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Abstract

Currently, there is not an effective therapy for cirrhosis of the liver except for liver transplant. However, finding a compatible liver is difficult due to the low supply and increased demand for healthy livers. Stem cell therapy may be a solution for liver cirrhosis. In our previous report, stem cells from Wharton's jelly and bone marrow were shown to improve liver function in a chemically induced liver fibrosis animal model. However, the immunological rejection of an allograft is always a risk for clinical application. In this study proposal, we suggest using human adipose-derived stem cells (ADSCs) because they are an immune-privileged cell type; they lack human leukocyte antigen-DR expression, and they also suppress the proliferation of activated allogenic lymphocytes and inhibit the production of inflammatory cytokines. In addition, ADSCs contain a sufficient amount of adult stem cells for autologous transplantation. Based on these benefits, ADSCs are promising candidates for clinical application when compared to other stem cell types. The aim of our study will be to investigate the safety and efficacy of autologous ADSCs for the clinical treatment of liver cirrhosis.

Keywords

Stem cell therapy Adipose-derived stem cells (ADSCs)Liver cirrhosis Clinical treatment

Introduction

Most cases of liver cirrhosis, despite the cause, progress from liver fibrosis to an irreversible final stage: liver cirrhosis. Liver injury can be caused by several factors, such as viral infection (hepatitis B or hepatitis C), autoimmune liver disease (autoimmune hepatitis), drug-induced liver damage, or sustained alcohol abuse (4). Currently, liver transplantation is considered the best option for patients with liver cirrhosis (13). However, there are considerations that affect a patient's curative possibilities, including organ shortage, graft failure, and rejection (13). Therefore, finding an alternative therapy for liver cirrhosis patients is critical and could save thousands of lives per year.

Mesenchymal stem cells (MSCs) are a potential source for autologous stem cell therapy. MSCs have the ability to self-renew and to differentiate into several cell types, including adipocytes, osteocytes, chondrocytes, hepatocytes, neurons, muscle cells, and epithelial cells (1,12,21,23). Petersen et al. first demonstrated that liver stem cells are derived from the bone marrow (BM) (20). Subsequently, BM-derived stem cells, including MSCs, were proposed to be reservoir cells capable of liver cell regeneration (6,15,28). Although MSCs were first identified in the BM, they can also be isolated from human umbilical cord blood, synovium, placenta, periosteum, skeletal muscle, and adipose tissue (25). Of the possible stem cell lines, we chose to use adult MSCs isolated from adipose tissue—adipose-derived stem cells (ADSCs). ADSCs are similar to BM-derived MSCs in that they can also differentiate into neurogenic, myogenic, osteogenic, and chondrogenic lineages (32,34). Moreover, these cells secrete multiple growth factors, including vascular endothelial growth factor, epithelial growth factor, and macrophage colony-stimulating factor. In addition, in our previous study, we showed that the transplanted ADSCs differentiated into albumin- and afetoprotein-secreting liver-like cells 1 week after transplantation. These results suggest that ADSC transplantation may facilitate recovery from chronic liver damage (10). More importantly, in comparison with other types of adult stem cells, ADSCs are easily accessible and abundant (about 1 × 10⁵ ADSCs per gram of fat) (24). Currently, there are ADSC-based clinical trials in specialties such as plastic surgery in addition to clinical trials related to diseases involving digestion, autoimmunity, cardiovascular and neurological function, as well studies focused on skeletal regeneration (9).

