ORIGINAL RESEARCH REPORT
STEM CELLS AND DEVELOPMENT Volume 00, Number 00, 2014 Mary Ann Liebert, Inc. DOI: 10.1089/scd.2013.0500
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Allogeneic Bone Marrow Mesenchymal Stem Cell Transplantation in Patients with UDCA-Resistant Primary Biliary Cirrhosis Li Wang,1,* Qin Han,2,* Hua Chen,1 Ke Wang,2 Guang-liang Shan,2 Fang Kong,1 Yun-jiao Yang,1 Yong-zhe Li,1 Xuan Zhang,1 Fen Dong,2 Qian Wang,1 Dong Xu,1 Zhao-jun Hu,1 Shi-hua Wang,2 Armand Keating,3 Ya-lan Bi,4 Feng-Chun Zhang,1 and Robert Chun-Hua Zhao 2
The objective of this study was to evaluate the safety and efficacy of allogeneic bone marrow mesenchymal stromal/stem cell transplantation (BM-MSCT) for patients with ursodeoxycholic acid (UDCA)-resistant primary biliary cirrhosis (PBC). Ten patients were enrolled in this trial of BM-MSCT. All patients were permitted to concurrently continue their previous UDCA treatment. The efficacy of BM-MSCT in UDCA-resistant PBC was assessed at various time points throughout the 12-month follow up. No transplantation-related side effects were observed. The life quality of the patients was improved after BM-MSCT as demonstrated by responses to the PBC-40 questionnaire. Serum levels of ALT, AST, g-GT, and IgM significantly decreased from baseline after BM-MSCT. In addition, the percentage of CD8 + T cells was reduced, while that of CD4 + CD25 + Foxp3 + T cells was increased in peripheral lymphocytic subsets. Serum levels of IL-10 were also elevated. Notably, the optimal therapeutic outcome was acquired in 3 to 6 months and could be maintained for 12 months after BMMSCT. In conclusion, allogeneic BM-MSCT in UDCA-resistant PBC is safe and appears to be effective.
of ductopenia and severe icteric cholestasis, progressing very quickly toward cirrhosis in less than 5 years [1]. Therefore, there is an urgent need for new therapeutic investigations for patients with UDCA-resistant PBC. Mesenchymal stromal/stem cells (MSCs) were originally isolated from bone marrow and widely studied for their ability to differentiate into multiple tissues, including bone, fat, cartilage, neurons, hepatocytes, and cardiocytes. We isolated a subpopulation of MSCs designated Flk-1 + MSCs from a variety of human tissues and demonstrated their wide differentiation potential and immunomodulatory functions [5–9]. Ex vivo expansion and in vivo infusion of bone marrow-derived Flk-1 + MSCs showed both feasibility and safety from monkey to human [10]. An important function of MSCs for autoimmune diseases is their immunomodulatory effect on various activated lymphoid cells such as T cells, B cells, natural killer cells, and dendritic cells. MSCs express low levels of HLA class I major histocompatibility complex (MHC) molecules and no class II MHC or costimulatory molecules [11]. Our
Introduction
P
rimary biliary cirrhosis (PBC) is a chronic autoimmune inflammatory liver disease that leads to the nonsuppurative destruction of small interlobular bile ducts, progressive cholestastis, and, eventually, liver fibrosis and cirrhosis. Ursodeoxycholic acid (UDCA) is currently the only drug for the treatment of PBC approved by FDA. Several lines of evidence indicate that UDCA improves biochemical liver tests, slows histological progression, and, therefore, may prolong patient survival without liver transplantation [1]. However, all of these benefits are only acquired in patients who have a perfect biochemical response to UDCA. An incomplete biochemical response to UDCA (UDCA resistant) at 1 year is more frequently observed in patients with more severe baseline biochemical and histological features and with poor prognosis [2,3]. We had even found that early biochemical response at 3 to 6 months could efficiently identify patients at a risk of poor outcome [4]. These patients, tragically, have a very rapid onset
1 Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Science, Beijing, China. 2 Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Peking Union Medical College Hospital, Center of Excellence in Tissue Engineering Chinese Academy of Medical Sciences, Beijing, China. 3 Institute of Medical Science, University of Toronto, Toronto, Canada. 4 Department of Pathology, Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Science, Beijing, China. *These authors contributed equally to this work.
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previous clinical trial with 33 patients demonstrated that a new transplantation strategy combining haploidentical peripheral blood stem cells (PBSCs) and Flk-1 + MSCs could prevent severe graft-versus-host disease (GVHD) [12]. In addition, we found that Flk-1 + MSCs infusion was therapeutically practicable, with no detectable side effects during the treatment of sclerodermatous chronic GVHD in a clinical report of four patients [13]. These results support the possibility of using MSCs for therapeutic applications in autoimmune diseases. In this study, we enrolled UDCA-resistant PBC patients to undergo bone marrow MSC transplantation (BM-MSCT) from their healthy family donors. This trial was approved by the Ethics Committee at Peking Union Medical College Hospital, China, in February 2010.
Patients and Methods Study design and follow-up protocol The study protocol was based on a design from the American Association for the Study of Liver Diseases Endpoints Conference [14]. All research on patients and donors in this study have been approved by the Ethical Review Board of Peking Union Medical College Hospital, and informed consents were obtained. The safety assessments included vital signs and discomfort records during BM-MSC infusion and at 1, 6, 12, 24, 48, and 72 h after that. Follow-up evaluations were conducted at 1, 3, 6, and 12 months after MSCT. The investigators recorded adverse events and assessed the quality of life and disease severity throughout the study. The quality of life assessment was evaluated using a PBC-40 questionnaire [15]. The disease severity was measured using the serum levels of the following biochemical markers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), g-glutamyl transpeptidase (g-GT), total bilirubin (TBIL), direct bilirubin (DBIL), and immunoglobulin (Ig) M; cytokines: interleukin (IL)-4, IL-6, IL-10, IL-17A, interferon (IFN)-g, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)-b1; and phenotypic analysis of peripheral blood lymphocytes and histological features of liver biopsies (before and 12 months after transplantation) [16].
