A Novel Diabetic Arthritic Model in Rats Induced by Streptozotocin, High-Fat Diet, and Complete Freund's Adjuvant.

Introduction: Ankle arthrodesis is one of the treatments of choice, particularly in late-stage and unstable diabetic Charcot arthropathy. Unfortunately, poor healing capacity might play a role in the high nonunion rate (10-40%). The advancement in regenerative medicine opens a new horizon for enhancing fusion after ankle arthrodesis in patients with poor healing capacity. However, a suitable small animal model is warranted to study the effectivity of these regenerative medicine approaches. Streptozotocin (STZ)-induced diabetes models and adjuvant-induced arthritis models with complete Freund's adjuvant are two established models. However, no study has combined those two models to make a diabetic arthritic model that more closely resembles the condition in Charcot arthropathy. Methods: Twenty male Sprague-Dawley rats were assigned into five groups, consisting of one control group, and four diabetic groups which were induced by STZ injection and a high-fat diet. Among these diabetic rats, two groups received complete Freund's adjuvant (CFA) injections to the left ankle of the hind limb. The control group, one of the diabetic-only groups, and one of the arthritic-diabetic-induced groups were euthanized at 4 weeks after STZ induction, and the remainder were euthanized 6 weeks after STZ induction. Clinical, radiological, and histological examinations were then compared in all five groups. Results: Diabetic status was successfully achieved in the model, which was maintained until the completion of the study. The CFA-induced ankles were significantly larger than the contralateral ankles in all groups (p<0.05). Histopathological evaluation confirmed arthritic changes in the CFA-induced group with less variability after 4 weeks of arthritis induction. Conclusion: This rat model of arthritic diabetic mimics the progressive and chronic nature of Charcot arthropathy in humans. This model can be further use to study treatments that might enhance the fusion rate in ankle arthrodesis in healing-defective patients such as those with diabetes. Level of Clinical Evidence: 5.

Evaluation of adipose-derived stem cells (ASCS) exosome implantation and platelet-rich fibrin (PRF) on critical long bone defects in Sprague-Dawley rats.

INTRODUCTION: This study aimed to assess the efficacy of adipose-derived mesenchymal stem cell exosomes (ASCs exosome) and platelet-rich fibrin (PRF) in treating critical long bone defects in Sprague-Dawley rats. Critical long bone defects, defined as exceeding 2 cm or 50% of the bone diameter, often pose a healing challenge. While autologous bone grafts have been considered, they have shown unreliable results and donor-site complications, necessitating alternative treatments. METHODS: The research followed a quasi-experimental post-test only control group design involving 30 male Sprague-Dawley rats. The rats were divided into five groups and subjected to femur bone defect creation, internally fixed with a 1.4 mm K-wire, and treated with various combinations of hydroxyapatite (HA), bone graft (BG), ASCs exosome, and PRF. Histomorphometry and BMP-2 gene expression analysis were performed to evaluate bone healing. RESULTS AND DISCUSSION: The results indicated that the group treated with HA + BG + ASCs exosome (group IV) exhibited the highest BMP-2 gene expression, while group III (HA + BG + ASCs exosome + PRF) had the highest chordin level. Overall, groups receiving ASCs exosome or PRF intervention showed elevated BMP-2 expression compared to the control group. The use of ASCs exosome and PRF showed comparable outcomes compared to bone graft administration in terms of histomorphometry analysis. CONCLUSION: The administration of adipose tissue derived mesenchymal stem cells and PRF has a comparable outcome with the use of bone graft in terms of osseus area and expression of BMP-2 in critical bone defect.

Effects of Dietary Restriction on PGC-1α Regulation in the Development of Age-associated Diseases.

Ageing is the most significant risk factor for a number of non-communicable diseases, manifesting as cognitive, metabolic, and cardiovascular diseases. Although multifactorial, mitochondrial dysfunction and oxidative stress have been proposed to be the driving forces of ageing. Peroxisome proliferator-activated receptor γ coactivator α (PGC-1α) is a transcriptional coactivator central to various metabolic functions, of which mitochondrial biogenesis is the most prominent function. Inducible by various stimuli, including nutrient limitations, PGC-1α is a molecule of interest in the maintenance of mitochondrial function and, therefore, the prevention of degenerative diseases. This review involves a literature search for articles retrieved from PubMed using PGC-1α, ageing, and dietary restriction as keywords. Dietary restriction has been shown to promote tissue-specific PGC-1α expression. Both dietary restriction and PGC-1α upregulation have been shown to prolong the lifespans of both lower and higher-level organisms; the incidence of non-communicable diseases also decreased in fasting mammals. In conclusion, dietary interventions may delay ageing by regulating healthy mitochondria in various organs, presenting the possibility of a new primary prevention for many age-related diseases.