ADSC-based stem cell therapy for liver disease is designed to replace diseased hepatocytes and to stimulate endogenous stem cell regeneration (10). We have developed a standardized protocol that we believe will provide autologous human ADSCs for clinical use. Our protocol includes instructions for the isolation, banking, and culture expansion of ADSCs, using a minimal amount of starting material (<5 g) from lipoaspiration. Through the culture expansion process, we can provide a sufficient amount of autologous ADSCs for multiple therapeutic procedures (10). In the previous study, we used human ADSCs and administered them directly into thioacetamide-treated livers. The ADSCs were obtained from Gwo Xi Stem Cell Applied Technology Co., Ltd. (Gwo Xi), IRB number: DMR100-IRB-202. Over 28 days of follow-up, immunohistochemistry showed that the transplanted ADSCs differentiated into hepatocyte-like cells 1 week after transplantation. In addition, liver function, including total bilirubin, prothrombin time, and albumin levels, recovered significantly (p < 0.01) (10). Using the Metavir score, we noted that both liver fibrosis and inflammatory activity decreased significantly after ADSC transplantation (p < 0.01) (10). To better understand the mechanism of how ADSCs aid in liver recovery, we also measured related markers and noted a reduction in the expression of α-smooth muscle actin and an increase in the expression of matrix metalloproteinase-9 (10). These results are the first to demonstrate that unmanipulated, transplanted human ADSCs can differentiate into hepatocyte-like cells in situ within a damaged liver and ameliorate liver fibrosis by secreting metalloproteinase, which decreases the activity of the cells that produce collagen fiber (stellate cells). Using our protocol for culture expansion, ADSCs maintained their potency and their ability to differentiate, independent of donor age (10). The preclinical efficacy studies indicate that ADSCs from older (60 years old) donors are useful for autologous cell therapy in liver disease and are comparable to those from young donors (27 years old) (10). This result suggests that our culture expansion protocol is suitable for different age donors.

Before culture-expanded stem cells could be used for clinical trials, the stem cells should be evaluated for aerobic and anaerobic bacteria, endotoxin, and mycoplasma. The in vivo safety of the stem cells also should be confirmed, including toxicity and tumorigenicity. The good tissue practice (GTP) guideline should be used to define the distinct biological characteristics of the cells; verify their stability throughout the storage, thawing, and shipping processes; and ensure the quality assurance and control of the components. Gwo Xi follows the Current GTP requirements, under the Code of Federal Regulations, Title 21, part 1271, and GTP guideline established by the Taiwan Food and Drug Administration (TFDA) to produce the ADSCs (27,29). Culture-expanded ADSCs from Gwo Xi showed the typical appearance, immunophenotype, and differentiation capacity of MSCs, and were genetically stable at least 12 passages in culture. Based on the toxicity and tumorigenicity test results, we conclude that the transplantation of up to 1 × 10⁸ cells/kg autologous ADSCs may be safe when given by intrahepatic injection (manuscript in preparation).

Aim and Hypothesis

The aim of this proposed study is to investigate the hypothesis that intrahepatic injection of autologous ADSCs is safe, feasible, and well tolerated as a therapeutic modality in liver cirrhosis patients. Our protocol uses naive ADSCs for direct administration into liver parenchymal tissue to restore liver function and to ameliorate liver fibrosis. Transplantation of autologous ADSCs into the diseased liver provides a less invasive method of transplantation than do those that rely on other types of stem cells. Our main hypothesis is that autologous ADSCs can be delivered to the cirrhosis-damaged liver by intrahepatic injection and that after delivery ADSCs can differentiate into hepatocyte-like cells in damaged liver tissue and can then repair liver damage.

Patients and Study Methodology

The study will be performed in accordance with the Institutional Review Board of China Medical University and Hospital Research Ethics Committee and the TFDA, Ministry of Health and Welfare. The original protocol has been approved by the China Medical University and Hospital Research Ethics Committee (CMUH102-REC1–064), and the Ministry of Health and Welfare approved the protocol (No. 28113265). This clinical trial is registered at Clinicaltrial.gov as NCT02297867.

Patients

Our Phase I/IIa clinical safety and efficacy study will enroll six liver cirrhosis patients (with both genders). Each patient will sign an informed consent form and also agree to the anonymous usage of their biological fluids for research purposes. Patients will undergo the stem cell therapy procedure using our liver cirrhosis protocol (Fig. 1).

Figure 1.

Flow chart for liver cirrhosis stem cell therapy protocol.

Inclusion Criteria

Liver cirrhosis patients aged 20 to 80 years (both inclusive)

Patients without mandatory communicable disease (HBV, HCV, HIV, syphilis)

Patients without rare disorder

Coagulation normalities

Patients without autoimmune disorder

Patients without Acquired Immune Deficiency Syndrome

Patients without cancer

Patients BMI > 15

Exclusion Criteria

Pregnant women

Patients with acute stroke in 1 month and unconsciousness

Patients with acute myocardial infarction or acute heart failure

Patients with serious liver dysfunction and coagulation dysfunction and ascites mild higher

Patients with acute respiratory failure or pneumonia

Kidney failure: BUN > 50

Anemia: Hematocrit < 25

Patients diagnosed with liver cancer or liver metastatic carcinoma

Patients with liver abscess

10.

