimas Aryo Kusumo

, Agung Putra

, Albertus Ari Adrianto

, Erik Prabowo

and Ignatius Riwanto

Departement of Digestive Surgery Subspecialist Program, Faculty of Medicine, Diponegoro University, Semarang,

Indonesia

Stem Cells and Cancer Research, Faculty of Medicine, Sultan Agung Islamic University, Semarang, Indonesia

Department of Digestive Surgery. Faculty of Medicine, Universitas Diponegoro, Semarang, Indonesia

Keywords: Post hepatectomy liver fibrosis, mesenchymal stem cell, bovine colostrum

Abstract: Post hepatectomy liver fibrosis (PHLF) is the most common cause of morbidity and mortality after liver

surgery. Recently, mesenchymal stem cells (MSCs) and bovine colostrum (BC) have been studied to exert an

anti-fibrotic. However, the effect of MSCs, BC, and their combination of tissue regeneration after PHLF

remains unclear. This study aimed to evaluate the effect of MSCs, BC and its combination in regulating

transforming growth factor-β (TGF-β) and serum glutamic pyruvic transaminase (SGPT) associated with liver

destruction inhibition on PHLF animal models. Eighteen Sprague-Dawley were injected with CCl4 for eight

weeks and 50% liver resection (LR). Mice were divided into three groups: group NaCl 0.9 % with

parenchymal injection, group MSC with parenchymal injection, and a group of MSCs with parenchymal

injection and BC oral. After administration, the level TGF β and SGPT were collected on the 3rd and 7th days.

This study showed that the TGF-β levels nit significant decrease under combination treatment compared to

the MSCs group until 34,11pg/mL ±4,18 and 34,5 pg/mL ±2.94, respectively, on day 7

. Besides, SPGT

levels of combination treatment also did not significantly decrease compared to the MSCs group until 93,6

U/L ±23,8 and 163,2U/l ±73,62, respectively, on day 3

. In conclusion, the combination therapy is no better

than single MSCs treatment in regenerating liver tissue on PHLF.

1 INTRODUCTION

Hepatocellular carcinoma (HCC) is the liver's

primary cancer, accounting for most liver cancers.

HCC is one of the leading causes of cancer-related

death worldwide and has high evidence in 2012. The

incidence of HCC found fourteen million patients,

and it grew to twenty-two million patients in the last

two decades. HCC causes infection of hepatitis B

virus infection, often accompanied by cholestasis. It

makes inflammatory processes that encourage liver

fibrosis. On normal liver can perform liver resection

https://orcid.org/0000-0003-4070-948X

https://orcid.org/0000-0002-9822-3119

https://orcid.org/0000-0002-2907-034X

https://orcid.org/0000-0002-2429-5419

https://orcid.org/0000-0003-3441-3738

up to 75% of the total liver volume by maintaining

25% of the remnant liver on large HCC. HCC cases

with fibrosis and less than 40% of liver remnants after

resection mostly progress into small for size liver

syndrome (SFSS) and liver failure (Wiliam and

Janagin, 2017; Xia, Lu, and Wang. 2008)

The progression of SFSS into liver failure is not

determined by the size of the liver remnant only but

also the hemodynamic liver circulation (

Golriz et

al.,2015)

. There was a disruption of normal hepatocyte

regeneration due to various events, including

parenchyma loss and hepatic vascular bed reduction.

The increasing portal pressure makes shear stress on

Kusumo, D., Putra, A., Adrianto, A., Prabowo, E. and Riwanto, I.

The Effectiveness of Mesenchymal Stem Cell and Colostrum Bovine Combination in Post Hepatectomy Liver Failure with Liver Fibrosis Animal Model.

DOI: 10.5220/0010491803050310

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 305-310

ISBN: 978-989-758-499-2

305

the sinusoid endothelial cell. Nitric oxide (NO) is then

released by liver sinusoidal endothelial cells

(LSECs), and a continuous pro-inflammatory

mediator exposure produced by the inflammatory

cell, particularly Kupffer cell (

Golriz et al.,2015, Ray et

al.,2015). During chronic inflammation, Kupffer cells

induce the prolonged release of TGF-β1 leading to LF

formation that contributed to liver failure following

liver resection (Hoffmann et al., 2020). Considering this

complicated process, finding an effective treatment in

resolving post hepatectomy liver fibrosis (PHLF)

could be very challenging. Only a few methods to

prevent SFSS in the preoperative, perioperative, and

post-operative due to liver transplantation as the lack

of liver donors (Kim et al., 2019).