The Effect of Intrahepatic and Intrasplenic Administration of Mesenchymal Stem Cell to Liver Function and Degree of Liver Fibrosis in Common Bile Duct Ligation Model in Rabbit.

BACKGROUND: Mesenchymal stem cells (MSC) is a promising alternative method in liver cirrhosis management. Several administration routes of MSC have been studied, but few studies compared one to another. The purpose of this study is to compare the intrahepatic and intrasplenic route of MSC administration in terms of liver function and degree of liver fibrosis in the bile duct ligation model in rabbits. METHOD: Experimental study was conducted using rabbits (Oryctolagus cuniculus) model undergoing bile duct ligation (BDL). The subjects were randomized into 4 groups: sham surgery; bile duct ligation; bile duct ligation followed by intrahepatic route of MSC (BDL + IH MSC), and bile duct ligation followed by intrasplenic route of MSC (BDL + IS MSC). Umbilical cord mesenchymal stem cell (UC MSC) was administered on the fifth day after bile duct ligation, and the subjects were observed until the fourteenth day after bile duct ligation. The liver function was evaluated with alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total and direct bilirubin. The degree of fibrosis was evaluated with Laennec score, fibrosis area fraction, the number of viable and necrosis hepatocytes, and the number of hepatic progenitor cells. RESULT: The subjects were randomized into 4 groups: 2 in sham surgery group, and 7 in each of the following groups: BDL, BDL + IH MSC and BDL + IS MSC groups. The mortality rate in BDL group was 57.1 %, while mortality in BDL + IH MSC and BDL + IS MSC groups were 14.3 % and 28.6 % respectively. No significant difference was found regarding liver function in each group, such as AST, ALT, total, and direct bilirubin. Histopathology examination in almost every subject undergone bile duct ligation (regardless of MSC administration) showed degree of fibrosis of Laennec 4B. Fibrosis area fraction, the number of viable and necrotic hepatocytes, and progenitor cells were analyzed; no significant difference was found between BDL + IH MSC and BDL + IS MSC groups, but the groups administered with MSC showed a larger number of viable hepatocytes compared to BDL group. No difference was found between BDL + IH MSC and BDL + IS MSC groups in terms of liver function and histologic parameters. CONCLUSION: Administration of MSC increases the number of viable hepatocytes, but no difference was found in terms of liver function and degree of liver fibrosis between the intrahepatic route and intrasplenic route of administration. TYPE OF STUDY: Animal Research, Randomized Controlled Study. LEVEL OF EVIDENCE: Level I? (animal research is not indicated in the levels of evidence table in the journal website).

Liver organoids cocultured on decellularized native liver scaffolds as a bridging therapy improves survival from liver failure in rabbits.

The present study aimed to develop viable liver organoids using decellularized native liver scaffolds and evaluate the efficacy of human liver organoid transplantation in a rabbit model of cirrhosis. Liver organoids were formed by coculture of hepatocyte-like cells derived from the human-induced pluripotent stem cells with three other cell types. Twelve 3-mo-old New Zealand White Rabbits underwent a sham operation, bile duct ligation, or biliary duct ligation followed by liver organoid transplantation. Liver organoid structure and function before and after transplantation were evaluated using histological and molecular analyses. A survival analysis using the Kaplan-Meier method was performed to determine the cumulative probability of survival according to liver organoid transplantation with significantly greater overall survival observed in rabbits that underwent liver organoid transplantation (P = 0.003, log-rank test). The short-term group had higher hepatic expression levels of ALB and CYP3A mRNA and lower expression levels of AST mRNA compared to the long-term group. The short-term group also had lower collagen deposition in liver tissues. Transplantation of human liver organoids cocultured in decellularized native liver scaffold into rabbits that had undergone bile duct ligation improved short-term survival and hepatic function. The results of the present study highlight the potential of liver organoid transplantation as a bridging therapy in liver failure; however, rejection and poor liver organoid function may limit the long-term efficacy of this therapeutic approach.

Induced Pluripotent Stem Cells (Ipscs) Based Liver Organoid: the Benefits and Challenges.

