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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I3 - Enhancing Expression of MMP-9 in Mesenchymal Stem Cells to Amplify Anti-Fibrotic Capacities to Treat Liver Fibrosis

Thursday, October 24, 2024

2:45 PM – 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Sarang Han

Undergraduate Student
University of Maryland - College Park
Ellicott Citt, Maryland, United States

Co-Author(s)

AV

Anjali Verma

Postdoctoral Fellow
University of Maryland, School of Medicine, United States
SK

Shani Kamberi

Medical Student
University of Maryland, School of Medicine, United States
Ks

Kasra shirini

Visiting Scholar
University of Maryland, School of Medicine, United States
DJ

Daniel Juan

Undergraduate researcher
University of Maryland, College Park, United States
AM

Alexandra Max

Student Researcher
University of Maryland, College Park, United States
VM

Valeria Mas, PhD

Division Head, Division of Surgical Sciences - Department of Surgery
Unversity of Maryland, School of Medicine, Maryland, United States

Primary Investigator(s)

RM

Raphael Meier

Assistant Professor
University of Maryland, School of Medicine, United States

Introduction: Liver fibrosis occurs due to the recruitment of extracellular matrix proteins, primarily collagen, in the result of inflammation, which can be the consequence of viral infection, alcohol abuse, autoimmune disease, and metabolic dysfunction-associated steatohepatitis (MASH). Persistent inflammation eventually fails liver regeneration, making liver stiffened and leave scar tissue, which disrupt the liver architecture, impairing the organ function. Since liver fibrosis can be progressed into severe, irreversible damage, known as liver cirrhosis, therapeutic investigation to treat liver fibrosis is urgently needed.

In this study, mesenchymal stem cells (MSCs)-based cell therapy was investigated to decrease liver fibrosis in mice model, by delivering Metalloproteinase-9 (MMP-9), a protein known to digest ECM proteins accumulated upon fibrosis.

Materials and

Methods: In this study, we utilized MSCs the multipotent stromal cells that is versatile and found in various tissues such as bone marrow, adipose tissues, umbilical cord etc. MSCs have inherent self-renewability, anti-inflammatory, immunomodulatory properties, which makes them an excellent cell therapy candidate. MMP-9 is a protein known to treat liver fibrosis in physiological environment, by digesting extracellular matrix proteins accumulated upon inflammation, and they are secreted from neutrophils or phagocytic stimuli. Therapeutic effect of MMP-9 was validated through in vitro conditioned media study to treat hepatic stellate cells (HSCs), activated with transforming growth factor-β (TGF-β), molecule secreted by monocytes to activate HSCs.

We genetically modified MSCs to enhance the Metalloproteinase-9 (MMP-9) expression in three different ways: SMAD4 silencing, CRISPRa/dCas9, and Lentiviral overexpression. Expression of MMP-9 has been quantitively investigated with quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA).

Then the MMP-9-enhanced MSCs were encapsulated in alginate using microfluidic device and crosslinked in calcium chloride, forming cellular alginate microparticle as a delivery vehicle. The cellular alginate microparticle were transplanted into mice with induced liver fibrosis, which were induced via bile duct ligation (BDL) method. The in vivo therapeutic effects were evaluated through qPCR, ELISA, western blot, and various staining methods.

Results, Conclusions, and Discussions: Successful MMP-9 amplifications in MSCs at both mRNA and protein level were confirmed by qPCR and ELISA in vitro.

Upon treating the LX2 cells (human hepatic stellate cell line) with conditioned media from MMP-9 enhanced MSCs in the presence or absence of TGF- β and, a significant reduction of fibrosis markers (α-SMA; α-smooth muscle actin and Fibronectin) was observed, compared to regular MSCs and control groups.

We also confirmed the role of MMP-9 in the resolution of fibrosis in mouse model of liver fibrosis. After 2 weeks of transplantation of encapsulated MMP-9 enhanced MSCs, MMP-9 enhanced MSCs showed decrease in expression of fibrotic makers, αSMA, Fibronectin, and collagen type I in mice liver tissue, compared to the no treatment groups and just MSCs encapsulated groups. There is a strong need to add biological replicates to the study, which is continuously being executed. This result gathered so far is highly convincing effective therapeutic effect of MMP-9 MSCs cell therapy on liver fibrosis.

Acknowledgements (Optional): This research was supported in part by the Maryland Stem Cell Research Fund, Lisa Dean Moseley Foundation, and University of Maryland Scholars Program, an initiative of the University of Maryland: MPowering the State.