On the other hand, previous studies reported that

MSC has the robust capability to suppress the release

of TGF secreted by inflammatory cells leading to

wound healing acceleration (Yo et al.,2013; Putra et

al., 2020). Other studies revealed that BC containing

growth factor and anti-oxidant also has an essential

role in liver regeneration (Sinn et al., 2017). MSCs

are multipotent cells characterized by the high

expressions of several surface markers such as

CD105, CD73, CD29, CD90, and CD44 and lack of

expressions of CD79a, CD11b. CD14, CD34, CD45,

and CD19 (Ly et al.,2014). These cells have low

immunogenicity, self-renewal, and multidirectional

differentiation properties (Zhang et al., 2018).

Previous studies have shown that MSCs have

immunomodulatory properties by secreting soluble

cytokines to inhibit inflammatory cells and prevent

excessive inflammatory damage to the liver tissue in

drug-induced liver failure animal model (Hu, Wu, and

Li, 2020). Bone marrow-derived mesenchymal stem

cells (BM-MSCs) reduced the expression of TGF β

lead to inhibition of the TGF β signalling pathway in

liver fibrosis formation

(Jang et al., 2014). A previous

study reported that the post hepatectomy LF animal

model, which received MSC treatment is successfully

survived with lower ALT and AST levels (Ding et al.,

2019)

BC is a complex mixture of protein, lipids,

lactose, vitamin, and mineral. It includes

immunoglobulin, multiple growth factors, and total

anti-oxidants capacity affecting liver injury due to

decreased TGF-β associated with fibrogenesis

inhibition and SGPT level decreased (Sinn et

al.,2017). Although MSCs and BC could resolve liver

injury in LF, the efficacy of both modalities in

decreasing the progressivity of liver damage, in this

case, need further investigation. Therefore, in this

study, we compare the effectiveness of MSCs with

BC combination compared to MSCs alone in

decreasing the progressivity of liver destruction in

PHLF by analyzing the regulation of TGF β and

SGPT level.

2 MATERIALS AND METHODS

2.1 Isolation of UC-MSCs

The umbilical cord (UC) derived MSCs (UC-MSCs)

were obtained from pregnant single Sprague-Dawley

(SD) rats under deep anaesthesia and transplanted

into an ALF rat model. The umbilical cord was cut

into pieces after the blood vessels were removed. It

was then transferred to a T25 culture flask containing

complete Dulbecco's Modified Eagle's medium

(DMEM) (Catalog #2192773 Sigma-Aldrich, Louis

St, MO) enriched with 10% Fetal Bovine Serum

(FBS) (Calatog #42A1190K GibcoTM Invitrogen,

NY, USA) and 100 IU/mL penicillin/streptomycin

(Catalog # 15070063 Sigma-Aldrich, USA). These

cells were incubated in a 5% CO2, 37ºC incubator,

and the medium was changed every three days. After

the cells reached 80% confluency, the MSC-like cells

were passaged with trypsin. The cells from the 4

passage were used for experiments. The Institutional

Review Board approved this study of the Medical

Department's Ethics Committee with number

254/VIII/2020/ commission bioethics, Sultan Agung

Islamic University, in Semarang, Indonesia.

2.2 PHLF Animal Models Induction of

PHLF by Made Remnant Liver

Fibrosis Animals and Experimental

Design

Eighteen Sprague-Dawley (SD) male rats were

randomly divided into three groups (n= 18). LF

induction was performed by injecting intraperitoneal

the carbon tetrachloride (CCl4) (Catalog #56235

Sigma–Aldrich, USA) with 1 ml/kg twice per week

for eight weeks. After six weeks, three rats in the

model group were sacrificed randomly, and the liver

tissue was obtained to verify LF with Sirius red. All

of the PHLF with LF rats undergoing liver resection

50% of the liver in the median and right lateral lobes.

The surgical procedure was performed within the

sterile condition under intravenous anaesthesia using

xylazine + ketamine (5 mg/kg + 100 mg/kg

intramuscularly) (

Constandinouu et al., 2005).

The Group of NaCl 0.9 % and parenchymal

injection NaCl 0,9% 500 μL, group MSC with

parenchymal injection with doses 1 x 10

cells

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dissolved in 500 μL of NaCl, and group combination:

MSCs parenchymal injection 1x 10

cells dissolved in

500 μL of NaCl and oral administration of BC doses

15μL/g per oral, daily with milk powder Good Health.

2.3 Flow Cytometry

Immunophenotyping of UC-MSCs

The immunophenotypes of MSCs were analyzed in

the fourth passage. MSCs were stained using

conjugated antibodies: fluorescein isothiocyanate

(FITC)-conjugated CD90, Allophycocyanin (APC)-

conjugated CD73, Peridinin Chlorophyll Protein

Complex (PerCP)-conjugated CD105 and

phycoerythrin (PE)-conjugated Lin monoclonal

antibodies for 30 min at 4ᴼC in the darkroom. The

cell's fluorescence intensity was evaluated through

flow cytometry (BD Bioscience, Franklin Lakes, NJ,

USA).