Patients and donors Ten patients (nine women and one man, whose median age was 50 years, with 4.5 years of disease duration and a
liver histological stage of I to III that was classified by Ludwig–Scheuer criteria [17,18]) were enrolled. These patients met the 2009 AASLD criteria [19] for PBC without evidence of decompensated liver disease, other connective tissue disease (CTD), or liver disease. All ten patients had an incomplete biochemical response to standard doses of UDCA (13–15 mg/kg/day) for more than 1 year according to the Paris criteria (ALP > 3 upper limit of normal (ULN), AST > 2 ULN, and bilirubin ‡ 1 mg/dL after 1 year of UDCA treatment) [3]. Permitted medications included prednisone ( £10 mg/d) and UDCA at a dose that was maintained at the pre-enrollment dose. Donors were first grade relative of the patients. They were considered healthy and qualified only if they had no disease history and normal test of blood routine, urine routine, liver and kidney function, and negative antinuclear antibody (ANA) and anti anti-mitochondrial antibody (AMA). The median age of the donors was 27.5 years old. No patient had withdrawn during a follow up of at least 12 months. Table 1 lists the clinical features of the PBC patients and the donor control for each of them.
BM-MSC preparation and transplantation All infused allogeneic MSCs were obtained from healthy family donors (eight participants from their own children and two from their brothers). The Flk-1 + CD31 - CD34 MSCs were collected and cultured as previously described [20]. Briefly, approximately 20 mL volunteers’ bone marrow aspirates were collected, and mononuclear cells were separated by Ficoll gradient centrifugation (density 1.077g/cm3). The interphase layer was collected and resuspended in Dulbecco’s modified Eagle’s medium (low-glucose DMEM/ F12), 40% MCDB-201 (Sigma) supplemented with 2% fetal bovine serum (FBS; Gibco Life Technologies), 1-insulin transferring selenium (Gibco Life Technologies), 10 - 9 M dexamethasone (Sigma), 10 - 4 M ascorbic acid 2-phosphate (Sigma), 10 ng/mL epidermal growth factor (Sigma), and 10 ng/mL platelet-derived growth factor BB (Sigma), and then plated in culture flasks (1 · 107 cells/75 cm2). The culture was maintained at 37C in a humidified environment containing 5% CO2. After 24–48 h of culture, nonadherent cells were removed, and the adherent layer was cultured until it reached 70%–80% confluence. The cells were then detached with 0.05% trypsin and 0.01% EDTA and replated. MSCs were harvested, used at passage 3 to 5,
Table 1. Patient and Donor Characteristics
No.
Sex/age (years)
Disease duration (years)
Baseline of ALP (U/L)
Baseline of TBIL (lM)
Baseline of AMA (x)
Baseline of AMA-M2 (U/mL)
Phase of histology
Donor/age (years)
Follow-up (months)
1 2 3 4 5 6 7 8 9 10
F/53 F/46 F/58 M/51 F/54 F/31 F/61 F/49 F/46 F/42
7 3 5 3 5 2 4 6 2 5
517 1149 240 407 522 766 390 441 1385 378
33.9 66.2 11.5 27.5 45.3 21.7 34.9 21 108.7 122
160 160 320 1280 640 1280 160 640 1280 1280
105 0 > 200 > 200 > 200 > 200 89 > 200 > 200 > 200
II II II I II I II II II III
Daughter/28 Son/21 Daughter/32 Daughter/23 Daughter/27 Brother/40 Son/35 Son/27 Son/18 Brother/45
16 16 16 16 16 15 15 14 13 12
BM-MSCT TREATMENT IN UDCA-RESISTANT PBC PATIENTS
and intravenously infused at a density of 3–5 · 105 cells per kg of body weight.
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Immunohistochemistry staining The 4 mm liver biopsy specimen sections were washed in PBS, incubated in 3% hydrogen peroxide for 10 min, and then washed in PBS. The nonspecific binding was blocked by incubating the sections in serum for 60 min. The slides were then incubated with the first antibody for 1 h, incubated with Biotin-conjugated secondary antibodies for 15 min at room temperature, colorated with DAB, counterstained with hematoxylin, and then observed under microscopy. The first antibodies used were goat monoclonal antibody (mAb) against Foxp3(Abcam; 1: 1000) and mouse mAb against CD8 (DAKO; 1: 50).
Statistical analysis Values were expressed as the mean – standard error of the mean (SEM). Paired Student’s t-tests and Signed Rank test were performed to compare the normal distribution data and non-normal distribution data, respectively, by statistical software (SAS). P < 0.05 was considered statistically significant.
Results Safety of BM-MSC transplantation No fever, chills, or vomiting appeared during the course of MSC infusion. We did not observe any fever, chills, or vomiting during the course of MSC infusion. Vital signs of the patients remained stable during a 72 h-observation. No adverse events were observed, and routine blood and urine studies remained normal at 1, 3, and 6 months after transplantation. No infusion-related instant reaction occurred. The infusion was well tolerated in patients, and no rejection occurred.
Efficacy of BM-MSC transplantation Life quality assessment. The PBC-40 questionnaire was used to estimate the patients’ quality of life by six criteria, including symptoms, itchiness, fatigue, cognition, and social and emotional function before and 12 months after MSCT. We observed improvements at 12 months after MSCT in itchiness (8.9 – 0.9 vs. 5.7 – 0.7, P = 0.001), fatigue (32.8 – 2.6 vs. 24.3 – 3.6, P = 0.022), and emotional function (9.9 – 0.9 vs. 7.1 – 0.6, P = 0.003). Table 2 lists the changes in the patients’ quality of life using the PBC-40 scale.
Table 2. Assessment of Quality of Life by PBC-40 scale Domains
Baseline
12 months after MSCT
Symptoms (7–35) Itch (7–35) Fatigue (11–55) Cognition (7–35) Social function (10–50) Emotional function (3–15)
21.3 – 1.8 8.9 – 0.9 32.8 – 2.6 16.3 – 1.9 27.4 – 3.3 9.9 – 0.9
19.7 – 1.5 5.7 – 0.7a 24.3 – 3.6a 15.2 – 1.7 25.4 – 3.1 7.1 – 0.6a
a
P < 0.05.