The liver is the main metabolic organ and functions to regulate many physiological functions in the human body. Approximately 70% of liver mass consists of hepatic cells (hepatocytes), which execute the liver's metabolic processes. When liver damage progresses to a chronic condition, such as end-stage liver disease (ESLD) or cirrhosis of the liver, the patient's only option for therapy is organ transplantation if the supply of available transplanted organs is insufficient to meet the patient's needs. The fundamental objective of the search for alternatives to organ transplantation has been to make liver tissue replacement more accessible and to produce hepatic and bioartificial liver tissue. Multiple hepatic cell lineages can be formed from human-induced pluripotent stem cells (hiPSCs) from embryoid bodies to become mature hepatocytes. hiPSCs also show a promising source for manufacturing human liver spheroids and are made to produce three-dimensional hepatobiliary organoids, and in some ways, it also briefly highlights important features of early hepatogenesis. Unquestionably, the art of cell culture has evolved to include the use of organoid technology as a resource for learning human biology in the context of health and illness. Organoids are essentially miniature organs that can grow in a three-dimensional matrix to resemble genuine organs in terms of both structure and function. This review summarized alternative protocols to differentiate hepatocytes from iPSC and to produce liver organoids based on iPSC in various ways. The growth of human iPSCs into liver organoids has been accomplished using several procedures.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Increase Human MCF7 Breast Cancer Cell Proliferation associated with OCT4 Expression and ALDH Activity.

OBJECTIVE: The aim of this study was to investigate the effect of mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) on the human MCF7 breast cancer cell proliferation that have been considered to contain limited CSC population and its association with the expression of OCT4 and ALDH1 stemness markers. METHODS: EVs were successfully isolated from the conditioned medium of umbilical cord MSCs using size exclusion chromatography. The isolated EV fraction was verified under a transmission electron microscope (TEM). Five and ten percent (v/v) concentration of MSC-EVs were then co-cultured with MCF7 cells. To investigate MSC-EV uptake by MCF7 cells, we performed confocal microscopy analysis. Subsequently, the proliferation of co-cultured MCF7 cells was determined using trypan blue exclusion assay, while their mRNA and protein expression of OCT4 as well as ALDH activity as the marker of stemness properties were analyzed using quantitative reverse transcription polymerase chain reaction, Western Blot, and Aldefluor™ assays, respectively. RESULT: MSC-EVs were detected as round-shaped, ~100 nm sized particles under TEM. We also demonstrate that MSC-EVs can be internalized by MCF7 cells. Notably, MSC-EVs of 5% concentration increased OCT4 mRNA expression and ALDH1 activity in MCF7 cells. At 10% concentration, MSC-EVs reduced the OCT4 expression and ALDH1 activity. CONCLUSION: MSC-derived EVs modulate the stemness of MCF7 cells, either OCT4 expression or ALDH1 activity, in a concentration dependent manner along with the increase of cell proliferation.

Effects of fasting on FOXO3 expression as an anti-aging biomarker in the liver.

Background: Aging is a multifactorial degenerative process that can be modulated by fasting through activation of the Fork-head transcription factor of the O class 3 (FOXO3), which plays an important role in increasing lifespans. However, the effects of different fasting durations on the expression of FOXO3 in the liver has not yet been reported. Objective: This study analyzed the effects of different fasting durations on the FOXO3 expression and its pathway by measuring sirtuin1 (SIRT1), insulin-like growth factor-1 (IGF-1), and superoxide dismutase (SOD) activity in the liver. Methods: New Zealand white rabbits were used to mimic the effects of fasting on humans. The rabbits were divided into the control, intermittent fasting (IF), and prolonged fasting (PF) groups. Both fasting groups were interspersed with the non-fasting phase for 8 h. This treatment was conducted for 6 days. On Day 7, all the rabbits were sacrificed, and their livers were taken to measure the FOXO3 and SIRT1 mRNA expressions, the IGF-1 protein level, and the SOD activity level. ANOVA, multiple comparison, and Pearson's correlation were performed for statistical analysis. Results: The FOXO3 and SIRT1 mRNA expressions were significantly higher in the IF group than in the control group. The FOXO3 expression was also 2.5 times higher in the IF group than in the PF group. There was a positive correlation between the FOXO3 and SIRT1 mRNA expressions. The IGF-1 protein level was significantly lower in the IF and PF groups than in the control group. The SOD-specific activity level was significantly higher in the IF group than in the control and PF groups. Conclusions: Intermittent fasting significantly increased the FOXO3 and SIRT1 mRNA expressions and the SOD activity level in the livers of the rabbits and significantly decreased the circulating and hepatic IGF-1. Therefore, intermittent fasting may give a protective intervention effect towards aging.