Patients with acute hepatitis

11.

Patients with acute infection

12.

Liver cirrhosis patients with HBV or HCV

13.

Patients diagnosed with carcinoma and receiving treatment

14.

Patients with schizophrenia or melancholia

15.

Patients received serious surgical operations in 3 months

16.

Patients unable to control hypertension (SBP > 180 mmHg, DBP > 110 mmHg) or diabetes (AC sugar > 200 mg/dl)

17.

Others cannot fit into the trial evaluated by investigator

Patient Withdrawal Criteria

Patients who are not able to maintain an appropriate follow-up schedule

Patients who opt to withdraw

Study Methodology

Physical and Laboratory Examination

All patients will undergo routine laboratory tests, including a complete blood count, biochemistry panel (including liver function tests glutamic oxaloacetic transaminase, glutamic pyruvic transaminase), bilirubin, albumin, blood urea nitrogen, estimated glomerular filtration rate (eGFR), creatine, α-fetoprotein, ammonia, prothrombin time/partial thromboplastin time, international normalization ratio, abdominal echo, and liver biopsy (Table 1).

Table 1.

Examination Chart for Stem Cell Therapy (Both Before and After the Procedure)

	4 Weeks (Pre)	1 Week (Post)	1 Month (Post)	2 Months (Post)	3 Months (Post)	4 Months (Post)	5 Months (Post)	6 Months (Post)
CBC
GOT
GPT
Bil(t)
Alb
BUN
eGFR
Creat
AFP
Ammonia
PT/PTT
INR
Abdominal echo
Liver biopsy

CBC, complete blood count; GOT, glutamic oxaloacetic transaminase; CPT, glutamic pyruvic transaminase; Bil(t), bilirubin; Alb, albumin; BUN, blood urea nitrogen; eGFR, estimated GFR; Creat, creatine; AFP, α-fetoprotein; PT/PTT, prothrombin time/partial thromboplastin time; INR, international normalize ratio; Pre, pretherapy; Post, posttherapy.

Isolation of Patient ADSCs

The patient adipose tissue will be harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery (2–5 g). Tissue samples will be placed in Ca²⁺/Mg²⁺-free phosphate-buffered saline (PBS) and immediately transferred to the Stem Cell R&D Center of Gwo Xi Stem Cell Applied Technology Co., Ltd. Adipose tissue will then be removed from the transport media, placed in a Petri dish, and cut into small pieces (1–2 mm³) in the presence of Ca²⁺/Mg²⁺-free PBS. The tissues will be dissociated with 0.1% collagenase I (Invitrogen-Gibco, Carlsbad, CA, USA) and incubated for 60 min at 37°C. After enzymatic digestion, the resultant cells will be collected and cultured in keratinocyte serum-free media (Invitrogen-Gibco) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), N-acetyl-L-cysteine (Sigma-Aldrich, St. Louis, MO, USA), L2 ascorbic acid, and phosphate (Sigma-Aldrich). The supernatant and debris will be removed from the culture dish after being cultured for 2 days. The ADSC culture will then be designated as passage zero.

ADSC Culture Suspension

The cells will be maintained in keratinocyte serum-free media (Invitrogen-Gibco) supplemented with 10% (v/v) FBS, N-acetyl-L-cysteine (Sigma-Aldrich), L2 ascorbic acid, and phosphate (Sigma-Aldrich). The cells will be incubated at 37°C with 5% CO₂. To prevent spontaneous differentiation, cultures will be maintained at subconfluent levels (<80% confluence). Culture-expanded ADSCs are genetically stable for at least 12 passages (22). In this clinical trial, culture expansion will be limited to four passages.

ADSC Preparation

Before cell transplantation, cells will be washed three times in sterile saline, and viability will be evaluated using the ADAM-MC^™ Automatic Cell Counter (Digital Bio, NanoEnTek Inc., Seoul, Korea). Cells will be suspended in physiological saline at a concentration of 1 × 10⁸ cells/ml.

ADSCs Surface Phenotype Characterization

ADSC surface markers will be characterized using a BD Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) after labeling with antibodies against the human clusters of differentiation: CD34, CD45, CD90, and CD105 (Becton Dickinson).

ADSC Transplantation

One milliliter of cell suspension will be injected intrahepatically under sonographic guidance using a gauge 18 needle.