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Biochemical response after transplantation We compared the serum levels of biomarkers at 1, 3, 6, and 12 months after BM-MSCT with baseline of the ten patients. The mean serum ALT was significantly decreased at 3 and 6 months, 66.8 – 16.1 and 71.5 – 18.4, respectively, compared with baseline, 106.4 – 30.8 (U/L). The mean serum AST was significantly decreased at 3 and 12 months, 65.5 – 11.4 and 64.5 – 11.8, respectively, compared with baseline, 80.7 – 14.5 (U/L). The mean serum g-GT was significantly decreased at 6 months, 365.0 – 127.6, compared with baseline, 508.3 – 194.2 (IU/L). The mean serum DBIL was significantly decreased at 6 months, 13.2 – 4.6, compared with baseline, 20.5 – 8.4 (mM). No significant changes were observed in either serum TBIL or ALP. We also analyzed the serum level of IgM. The mean serum IgM was significantly decreased at 3 and 12 months, 3.39 – 0.37 and 3.56 – 0.38, respectively, compared with baseline, 4.03 – 0.40 (g/L). (Fig. 1).
Phenotypic analysis of lymphocytes To examine the effects of MSCT on lymphocytes, we analyzed lymphocyte subsets in peripheral blood mononuclear cells (PBMCs) by flow cytometry. The percentage of CD4 + T and CD19 + B cells did not change significantly during the follow-up period (Fig. 2A, B). The proportion of CD8 + T cells significantly decreased at 3 and 6 months after MSCT (16.8 – 2.4 vs. 11.2 – 1.9, P = 0.014; 10.1 – 1.7, P = 0.001; Fig. 2C). BM-MSCT treatments increased the CD4 + CD25 + Foxp3 + T cells percentage, which were identified as regulatory T cells (Treg cells) in the T-cell population at 6 months (0.44 – 0.11 vs. 1.23 – 0.26, P = 0.030; Fig. 2D).
Serum cytokine levels Serum IL-10 increased (1.83 – 0.23 pg/mL vs. 2.67 – 0.28 pg/ mL, P = 0.004) at 3 months after MSCT and steadily decreased thereafter. No significant alteration of serum IL-4, IL-6, IL17A, TGF-b1, TNF-a, or IFN-g was detected during the followup period. Figure 3 shows the serum cytokine profile.
Histological changes and immunohistochemistry staining of liver The liver biopsy specimens were obtained from three patients through a percutaneous needle puncture conducted under the guidance of CT scan before and 12 months after BM-MSCT. Blinded analysis by two pathologic experts revealed that the patients with BM-MSCT demonstrated no histological liver deterioration, including fibrosis (grade 0–3), portal and periportal inflammation (grade 0–3), lymphocytic periportal piecemeal necrosis (grade 0–2), ductular proliferation (grade 0–2), intralobular hepatocyte necrosis (grade 0–2), and degree of ductopenia (grade 0–2). Table 3 lists the changes to the histological features of the patients. We also detected CD8 + T cells and Treg-FoxP3 + cells in liver biopsy specimens through immunohistochemistry. CD8 + T cells and FoxP3 + Treg cells were mainly found in inflammatory infiltration sites (Fig. 4). The rate of CD8 + T cells and FoxP3 + Treg cells shown in immunohistochemistry was similar to that in the peripheral blood detected at the same time point.
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FIG. 1. Serial evaluation of serum biochemical markers (months). ALT (A), AST (B), ALP (C), g-GT (D), serum total bilirubin (E), serum direct bilirubin (F), and IgM (G) levels of UDCA-resistant PBC patients at 0, 1, 3, 6, and 12 months after BM-MSCT. Data are expressed as mean – standard error of mean (SEM). Asterisks indicate statistically significant differences (P < 0.05) from the values at baseline. ALT, aminotransferase; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BM-MSCT, bone marrow mesenchymal stromal/stem cell transplantation; g-GT, g-glutamyl transpeptidase; PBC, primary biliary cirrhosis; UDCA, ursodeoxycholic acid.
Follow up During the 12 month follow up, no case had progressed rapidly to decompensated liver disease (eg, ascites, jaundice, coagulopathy, hepatic encephalopathy, or varices) in the trial group.
Discussion Many tissues contain MSCs, including bone marrow, umbilical cord, and adipose tissue. BM-MSCs are readily available and easy to obtain. Based on their low immunogenic potential and immunomodulatory effects on immune responses, alloge-
FIG. 2. Phenotypic analysis of lymphocytes after MSCT (months). Percentage of CD4 + T cells (A), CD19 + B cells (B), CD8 + T cells (C), and CD4 + CD25 + Foxp3 + cells (D) in PBMC of subject patients at 0, 1, 3, 6, and 12 months after BM-MSCT. Data are expressed as mean – standard error of mean (SEM). Asterisks indicate statistically significant differences (P < 0.05) from the values at baseline. PBMC, peripheral blood mononuclear cells.