The Impact of Prolonged and Intermittent Fasting on PGC-1α, Oct-4, and CK-19 Liver Gene Expression.

BACKGROUND: Liver stemness refers to the high regenerative capacity of the organ. This intrinsic regeneration capacity allows the restoration of post-resection liver function in up to 50% of liver donors. Liver cirrhosis is one of the terminal liver diseases with a defect in the intrinsic regeneration capacity. Several attempts to restore intrinsic regeneration capacity by conducting in vivo studies on stem cells in various organs have shown the positive impact of fasting on stemness. An increased capacity for stem cell proliferation and regeneration was reported due to fasting. Prolonged fasting (PF) has been reported to maintain the long-term proliferative ability of hematopoietic stem cells. However, clinical trials on intermittent fasting (IF) have not conclusively given positive results for fasting individuals. OBJECTIVES: This research aims to investigate the effect of fasting on liver stemness by comparing the expression of octamer-binding transcription factor 4 (Oct-4), cytokeratin 19 (CK-19), and peroxisome proliferator-activated receptor γ co-activator α (PGC-1α) in liver cells of fasted rabbits with rabbits fed ad libitum. This study compares two types of fasting, which are intermittent (16 hours) and prolonged (40 hours) fasting, for liver stemness and intrinsic regenerative capacity. METHODS: A total of 18 rabbits were conditioned into 3 different groups. The first group was subjected to an ad libitum diet, the second to intermittent fasting (16-hour fasting), and the third to prolonged fasting (40-hour fasting). Afterward, the RNA was extracted from the liver tissues of each rabbit and analyzed via real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Relative expression was calculated using the Livak method. RESULTS: Compared to the ad libitum diet, a greater increase was reported in PGC-1α, upregulated Oct4, and steady CK-19 gene expressions in the livers of intermittent fasting rabbits. Prolonged fasting increased PGC1α, reduced liver stemness, and a statistically insignificant decrease in intrinsic liver regenerative capacity. CONCLUSION: Intermittent fasting indicates preferable molecular alterations in liver stemness and intrinsic regenerative capacity compared to prolonged fasting.

The Effect of Fasting on Oxidative Stress in the Vital Organs of New Zealand White Rabbit.

Background: Oxidative stress is defined as the condition in which balance between the synthesis and detoxification of reactive oxygen species in cells is disrupted. This research explored the effects of intermittent and prolonged fasting on malondialdehyde (MDA), carbonyl, reduced glutathione (GSH), and specific activity of catalase as biomarkers for oxidative stress in hearts, brains, and kidneys of New Zealand White (NZW) rabbits. Methods: Fifteen NZW rabbits were divided into control, intermittent fasting (IF), and prolonged fasting (PF) groups. The controls were fed ad lib. IF and PF groups were fasted for 16 and 40 hours, respectively, followed by eight hours of non-fasting, for six days and were sacrificed on the 7th day. One hundred mg of heart, brain, and kidney tissues were homogenized in 1 ml of phosphate-buffered saline. MDA, carbonyl, GSH, and catalase were analyzed by spectrophotometry. Data were analyzed using One-way ANOVA and post hoc test. Results: In heart, MDA was significantly greater in the control than in the IF and PF groups. In brain, GSH was greater in the IF than in the PF and control groups. Also, in brain, catalase specific activity was significantly greater in the control than in the IF and PF groups. In kidney, catalase specific activity was significantly less in the PF than in the control group. Conclusion: The effect of fasting on oxidative stress in various organs showed various responses, however fasting reduced oxidative stress based on MDA and GSH levels in the heart and brain, respectively.

Hepatocyte Differentiation from iPSCs or MSCs in Decellularized Liver Scaffold: Cell-ECM Adhesion, Spatial Distribution, and Hepatocyte Maturation Profile.