2.4 In Vitro Differentiation

MSCs differentiation potential was determined to

characterize the isolated cells. These cells were

cultured in DMEM medium supplemented with 10%

FBS, ten mmol/L β-glycerophosphate, 10



mol/L/ 0.1

µM dexamethasone, 50 µmol/L ascorbate-2-

phosphate (Catalog #SLBL4673V Sigma-Aldrich,

Louis St, MO), at 37ᴼC and 5% CO

. The fixed cells

were stained with 0.2 % Alizarin Red solution

(Catalog #MKBS9114V Sigma-Aldrich) to represent

calcium deposition (the cells were used from the

fourth passage).

2.5 Elisa TGF- β

The rat's blood was harvested via peri-orbital venous

plexus bleeding under general anaesthesia on day 3

day after treatment, and the serum was collected

by centrifugation at 4⁰C. The TGF-β levels were

measured by ELISA kits, based on the manufacturer's

instructions (Abbkine) and according to a standard

curve constructed for each assay. The colourimetric

absorbance was recorded at a wavelength of 450 nm.

2.6 SGPT Levels

The levels of SGPT were measured on the 3

, 7

days

after treatment to determine liver function. Blood

samples were collected from the peri-orbital vein

under anaesthesia, using xylazine + ketamine (5

mg/kg + 100 mg/kg intramuscularly) (Alfasan,

Netherlands). The serum level of SGPT was

measured using automatic analyzers (BT 3000 PLUS,

Italy).

2.7 Statistical Analysis

All data were presented as mean ± standard deviation

with differences between groups analyzed by a one-

way ANOVA and least significant difference

comparison post hoc LSD test. Analysis using a p <

0.05 significant statistical value.

3 RESULTS

3.1 Characteristics of UC-MSCs and

Differentiation Test

The isolated cells showed peculiar fibroblast-like

(spindle shape) morphology. To determine and verify

the MSCs marker, we assessed MSC's marker

expression using flow cytometry after the fourth

passages (Figure 1). The results showed that the

isolated cells expressed an MSC-specific marker

including positive expression of CD105 (96.7%),

CD73 (99.2%), and CD90 (96.7%) and lack of Lin

(0.03%). In line with the flow cytometry analysis, we

also examined the osteogenic capabilities of MSCs.

We found the differentiation of MSC into osteogenic

has occurred, indicated by calcium deposits as red

appearance using the Alizarin red dye staining

method (Figure 2). It is according to the International

Society of Cellular Therapy (ISCT).

Figure 1: MSCs characterization. The graph displayed the

expression MSC, positive markers (CD105, CD73, and

CD90), and lack of the negative marker Lin (Lin̄)

expression.

The Effectiveness of Mesenchymal Stem Cell and Colostrum Bovine Combination in Post Hepatectomy Liver Failure with Liver Fibrosis

Animal Model

307

Figure 2. The morphology and differentiation of MSCs.

(A)MSC Differentiation. Alzarin red dye showed a red

colour appearance in the MSC differentiation test

(magnification x40, scale bar 50 μm). (B) Morphological

characterization. MSCs appearance; homogeneous,

spindle-shaped, fibroblast-like cells (magnification x10,

scale bar 200 μm).

3.2 MSCs Suppress TGF-β and SGPT

Levels in PHLF Animal Model

The TGF- is one of the primary growth factors with

pleiotropic capability particularly associated with

fibrosis formation due to its ability to changes

activate hepatic stellate cell (HSC) into myofibroblast

(MF) in producing collagen type III. To determine the

role of MCS in decreasing TGF- level in PHLF, we

analyzed using ELISA. In this study we found, there

is a significant decreased (p<0,05) of TGF- level on

day 7

in MSC treatment group (day 3

29,66 pg/mL

±0,76; day 7

34,11 pg/mL ±4,18, respectively) and

Combination group (day 3

36,54 pg/mL ±3,72; day

34,5 pg/mL ±2.94) compared with the control

group NaCl Injection (day 3

36.33 pg/ml±3.04, day

56.7pg/ml ± 6.22 ) (Figure 3).

Figure 3. The level of TGF-β at 3

, and 7

day in the NaCl,

MSC, and combination group. *The level of TGF- day

MSC (34,11pg/mL ±4,18) and combination group,

(34,5

pg/mL ±2.94) on day 7

was decreased significantly

compared to NaCl group (56.7pg/ml± 6.22) p<0,05. C:

Colostrum

The increase of SGPT level indicated that there

was a hepatocellular injury, including on PHLF. We

examined the level of SGPT after MSC treatment

compared to a combination of both treatments to

regenerate the damaged liver tissue in PHLF. In this

study, we found a significant decrease (p:0,024) of

SGPT level in the MSC group only on day 3rd (day 3

rd 93,6 U/L ±23,8, day 7th 58,8 U/L±2.68). We did

not found a significant decrease in the combination

group on day 3rd and 7th (day 3 rd 131,4 U/L±41,2,

day 7th 52,8 U/l±10,57) compared with NaCl group

(day 3 rd 163,2U/l ±73,62, day 7th 66U/l±17.01)

(Figure 4).