neic MSCs are used to treat GVHD [12,21] and some CTDs, including systemic lupus erythematosus [22], scleroderma [23], and polymyositis/dermatomyositis [24]. Recently, experimental evidence showed that BM-MSCs might be effective in a Poly I:C -induced PBC mouse model [25]. Therefore, we designed this study to evaluate the safety and clinical and immunologic efficacy of allogeneic BM-MSCT in PBC patients with an incomplete response to UDCA. The liver histologic features of PBC are from nonsuppurative inflammation of small interlobular bile ducts to fibrosis and cirrhosis. Given the irreversibility of fibrosis and cirrhosis, and the poor efficacy of BM-MSCT to
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BM-MSCT TREATMENT IN UDCA-RESISTANT PBC PATIENTS
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FIG. 3. Serial serum cytokine levels after MSCT (months). Serum IL-10 (A), IL-4 (B), IL-6 (C), IL-17A (D), TGF-b (E), TNF-a (F), and INF-g (G) levels of BM-MSCT-recipients at 0, 1, 3, 6, and 12 months after the therapy. Data are expressed as mean – standard error of mean (SEM). Asterisks indicate statistically significant differences (P < 0.05) from the values at baseline. decompensated liver cirrhosis at the late stage of fibrosis [26], we enrolled PBC patients who were in early-to-moderate stages of liver diseases (histological stage IV was excluded) to accept the new therapy. First, we investigated the safety of this treatment. The vital signs of all the patients remained stable during and at 1, 6, 12, 24, 48, and 72 h after infusion. No adverse events were detected during the follow-up period. Routine blood and urine remained normal at 1, 3, 6, and 12 months after MSCT. Together, these data show that allogeneic MSCT was a safe approach. We next assessed the efficacy of BM-MSCT. PBC-40 form is a disease-specific health-related quality of life assessment that is developed and validated exclusively for PBC. It consists of six symptom criteria related to ‘‘fatigue,’’ ‘‘itch,’’ ‘‘cognition,’’ ‘‘symptoms,’’ ‘‘social’’ and ‘‘emotional,’’ and it has been used in PBC to assess severity of fatigue and response to treatment [27]. An improvement in the quality of life, including fatigue and pruritus, is an increasingly important therapeutic goal in the studies of patients with PBC. Although we could not exclude the possible influence of psychological factors, we observed that pruritus, fatigue, and emotional function of the patients significantly improved in this study. Serum biochemical markers such as ALP and g-GT are commonly elevated and defined, along with AMA, to di-
agnose PBC. Mildly elevated serum ALT and AST levels are usually observed in PBC but are not diagnostic. The increased serum level of bilirubin is a late phenomenon in PBC and is an independent predictor of survival. The complete remission of the biochemical markers indicates better prognosis and longer survival without liver transplantation [3]. The biochemical response to UDCA has been defined by numerous criteria: the French criteria [3], which are strongly recommended by AASLD and utilized in our trial, the Spanish criteria [2], and the Dutch criteria [28]. From them, we concluded that ALP, AST, and TBIL/DBIL are important biochemical markers for the evaluation of PBC during clinical trials. In this study, serum ALT and AST significantly diminished at 3 months after MSCT. The downregulation of ALT and AST was maintained at 6 and 12 months after MSCT, respectively. In addition, g-GT and DBIL were reduced 6 months after MSCT. No significant changes were observed in either serum TBIL or ALP. Altogether, these data suggest that MSCT improves liver function of the UDCA-resistant PBC patients. Although AMA and AMA-M2 are useful in diagnosing PBC and may be correlated with disease progression [29], most experts consider it is too rush to draw the conclusion that a high titer of these markers is related to disease severity or progressively worsening histology [30]. Moreover,
Table 3. Analysis of Histological Features by Two Pathology Experts (A&B) Fibrosis
Portal and periportal Lymphocytic periportal Intralobular Ductular inflammation piecemeal necrosis hepatocyte necrosis proliferation Ducto-penia
No. Months
A
B
A
B
A
B
A
B
A
B
A
B
1
1 2 1 1 1 1
1 1 1 1 0 1
2 2 2 2 2 3
1 1 1 1 1 2
2 2 0 0 0 1
1 0 1 0 0 1
0 0 1 1 0 0
0 1 0 1 0 0
0 0 0 0 0 0
0 0 0 0 0 1
1 1 1 0 0 0
0 1 1 1 0 0
3 4
0 12 0 12 0 12
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FIG. 4. CD8 + T cells and TregsFoxP3 + cells immunohistochemistry staining in liver biopsy specimens. (A, B) are CD8 staining before and after MSCT, respectively. (C, D) are FoxP3 staining before and 12 months after MSCT, respectively. Positive cells have brown cytoplasm. Color images available online at www.liebertpub.com/scd
during the American Association for the Study of Liver Diseases endpoints conference, antibody detection was not recommended to be used as an endpoint [14]. Therefore, we did not use AMA/AMA-M2 titers as an endpoint. An important serology feature observed in PBC is the elevation of serum IgM. In this trial, we found that IgM levels were significantly reduced at 3 and 12 months after MSCT, further supporting the efficacy of MSCT. To investigate the role of MSCs in the recipients, we analyzed the lymphocyte phenotype of the PBMCs and several relevant serum cytokines. MSCT was associated with certain changes to the lymphocytes. Levels of CD8 + T cells decreased after MSCT, and were significantly less at month 3 and 6. Given that CD8 + T cells are the primary contributors to autoimmune cholangitis in the adoptive transfer models [31,32] and our observed reduction of CD8 + T cells in the PBMC, we suggest that MSCs might suppress the proliferation and hepatic infiltration of CD8 + T cells. The percentages of CD4 + T and CD19 + B cells did not significantly change during the follow up. CD4 + CD25 + Foxp3 + T cells are widespreadly identified as regulatory T cells (Treg cells) and crucial for the maintenance of immunologic self-tolerance and negative control of overactive autoimmune responses [33]. Lower levels of Treg cells have been observed in the peripheral blood of PBC patients and were attributed to the pathogenesis of the disease [34]. Roord et al. showed that MSCs restored immune homeostasis through activation of CD4 + CD25 + Foxp3 + T cells [35]. In our studies, the treatment of MSCT increased the percentage of CD4 + CD25 + Foxp3 + T cells after 3 months and maintained the levels at 6 months after MSCT, suggesting that upregulation of Treg cells by MSC was one of the mechanisms by which the disease was improved [25]. Previous reports indicate that PBC is a Th1-dominant disease [36]. We evaluated the change of serum cytokines of Th1 and Th2, including IFN-g, IL-4, IL-6, IL-10, and TGFb1 before and after MSCT. Among these cytokines, only IL-