Mesenchymal stem cells (MSC) and induced pluripotent stem cells (iPSC) have been reported to be able to differentiate to hepatocyte in vitro with varying degree of hepatocyte maturation. A simple method to decellularize liver scaffold has been established by the Department of Histology, Faculty of Medicine, Universitas Indonesia, in SCTE IMERI lab.15 This study aims to evaluate hepatocyte differentiation from iPSCs compared to MSCs derived in our decellularized liver scaffold. The research stages started with iPSC culture, decellularization, seeding cell culture into the scaffold, and differentiation into hepatocytes for 21 days. Hepatocyte differentiation from iPSCs and MSCs in the scaffolds was characterized using hematoxylin-eosin, Masson Trichrome, and immunohistochemistry staining to determine the fraction of the differentiation area. RNA samples were isolated on days 7 and 21. Expression of albumin, CYP450, and CK-19 genes were analyzed using the qRT-PCR method. Electron microscopy images were obtained by SEM. Immunofluorescence examination was done using HNF4-α and CEBPA markers. The results of this study in hepatocyte-differentiated iPSCs compared with hepatocyte-differentiated MSCs in decellularized liver scaffold showed lower adhesion capacity, single-cell-formation and adhered less abundant, decreased trends of albumin, and lower CYP450 expression. Several factors contribute to this result: lower initial seeding number, which causes only a few iPSCs to attach to certain parts of decellularized liver scaffold, and manual syringe injection for recellularization, which abruptly and unevenly creates pattern of single-cell-formation by hepatocyte-differentiated iPSC in the scaffold. Hepatocyte-differentiated MSCs have the advantage of higher adhesion capacity to collagen fiber decellularized liver scaffold. This leads to positive result: increase trends of albumin and higher CYP450 expression. Hepatocyte maturation is shown by diminishing CK-19, which is more prominent in hepatocyte-differentiated iPSCs in decellularized liver scaffold. Confirmation of mature hepatocyte-differentiated iPSCs in decellularized liver scaffold maturation is positive for HNF4-a and CEBPA. The conclusion of this study is hepatocyte-differentiated iPSCs in decellularized liver scaffold is mature with lower cell-ECM adhesion, spatial cell distribution, albumin, and CYP450 expression than hepatocyte-differentiated MSCs in decellularized liver scaffold.

A Review of the Impact of Calorie Restriction on Stem Cell Potency.

Calorie restriction (CR) prolongs lifespan in various species and also minimises pathologies caused by aging. One of the characteristics seen in age-related pathologies is stem cell exhaustion. Here, we review the various impacts of CR on mammalian health mediated through stem cell potency in various tissues. This study comprised of a literature search through NCBI, Science Direct, Google Scholar and PubMed, focusing on the impact of CR on pluripotency. In the skeletal muscle, CR acts as an anti-inflammatory agent and increases the presence of satellite cells endogenously to improve regeneration, thus causing a metabolic shift to oxidation to meet oxygen demand. In the intestinal epithelium, CR suppresses the mechanistic target of rapamycin complex 1 (mTORC1) signalling in Paneth cells to shift the stem cell equilibrium towards self-renewal at the cost of differentiation. In haematopoiesis, CR prevents deterioration or maintains the function of haematopoietic stem cells (HSCs) depending on the genetic variation of the mice. In skin and hair follicles, CR increases the thickness of the epidermis and hair growth and improves hair retention through stem cells. CR mediates the proliferation and self-renewal of stem cells in various tissues, thus increasing its regenerative ability.

Reconstructing an ovary cancer microenvironment for in vitro 3D drug testing: A new avenue for ovary cancer research.

OBJECTIVE: To elaborate on in vitro 3D ovary cancer models that can mimic the in vivo condition of an ovary cancer microenvironment for anticancer drug discovery. METHODS: Literature research was conducted in NCBI, ScienceDirect, and Pubchem databases. A total of 19 articles relevant to the search terms were included in this review. RESULTS: The ovary cancer niche has been described as the home of cancer stem cells, multipotent stromal cells, fibroblasts, blood and lymphatic vessels, growth factors, extracellular matrix protein (ECM). Cell cancer behaviour toward anticancer drugs can also be altered by deposition of ECM. A human ovary decellularized protocol generates ovary scaffold. Incorporation of cancer stem cells (CSC) in the ovary cancer niche thus yield 3D reconstructed microenvironment for further anticancer drug testing. CONCLUSIONS: The 3D reconstructed microenvironment provides a platform for further anticancer drug testing.

Enhancement of the Therapeutic Capacity of Mesenchymal Stem Cells by Genetic Modification: A Systematic Review.