Follow-Up

All patients will be followed regularly according to our clinical schedule (Table 2). Patients will be evaluated by Model For End-Stage Liver Disease Score (MELD score), child score (Table 3), and routine laboratory tests (Table 1). MELD uses the patient's values for serum bilirubin, serum creatinine, and the international normalized ratio (INR) for prothrombin time to predict survival. It is calculated according to the following formula (14): MELD = 3.78[Ln serum bilirubin (mg/dl)] + 11.2[Ln INR] + 9.57[Ln serum creatinine (mg/dl)] + 6.43.

Table 2.

Patient Follow-Up Schedule

Visit	Enrollment	Treatment	Follow-Up
Evaluation Time	4 Weeks Prior	Day 0	1 Week	1 Month	2 Months	3 Months	4 Months	5 Months	6 Months
Sign informed consent form	□
Inclusion/exclusion criteria	□
Diagnosis of liver cirrhosis	□		□	□	□	□	□	□	□
Physical and laboratory examination	□		□	□	□	□	□	□	□
ADSC isolation	□
ADSC transplantation		□
Follow-up			□	□	□	□	□	□	□
Liver biopsy		□							□
National ADR Reporting System	▴	▴	▴	▴	▴	▴	▴	▴	▴

ADR, adverse drug reaction.

□

Patients will undergo the marked procedures according to this schedule.

▴

When unexpected serious adverse drug reactions occur during clinical trials, investigator should report the findings to the TFDA.

Table 3.

Child-Pugh Classification

Name of the Factor	Score: 1 Point	Score: 2 Points	Score: 3 Points
Serum albumin (g/dl)	Higher than 3.5	3.0–3.5	Lower than 3.0
Serum bilirubin (mg/dl)	Lower than 2.0	2.0–3.0	Higher than 3.0
Prothrombin time (s)	1–4	4–6	Higher than 6.0
Ascites	None	Moderate	Severe
Encephalopathy	None	Mild	Severe
The three classes are formed and their scores are:
Class A	Score of 5–6
Class B	Score of 7–9
Class C	Score higher than 9

The Child-Pugh classification is a scoring system that helps determine the cirrhosis of the liver prognosis. Here, scoring is based upon several factors: albumin, ascites, total bilirubin, prothrombin time, and encephalopathy.

Statistical Analysis

Statistical analysis will be performed using the SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Safety and efficacy endpoints will be observed for each patient by comparing baseline values to postprocedure values. The statistical analysis will be performed by analysis of unpaired t-test or by analysis of variance (ANOVA followed by Tukey's post hoc test). A value of p < 0.05 will be considered as statistically significant.

Discussion

Autologous stem cell transplantation appears to be a beneficial replacement for orthotopic liver transplantation or hepatocyte transplantation. Human ADSCs are multipotent MSCs (26) and are a suitable source for autologous cell therapy. In clinical applications, an increasing number of reports indicate that ADSCs are a promising treatment for diseases that are difficult to cure or for the reconstruction of anatomical defects (9). However, barriers to the implementation of this application include both the quality and the quantity of available stem cells. To obtain therapeutic doses (i.e., 1 × 10⁸ cells), stem cells must undergo culture expansion ex vivo. Therefore, to obtain human autologous ADSCs for clinical usage, we developed a protocol to isolate, bank, and expand ADSCs from minimal amounts of fat tissue according to the GTP guideline (27). The GTP is a guideline established by TFDA for researchers and manufacturers to follow. The regulations for human organ banks were then announced by the Department of Health of Taiwan in February, 2009. These are the regulations for institutions that are engaged in the processing or the preservation of human organs, tissues, and cells for the purpose of transplantation. Gwo Xi Stem Cell Applied Technology was inspected according to GTP by the TFDA, Ministry of Health and Welfare in November 2013. This clinical trial has been approved by the TFDA. Gwo Xi will follow the standardized protocol for the isolation, banking, and culture expansion of ADSCs (Fig. 1).