10 levels increased after infusion, while the other cytokine levels did not change. IL-10 is known to have potent immunosuppressive properties [37–39]. It inhibits the growth and differentiation of B cells, T cells, NK cells, and other cells that influence inflammatory responses and stimulates naı¨ve T cells to become Treg cells, which can again produce IL-10 to generate positive feedback [40]. Therefore, upregulation of the IL-10 pathway may be due to MSCT and result in the augmentation of Treg cells, which is the underlying therapeutic pathway for PBC patients. In addition, MSCs may also secrete TGF-b1 to promote the generation of Treg cells [41] and obstruct the differentiation of Th17 [42], thus mediating immune-regulation. However, we did not detect increased serum TGF-b1 levels in this trial in contrast to a previously reported animal experiment [25]. The examination of serum IL-17A levels revealed a slight (but not significant) reduction after MSCT, which may be helpful to PBC patients. No significant alterations of serum IL-4, IL-6, TNF-a, or IFN-g were detected during the follow-up period. Histological evaluation focused on the lesions, and fibrosis has been identified as a predictor of survival [43]. Improvement in liver histology is a desirable outcome in clinical trials of the treatment of PBC [14]. Twelve months after BM-MSCT, all but three patients were reluctant to have two liver biopsies because of surgical discomfort. Interestingly, no histological progression was observed in the three re-biopsied cases, although we did not find any improvement in ductopenia, extent of periportal cell necrosis, interface hepatitis, or fibrosis. The frequency of CD8 + T cells and FoxP3 + Tregs cells was also analyzed in these liver biopsy specimens, and no significant difference was observed. The percentage of CD8 + T cells and Treg-FoxP3 + cells in peripheral blood was also analyzed pretreatment and at 1, 3, 6, and 12 months post-treatment. The percentage of CD8 + T cells was significantly decreased at 3 and 6 months, and that of Treg-FoxP3 + cells increased at 6 months, though these favorable changes did not continue at 12 months
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BM-MSCT TREATMENT IN UDCA-RESISTANT PBC PATIENTS
after stem cell therapy. These results may advise that, for further studies, the second liver biopsy and the analysis of CD8 + T cells and FoxP3 + Treg cells be performed at an earlier posttreatment stage. All patients enrolled in our study had an incomplete response to UDCA, and were destined to have a poor prognosis and more rapid histological progression [1]. During 1 year follow up, no patient progressed rapidly to decompensated liver disease in the trial group. Therefore, these results demonstrate a relatively satisfying outcome because of the minimal histological and clinical deterioration. Our results need confirmation in a prospective randomized trial to confirm efficacy. In summary, this study provides evidence of the safety and efficacy of allogeneic BM-MSCT for the treatment of UDCAresistant PBC patients. BM-MSCT improved the life quality of the patients as demonstrated by responses to the well-established PBC-40 questionnaire. Serum levels of ALT, AST, g-GT, DBIL, and IgM significantly decreased. The immunoregulatory role of BM-MSC administration in PBC patients is likely attributable to an increase of CD4 + CD25 + Foxp3 + T cells, an increase in IL-10 levels, and a reduction of CD8 + T cells. PBC patients undergoing BM-MSCT had no histological liver deterioration such as fibrosis. The optimal therapeutic effect was observed at 3 to 6 months after MSCT and could be maintained for 12 months. Further studies are required to determine the optimal frequency of infusions.
Acknowledgments The authors thank Cui Quan-cai, Lu Zhao-hui (Department of Pathology, PUMCH) for the reading of liver biopsy and analyzing the changes of histological features. This study is supported by the Research Special Fund for Public Welfare Industry of Health (201202004), National Major Scientific and Technological Special Project for ‘‘Significant New Drugs Development’’ (2012ZX09303006-002), National High Technology Research and Development Program of China (2011AA020111), ‘‘863 Projects’’ of Ministry of Science and Technology of PRChina (no. 2011AA020100), the National Key Scientific Program of China (no. 2011CB964901), and Program for Changjiang Scholars and Innovative Research Team in University-PCSIRT (no. IRT0909).
Author Disclosure Statement The authors declare that there is no conflict of interest.
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differentiation, maturation, and function of human monocytederived dendritic cells. Stem Cells Dev 13:263–271. Le Blanc K, F Frassoni, L Ball, F Locatelli, H Roelofs, I Lewis, E Lanino, B Sundberg, ME Bernardo, et al. (2008). Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 371:1579–1586. Sun L, D Wang, J Liang, H Zhang, X Feng, H Wang, B Hua, B Liu, S Ye, et al. (2010). Umbilical cord mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus. Arthritis Rheum 62:2467–2475. Christopeit M, M Schendel, J Foll, LP Muller, G Keysser and G Behre. (2008). Marked improvement of severe progressive systemic sclerosis after transplantation of mesenchymal stem cells from an allogeneic haploidentical-related donor mediated by ligation of CD137L. Leukemia 22:1062–1064. Wang D, H Zhang, M Cao, Y Tang, J Liang, X Feng, H Wang, B Hua, B Liu and L Sun. (2011). Efficacy of allogeneic mesenchymal stem cell transplantation in patients with drug-resistant polymyositis and dermatomyositis. Ann Rheum Dis 70:1285–1288. Wang D, H Zhang, J Liang, Z Gu, X Ma, J Huang, J Lin, Y Hou, L Lu and L Sun. (2011). Effect of allogeneic bone marrow-derived mesenchymal stem cells transplantation in a polyI:C-induced primary biliary cirrhosis mouse model. Clin Exp Med 11:25–32. Mohamadnejad M, K Alimoghaddam, M MohyeddinBonab, M Bagheri, M Bashtar, H Ghanaati, H Baharvand, A Ghavamzadeh and R Malekzadeh. (2007). Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Arch Iran Med 10:459–466. Al-Harthy N, T Kumagi, C Coltescu and GM Hirschfield. (2010). The specificity of fatigue in primary biliary cirrhosis: evaluation of a large clinic practice. Hepatology 52: 562–570. Kuiper EM, BE Hansen, RA de Vries, JW den OudenMuller, TJ van Ditzhuijsen, EB Haagsma, MH Houben, BJ Witteman, KJ van Erpecum and HR van Buuren. (2009). Improved prognosis of patients with primary biliary cirrhosis that have a biochemical response to ursodeoxycholic acid. Gastroenterology 136:1281–1287. Gatselis NK, K Zachou, GL Norman, S Gabeta, P Papamichalis, GK Koukoulis and GN Dalekos. (2013). Clinical significance of the fluctuation of primary biliary cirrhosisrelated autoantibodies during the course of the disease. Autoimmunity 46:471–479. Hu CJ, FC Zhang, YZ Li and X Zhang. (2010). Primary biliary cirrhosis: what do autoantibodies tell us? World J Gastroenterol 16:3616–3629. Itoh S, Y Matsuzaki, T Kimura, T Ikegami, J Shoda, M Fujiwara and N Tanaka. (2000). Cytokine profile of liverinfiltrating CD4 + T cells separated from murine primary biliary cirrhosis-like hepatic lesions induced by graft-versushost reaction. J Gastroenterol Hepatol 15:443–451. Moritoki Y, W Zhang, K Tsuneyama, K Yoshida, K Wakabayashi, GX Yang, C Bowlus, WM Ridgway, Y Ueno, et al. (2009). B cells suppress the inflammatory response in a mouse model of primary biliary cirrhosis. Gastroenterology 136:1037–1047. Maccario R, M Podesta, A Moretta, A Cometa, P Comoli, D Montagna, L Daudt, A Ibatici, G Piaggio, et al. (2005). Interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the