BACKGROUND: The therapeutic capacity of mesenchymal stem cells (also known as mesenchymal stromal cells/MSCs) depends on their ability to respond to the need of the damaged tissue by secreting beneficial paracrine factors. MSCs can be genetically engineered to express certain beneficial factors. The aim of this systematic review is to compile and analyze published scientific literatures that report the use of engineered MSCs for the treatment of various diseases/conditions, to discuss the mechanisms of action, and to assess the efficacy of engineered MSC treatment. METHODS: We retrieved all published studies in PubMed/MEDLINE and Cochrane Library on July 27, 2019, without time restriction using the following keywords: "engineered MSC" and "therapy" or "manipulated MSC" and "therapy." In addition, relevant articles that were found during full text search were added. We identified 85 articles that were reviewed in this paper. RESULTS: Of the 85 articles reviewed, 51 studies reported the use of engineered MSCs to treat tumor/cancer/malignancy/metastasis, whereas the other 34 studies tested engineered MSCs in treating non-tumor conditions. Most of the studies reported the use of MSCs in animal models, with only one study reporting a trial in human subjects. Thirty nine studies showed that the expression of beneficial paracrine factors would significantly enhance the therapeutic effects of the MSCs, whereas thirty three studies showed moderate effects, and one study in humans reported no effect. The mechanisms of action for MSC-based cancer treatment include the expression of "suicide genes," induction of tumor cell apoptosis, and delivery of cytokines to induce an immune response against cancer cells. In the context of the treatment of non-cancerous diseases, the mechanism described in the reviewed papers included the expression of angiogenic, osteogenic, and growth factors. CONCLUSION: The therapeutic capacity of MSCs can be enhanced by inducing the expression of certain paracrine factors by genetic modification. Genetically engineered MSCs have been used successfully in various animal models of diseases. However, the results should be interpreted cautiously because animal models might not perfectly represent real human diseases. Therefore, further studies are needed to explore the translational potential of genetically engineered MSCs.

Alpha mangostin Inhibits Hepatic Stellate Cells Activation Through TGF-ß/Smad and Akt Signaling Pathways: An in vitro Study in LX2.

BACKGROUND: Alpha mangostin has been reported to have activity for the treatment of liver fibrosis in the rats. However, the mechanisms of action are poorly understood. This study was aimed to investigate the effect of alpha mangostin on hepatic stellate cells (HSC) activation and proliferation through TGF-ß/Smad and Akt signaling pathways. METHODS: Immortalized HSC, LX2 cells, were incubated with TGF-ß with or without alpha mangostin (5 or 10 µM). Sorafenib 10 µM was used as positive control. LX2 viability was counted using trypan blue exclusion method. The effect of alpha mangostin on TGF-ß concentrations, and the expressions of proliferation and fibrogenic markers were evaluated. RESULTS: Alpha mangostin treatment resulted in a reduced proliferation of HSC, decreased Ki-67 and p-Akt expressions. These findings were followed with decreased concentrations of TGF-ß in the medium of cells treated with alpha mangostin, decreased expressions of COL1A1, TIMP1, PAI1, α-SMA, and p-Smad3 as fibrogenic markers. These effects were shown to be dose-dependent. CONCLUSIONS: Alpha mangostin inhibits hepatic stellate cells proliferation and activation through TGF-ß/Smad and Akt signaling pathways in dose dependent manner.

Bone Marrow Stem Cells Anti-liver Fibrosis Potency: Inhibition of Hepatic Stellate Cells Activity and Extracellular Matrix Deposition.

Transplantation of bone marrow derived stem cells (BMSCs) has been reported inhibits liver fibrosis. Several in vitro studies by co-culturing BMSCs and hepatic stellate cells (HSCs) indirectly or directly in 2D models showed inhibition of HSC as the key player in liver fibrosis. In this study, we investigated direct effect of BMSCs on HSCs by co-culturing BMSCs and HSCs in 3D model as it represents the liver microenvironment with intricate cell-cell and cell-matrix interactions. Primary isolated rat HSCs and BMSCs were directly co-cultured at 1:1 ratio with hanging drop method. The monoculture of rat HSCs served as positive control. Mono-culture and co-culture samples were harvested on day 3, 5 and 7 for histological analysis. The samples were analyzed for extracellular matrix deposition by Masson's Trichrome staining, tenascin-C immunocytochemistry, resting HSC's state as shown by positive Oil Red O stained cells. Our results indicated CD90＋CD34- BMSCs anti-liver fibrosis potency as evidenced by higher proportion of Oil Red O-positive cells in the co-culture group compared to the monoculture group and the significant decrease in extracellular matrix deposition as well as the decrease in tenascin-C expression in the co-culture group (p<0.05) compared to the monoculture group. These findings demonstrate that BMSCs have a potential therapeutic effect against liver fibrotic process through their capacity to inhibit HSCs activation and their effect in minimizing extracellular matrix deposition.