Various routes of ADSC administration for liver disease, including intravenous (IV) (3), hepatic portal vein (16,17,30), intrasplenic (8), and intrahepatic (10) have been reported for stem cell delivery. Of these routes, IV is a convenient strategy for cell delivery and has been shown to produce therapeutic effects to the delivery site. However, IV-injected MSCs may be transiently trapped in the lungs, sequestered in the spleen, and are often eliminated by kidneys (7). Initial accumulation of MSCs in the lungs may induce secretion of secondary anti-inflammatory effector molecules (15). Despite these issues, the recent demonstration of the in vivo homing properties of BM-derived MSCs and ADSCs has further stimulated the IV introduction of MSCs for therapy (19). Terai et al. used IV delivery for BM mononuclear cells in liver cirrhosis patients (28) and report significant improvement in liver function after therapy (11). In that study, the approach was based on the homing property of circulating stem cells to injured areas and subsequent differentiation into hepatocytes. However, the liver cirrhotic microenvironment may not be optimal for supporting homing and differentiation (18). Chen et al. compared the injection of 1 × 10⁶ BMMSCs/ml through the portal vein, the femoral vein, and intrahepatic into the allogenic rat liver (6). Of these, the option with the greatest number of delivered cells was the intrahepatic group, followed by the portal vein group, with the femoral vein group having the lowest efficiency. These data suggest that direct intrahepatic injection results in a better effect for liver cirrhotic therapy (6). Amer et al. treated end-stage liver failure patients with autologous BMMSCs. In comparing intrahepatic with intrasplenic injection, the intrahepatic group showed more pronounced improvement in the fatigue impact scale and in the MELD score during the first month. This suggests faster engraftment and function of those cells that were injected intrahepatically (2). We previously reported that the transplantation of human BMMSCs and Wharton's jelly-derived stem cells through the portal vein can promote liver recovery after liver cirrhosis has occurred (5,17).

There are about 350 million hepatitis B virus (HBV) carriers in the world, and 75% are living in Asia. In Taiwan, we have 3 million HBV carriers and exposure in the high risk about HBV causes liver cirrhosis and liver carcinoma. In 2010, Zhong et al. isolated BMMSCs from patients with chronic HBV infections and compared the cell properties (33). This study demonstrated that BMMSCs from chronic hepatitis B patients proliferate defectively and decrease expression of growth factor receptors (33). On the other hand, Wang et al. isolated BMMSCs and ADSCs from chronic HBV patients and characterized the morphology, growth potency, surface phenotype, and differentiation (31). Their study demonstrated that ADSCs from chronic hepatitis B patients have similar cell properties and differentiation potential as BMSCs. However, compared with cell abundance, accessibility and proliferation capacity, ADSCs may be alternative stem cells for chronic hepatitis B patients (31). According to the aforementioned, we consider more about the safety issue in this clinical trial. Thus, this is the reason why we exclude liver cirrhosis patients with HBV or HCV in this phase I/IIa protocol.

In our recent study, we showed that undifferentiated ADSCs injected intrahepatically differentiate into hepatocyte-like cells in damaged liver tissue and can repair liver fibrosis (10). Both the biological mechanism of ADSC-based liver repair and our study observations support the hypothesis that intrahepatic injection of autologous ADSCs is safe, feasible, and well tolerated as a therapeutic modality in liver cirrhosis patients.

The promising preclinical study data as well as the positive outcomes observed in clinical trials indicate that ADSCs are a feasible potential cell-based therapy, which has been shown to be safe in many applications. Thus, we propose direct intrahepatic injection of autologous ADSCs (10) for our clinical trials to treat patients with liver cirrhosis.

Footnotes

Acknowledgments

This study was funded by Ministry of Science and Technology, Taiwan (MOST103–2320-B-039–021-MY3), Ministry of Economic Affairs (102-EC-17-A-19-I1–0051), Taiwan Ministry of Health and Welfare, China Medical University Hospital Cancer Research Center of Excellence (MOHW103-TD-B-111–03), Clinical Trial and Research Center of Excellence (MOHW103-TDU-B-212–113002), and CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan. The authors declare no conflicts of interest.

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A Proposed Novel Stem Cell Therapy Protocol for Liver Cirrhosis

Abstract

Keywords

Introduction

Aim and Hypothesis

Patients and Study Methodology

Patients

Inclusion Criteria

Exclusion Criteria

Patient Withdrawal Criteria

Study Methodology

Physical and Laboratory Examination

Isolation of Patient ADSCs

ADSC Culture Suspension

ADSC Preparation

ADSCs Surface Phenotype Characterization

ADSC Transplantation

Follow-Up

Statistical Analysis

Discussion

Footnotes

Acknowledgments

References