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Address correspondence to: Feng-chun Zhang, MD Department of Rheumatology and Clinical Immunology Peking Union Medical College Hospital (PUMCH) Chinese Academy of Medical Science Beijing 100730 China E-mail: [email protected] Robert Chun-hua Zhao, MD, PhD Institute of Basic Medical Sciences Chinese Academy of Medical Sciences School of Basic Medicine Peking Union Medical College Peking Union Medical College Hospital Center of Excellence in Tissue Engineering Chinese Academy of Medical Sciences Beijing 100005 China E-mail: [email protected] Received for publication October 13, 2013 Accepted after revision May 15, 2014 Prepublished on Liebert Instant Online XXXX XX, XXXX
STEM CELLS AND DEVELOPMENT Volume 00, Number 00, 2014 Mary Ann Liebert, Inc. DOI: 10.1089/scd.2013.0500
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Allogeneic Bone Marrow Mesenchymal Stem Cell Transplantation in Patients with UDCA-Resistant Primary Biliary Cirrhosis Li Wang,1,* Qin Han,2,* Hua Chen,1 Ke Wang,2 Guang-liang Shan,2 Fang Kong,1 Yun-jiao Yang,1 Yong-zhe Li,1 Xuan Zhang,1 Fen Dong,2 Qian Wang,1 Dong Xu,1 Zhao-jun Hu,1 Shi-hua Wang,2 Armand Keating,3 Ya-lan Bi,4 Feng-Chun Zhang,1 and Robert Chun-Hua Zhao 2
The objective of this study was to evaluate the safety and efficacy of allogeneic bone marrow mesenchymal stromal/stem cell transplantation (BM-MSCT) for patients with ursodeoxycholic acid (UDCA)-resistant primary biliary cirrhosis (PBC). Ten patients were enrolled in this trial of BM-MSCT. All patients were permitted to concurrently continue their previous UDCA treatment. The efficacy of BM-MSCT in UDCA-resistant PBC was assessed at various time points throughout the 12-month follow up. No transplantation-related side effects were observed. The life quality of the patients was improved after BM-MSCT as demonstrated by responses to the PBC-40 questionnaire. Serum levels of ALT, AST, g-GT, and IgM significantly decreased from baseline after BM-MSCT. In addition, the percentage of CD8 + T cells was reduced, while that of CD4 + CD25 + Foxp3 + T cells was increased in peripheral lymphocytic subsets. Serum levels of IL-10 were also elevated. Notably, the optimal therapeutic outcome was acquired in 3 to 6 months and could be maintained for 12 months after BMMSCT. In conclusion, allogeneic BM-MSCT in UDCA-resistant PBC is safe and appears to be effective.
of ductopenia and severe icteric cholestasis, progressing very quickly toward cirrhosis in less than 5 years [1]. Therefore, there is an urgent need for new therapeutic investigations for patients with UDCA-resistant PBC. Mesenchymal stromal/stem cells (MSCs) were originally isolated from bone marrow and widely studied for their ability to differentiate into multiple tissues, including bone, fat, cartilage, neurons, hepatocytes, and cardiocytes. We isolated a subpopulation of MSCs designated Flk-1 + MSCs from a variety of human tissues and demonstrated their wide differentiation potential and immunomodulatory functions [5–9]. Ex vivo expansion and in vivo infusion of bone marrow-derived Flk-1 + MSCs showed both feasibility and safety from monkey to human [10]. An important function of MSCs for autoimmune diseases is their immunomodulatory effect on various activated lymphoid cells such as T cells, B cells, natural killer cells, and dendritic cells. MSCs express low levels of HLA class I major histocompatibility complex (MHC) molecules and no class II MHC or costimulatory molecules [11]. Our
Introduction
P
rimary biliary cirrhosis (PBC) is a chronic autoimmune inflammatory liver disease that leads to the nonsuppurative destruction of small interlobular bile ducts, progressive cholestastis, and, eventually, liver fibrosis and cirrhosis. Ursodeoxycholic acid (UDCA) is currently the only drug for the treatment of PBC approved by FDA. Several lines of evidence indicate that UDCA improves biochemical liver tests, slows histological progression, and, therefore, may prolong patient survival without liver transplantation [1]. However, all of these benefits are only acquired in patients who have a perfect biochemical response to UDCA. An incomplete biochemical response to UDCA (UDCA resistant) at 1 year is more frequently observed in patients with more severe baseline biochemical and histological features and with poor prognosis [2,3]. We had even found that early biochemical response at 3 to 6 months could efficiently identify patients at a risk of poor outcome [4]. These patients, tragically, have a very rapid onset
1 Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Science, Beijing, China. 2 Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Peking Union Medical College Hospital, Center of Excellence in Tissue Engineering Chinese Academy of Medical Sciences, Beijing, China. 3 Institute of Medical Science, University of Toronto, Toronto, Canada. 4 Department of Pathology, Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Science, Beijing, China. *These authors contributed equally to this work.
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previous clinical trial with 33 patients demonstrated that a new transplantation strategy combining haploidentical peripheral blood stem cells (PBSCs) and Flk-1 + MSCs could prevent severe graft-versus-host disease (GVHD) [12]. In addition, we found that Flk-1 + MSCs infusion was therapeutically practicable, with no detectable side effects during the treatment of sclerodermatous chronic GVHD in a clinical report of four patients [13]. These results support the possibility of using MSCs for therapeutic applications in autoimmune diseases. In this study, we enrolled UDCA-resistant PBC patients to undergo bone marrow MSC transplantation (BM-MSCT) from their healthy family donors. This trial was approved by the Ethics Committee at Peking Union Medical College Hospital, China, in February 2010.