Figure 4. SGPT level on 3

, and 7

day in NaCl, MSC

and combine group. Data are presented as the mean ±

standard deviation. *The level of SGPT decreased

significantly in MSCs group only on day 3

(93,6 U/L

±23,8) compared to NaCl group (163,2U/l ±73,62) p < 0,05.

C: Colostrum

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4 DISCUSSION

The aims of this study were evaluated the

effectiveness of MSCs and combination of MSCs and

BC in the regeneration of liver tissues on PHLF by

analyzing the regulation of TGF β and SGPT level.

The ability of MSCs decrease fibrosis formation by

controlling the prolonged release of TGF-β produced

by MF as associate cell product fibrosis deposits. BC

containing anti-oxidant also can improve LF and

restore the damaged structure and function of liver

tissue. As the potent mediators, the TGF-β initiates

consistently with the activation and differentiation of

HSCs to be active MFs resulting in collagen type III

deposition. We injected CCl4 as a hepatotoxic

chemical to induce LF and performed liver resection

50 % to induce PHLF by major resection liver to

make a small liver remnant (Golriz et al., 2015)

We present here that MSCs and combination group

can suppress the release of TGF-β in PHLF model

animals. We believe this is a novel discovery to

demonstrate the mechanism of how the PHLF with

fully developed TGF-β-dependent fibrosis can be

disrupted by a combination of BC and MSCs

application. We found that MSC has two mechanisms

of action. First, MSCs can decrease TGF-β levels by

depolarizing macrophage type I (M1) into

macrophage type II (M2) (Darlan et al., 2020).

Second, we suggest that MSCs affect

immunomodulatory mechanisms by releasing IL-10.

The receptor of Kupffer-IL-10 binding might activate

Janus tyrosine kinase 1 (JAK1) and tyrosine kinase-2,

leading to activation of signal transducer and

transcription 3 (STAT3), then translocated into the

nucleus to binds the promoters of target genes, the

suppressor of cytokine signalling 3 (SOCS3)

correlated with the decreased expression of tumour

necrosis factor (TNF)-α, IL-1β, and TGF-β (

Sziksz et

al., 2015). Our findings were in line with a previous

study that showed MSCs could prevent peritoneal

fibrosis by releasing IL-10 ( Muhar et al., 2018).

Our

finding MF cell produced TGFβ for autocrine to stay

active production collagen type III. MSC Group and

combine group has released immunomodulatory

properties, leading to the decreasing of TFGβ longer

day 7

in PHLF

In line with the decrease of TGFβ level, we also

found SGPT level decreasing just in the MSCs group

only on day 3

. Phase inflammation on PHLF

disrupts hepatocyte cells because injury from liver

resection and stagnant portal venous blood stimulates

inflammation with collagen deposition on the portal

venous release by MF (

Golriz et al.,2015, Ray et

al.,2015). MSC has an effect immunomodulator and

enhances regeneration hepatocyte by release

secretome. Our previous study releases that

intraparenchymal of MSCs administration in the

damaged liver tissue leads to the MSC migration to

the injured areas for repairing and restoring the

damaged liver structure and its function. BC previous

study can decrease SGPT level by release anti-

oxidants in acute liver injury (Sinn et al., 2017).

We assume the combination group can not affect

inflammatory phase PHLF because BC has a product

growth factor pro-inflammation and disrupts

immunomodulator from MSC (

Quiles et al., 2006). In

repairing and restoring damaged tissue, MSCs should

previously control inflammation by releasing IL-10 to

inhibit the prolonged release of TGF, leading to

healing process acceleration (Putra et al., 2019).

Under prolonged controlled inflammation, MSC

induces decreased remnant liver destruction and

restored liver function (

Fiore, 2018). Taken together,

the single MSCs treatment more effective than the

combination of MSCs and BC to regenerating liver

tissues on PHLF.

5 CONCLUSIONS

We conclude that MSC and BC's combination is not

better than MSCs alone in decreasing TGFβ and

SGPT. We suggest further research about the

regeneration of the PHLF by examining marker

regeneration and portal flow using MSC to explain

the liver's healing process.

Conflict of Interest

The authors declare that they have no conflict of

interests.

ACKNOWLEDGMENTS

We acknowledge the Digestive Surgery Subspecialist

Program of Diponegoro University, and thank the

Stem Cell and Cancer Research (SCCR) Laboratory,

the medical faculty at Sultan Agung Islamic

University (UNISSULA), Semarang, and who

contributed to this research.

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