Patients and Methods Study design and follow-up protocol The study protocol was based on a design from the American Association for the Study of Liver Diseases Endpoints Conference [14]. All research on patients and donors in this study have been approved by the Ethical Review Board of Peking Union Medical College Hospital, and informed consents were obtained. The safety assessments included vital signs and discomfort records during BM-MSC infusion and at 1, 6, 12, 24, 48, and 72 h after that. Follow-up evaluations were conducted at 1, 3, 6, and 12 months after MSCT. The investigators recorded adverse events and assessed the quality of life and disease severity throughout the study. The quality of life assessment was evaluated using a PBC-40 questionnaire [15]. The disease severity was measured using the serum levels of the following biochemical markers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), g-glutamyl transpeptidase (g-GT), total bilirubin (TBIL), direct bilirubin (DBIL), and immunoglobulin (Ig) M; cytokines: interleukin (IL)-4, IL-6, IL-10, IL-17A, interferon (IFN)-g, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)-b1; and phenotypic analysis of peripheral blood lymphocytes and histological features of liver biopsies (before and 12 months after transplantation) [16].
Patients and donors Ten patients (nine women and one man, whose median age was 50 years, with 4.5 years of disease duration and a
liver histological stage of I to III that was classified by Ludwig–Scheuer criteria [17,18]) were enrolled. These patients met the 2009 AASLD criteria [19] for PBC without evidence of decompensated liver disease, other connective tissue disease (CTD), or liver disease. All ten patients had an incomplete biochemical response to standard doses of UDCA (13–15 mg/kg/day) for more than 1 year according to the Paris criteria (ALP > 3 upper limit of normal (ULN), AST > 2 ULN, and bilirubin ‡ 1 mg/dL after 1 year of UDCA treatment) [3]. Permitted medications included prednisone ( £10 mg/d) and UDCA at a dose that was maintained at the pre-enrollment dose. Donors were first grade relative of the patients. They were considered healthy and qualified only if they had no disease history and normal test of blood routine, urine routine, liver and kidney function, and negative antinuclear antibody (ANA) and anti anti-mitochondrial antibody (AMA). The median age of the donors was 27.5 years old. No patient had withdrawn during a follow up of at least 12 months. Table 1 lists the clinical features of the PBC patients and the donor control for each of them.
BM-MSC preparation and transplantation All infused allogeneic MSCs were obtained from healthy family donors (eight participants from their own children and two from their brothers). The Flk-1 + CD31 - CD34 MSCs were collected and cultured as previously described [20]. Briefly, approximately 20 mL volunteers’ bone marrow aspirates were collected, and mononuclear cells were separated by Ficoll gradient centrifugation (density 1.077g/cm3). The interphase layer was collected and resuspended in Dulbecco’s modified Eagle’s medium (low-glucose DMEM/ F12), 40% MCDB-201 (Sigma) supplemented with 2% fetal bovine serum (FBS; Gibco Life Technologies), 1-insulin transferring selenium (Gibco Life Technologies), 10 - 9 M dexamethasone (Sigma), 10 - 4 M ascorbic acid 2-phosphate (Sigma), 10 ng/mL epidermal growth factor (Sigma), and 10 ng/mL platelet-derived growth factor BB (Sigma), and then plated in culture flasks (1 · 107 cells/75 cm2). The culture was maintained at 37C in a humidified environment containing 5% CO2. After 24–48 h of culture, nonadherent cells were removed, and the adherent layer was cultured until it reached 70%–80% confluence. The cells were then detached with 0.05% trypsin and 0.01% EDTA and replated. MSCs were harvested, used at passage 3 to 5,
Table 1. Patient and Donor Characteristics
No.
Sex/age (years)
Disease duration (years)
Baseline of ALP (U/L)
Baseline of TBIL (lM)
Baseline of AMA (x)
Baseline of AMA-M2 (U/mL)
Phase of histology
Donor/age (years)
Follow-up (months)
1 2 3 4 5 6 7 8 9 10
F/53 F/46 F/58 M/51 F/54 F/31 F/61 F/49 F/46 F/42
7 3 5 3 5 2 4 6 2 5
517 1149 240 407 522 766 390 441 1385 378
33.9 66.2 11.5 27.5 45.3 21.7 34.9 21 108.7 122
160 160 320 1280 640 1280 160 640 1280 1280
105 0 > 200 > 200 > 200 > 200 89 > 200 > 200 > 200
II II II I II I II II II III
Daughter/28 Son/21 Daughter/32 Daughter/23 Daughter/27 Brother/40 Son/35 Son/27 Son/18 Brother/45
16 16 16 16 16 15 15 14 13 12
BM-MSCT TREATMENT IN UDCA-RESISTANT PBC PATIENTS
and intravenously infused at a density of 3–5 · 105 cells per kg of body weight.
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Immunohistochemistry staining The 4 mm liver biopsy specimen sections were washed in PBS, incubated in 3% hydrogen peroxide for 10 min, and then washed in PBS. The nonspecific binding was blocked by incubating the sections in serum for 60 min. The slides were then incubated with the first antibody for 1 h, incubated with Biotin-conjugated secondary antibodies for 15 min at room temperature, colorated with DAB, counterstained with hematoxylin, and then observed under microscopy. The first antibodies used were goat monoclonal antibody (mAb) against Foxp3(Abcam; 1: 1000) and mouse mAb against CD8 (DAKO; 1: 50).
Statistical analysis Values were expressed as the mean – standard error of the mean (SEM). Paired Student’s t-tests and Signed Rank test were performed to compare the normal distribution data and non-normal distribution data, respectively, by statistical software (SAS). P < 0.05 was considered statistically significant.
Results Safety of BM-MSC transplantation No fever, chills, or vomiting appeared during the course of MSC infusion. We did not observe any fever, chills, or vomiting during the course of MSC infusion. Vital signs of the patients remained stable during a 72 h-observation. No adverse events were observed, and routine blood and urine studies remained normal at 1, 3, and 6 months after transplantation. No infusion-related instant reaction occurred. The infusion was well tolerated in patients, and no rejection occurred.
Efficacy of BM-MSC transplantation Life quality assessment. The PBC-40 questionnaire was used to estimate the patients’ quality of life by six criteria, including symptoms, itchiness, fatigue, cognition, and social and emotional function before and 12 months after MSCT. We observed improvements at 12 months after MSCT in itchiness (8.9 – 0.9 vs. 5.7 – 0.7, P = 0.001), fatigue (32.8 – 2.6 vs. 24.3 – 3.6, P = 0.022), and emotional function (9.9 – 0.9 vs. 7.1 – 0.6, P = 0.003). Table 2 lists the changes in the patients’ quality of life using the PBC-40 scale.
Table 2. Assessment of Quality of Life by PBC-40 scale Domains
Baseline
12 months after MSCT
Symptoms (7–35) Itch (7–35) Fatigue (11–55) Cognition (7–35) Social function (10–50) Emotional function (3–15)
21.3 – 1.8 8.9 – 0.9 32.8 – 2.6 16.3 – 1.9 27.4 – 3.3 9.9 – 0.9
19.7 – 1.5 5.7 – 0.7a 24.3 – 3.6a 15.2 – 1.7 25.4 – 3.1 7.1 – 0.6a
a
P < 0.05.
3
Biochemical response after transplantation We compared the serum levels of biomarkers at 1, 3, 6, and 12 months after BM-MSCT with baseline of the ten patients. The mean serum ALT was significantly decreased at 3 and 6 months, 66.8 – 16.1 and 71.5 – 18.4, respectively, compared with baseline, 106.4 – 30.8 (U/L). The mean serum AST was significantly decreased at 3 and 12 months, 65.5 – 11.4 and 64.5 – 11.8, respectively, compared with baseline, 80.7 – 14.5 (U/L). The mean serum g-GT was significantly decreased at 6 months, 365.0 – 127.6, compared with baseline, 508.3 – 194.2 (IU/L). The mean serum DBIL was significantly decreased at 6 months, 13.2 – 4.6, compared with baseline, 20.5 – 8.4 (mM). No significant changes were observed in either serum TBIL or ALP. We also analyzed the serum level of IgM. The mean serum IgM was significantly decreased at 3 and 12 months, 3.39 – 0.37 and 3.56 – 0.38, respectively, compared with baseline, 4.03 – 0.40 (g/L). (Fig. 1).
Phenotypic analysis of lymphocytes To examine the effects of MSCT on lymphocytes, we analyzed lymphocyte subsets in peripheral blood mononuclear cells (PBMCs) by flow cytometry. The percentage of CD4 + T and CD19 + B cells did not change significantly during the follow-up period (Fig. 2A, B). The proportion of CD8 + T cells significantly decreased at 3 and 6 months after MSCT (16.8 – 2.4 vs. 11.2 – 1.9, P = 0.014; 10.1 – 1.7, P = 0.001; Fig. 2C). BM-MSCT treatments increased the CD4 + CD25 + Foxp3 + T cells percentage, which were identified as regulatory T cells (Treg cells) in the T-cell population at 6 months (0.44 – 0.11 vs. 1.23 – 0.26, P = 0.030; Fig. 2D).
Serum cytokine levels Serum IL-10 increased (1.83 – 0.23 pg/mL vs. 2.67 – 0.28 pg/ mL, P = 0.004) at 3 months after MSCT and steadily decreased thereafter. No significant alteration of serum IL-4, IL-6, IL17A, TGF-b1, TNF-a, or IFN-g was detected during the followup period. Figure 3 shows the serum cytokine profile.
Histological changes and immunohistochemistry staining of liver The liver biopsy specimens were obtained from three patients through a percutaneous needle puncture conducted under the guidance of CT scan before and 12 months after BM-MSCT. Blinded analysis by two pathologic experts revealed that the patients with BM-MSCT demonstrated no histological liver deterioration, including fibrosis (grade 0–3), portal and periportal inflammation (grade 0–3), lymphocytic periportal piecemeal necrosis (grade 0–2), ductular proliferation (grade 0–2), intralobular hepatocyte necrosis (grade 0–2), and degree of ductopenia (grade 0–2). Table 3 lists the changes to the histological features of the patients. We also detected CD8 + T cells and Treg-FoxP3 + cells in liver biopsy specimens through immunohistochemistry. CD8 + T cells and FoxP3 + Treg cells were mainly found in inflammatory infiltration sites (Fig. 4). The rate of CD8 + T cells and FoxP3 + Treg cells shown in immunohistochemistry was similar to that in the peripheral blood detected at the same time point.
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FIG. 1. Serial evaluation of serum biochemical markers (months). ALT (A), AST (B), ALP (C), g-GT (D), serum total bilirubin (E), serum direct bilirubin (F), and IgM (G) levels of UDCA-resistant PBC patients at 0, 1, 3, 6, and 12 months after BM-MSCT. Data are expressed as mean – standard error of mean (SEM). Asterisks indicate statistically significant differences (P < 0.05) from the values at baseline. ALT, aminotransferase; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BM-MSCT, bone marrow mesenchymal stromal/stem cell transplantation; g-GT, g-glutamyl transpeptidase; PBC, primary biliary cirrhosis; UDCA, ursodeoxycholic acid.
Follow up During the 12 month follow up, no case had progressed rapidly to decompensated liver disease (eg, ascites, jaundice, coagulopathy, hepatic encephalopathy, or varices) in the trial group.
Discussion Many tissues contain MSCs, including bone marrow, umbilical cord, and adipose tissue. BM-MSCs are readily available and easy to obtain. Based on their low immunogenic potential and immunomodulatory effects on immune responses, alloge-
FIG. 2. Phenotypic analysis of lymphocytes after MSCT (months). Percentage of CD4 + T cells (A), CD19 + B cells (B), CD8 + T cells (C), and CD4 + CD25 + Foxp3 + cells (D) in PBMC of subject patients at 0, 1, 3, 6, and 12 months after BM-MSCT. Data are expressed as mean – standard error of mean (SEM). Asterisks indicate statistically significant differences (P < 0.05) from the values at baseline. PBMC, peripheral blood mononuclear cells.
neic MSCs are used to treat GVHD [12,21] and some CTDs, including systemic lupus erythematosus [22], scleroderma [23], and polymyositis/dermatomyositis [24]. Recently, experimental evidence showed that BM-MSCs might be effective in a Poly I:C -induced PBC mouse model [25]. Therefore, we designed this study to evaluate the safety and clinical and immunologic efficacy of allogeneic BM-MSCT in PBC patients with an incomplete response to UDCA. The liver histologic features of PBC are from nonsuppurative inflammation of small interlobular bile ducts to fibrosis and cirrhosis. Given the irreversibility of fibrosis and cirrhosis, and the poor efficacy of BM-MSCT to
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BM-MSCT TREATMENT IN UDCA-RESISTANT PBC PATIENTS
5
FIG. 3. Serial serum cytokine levels after MSCT (months). Serum IL-10 (A), IL-4 (B), IL-6 (C), IL-17A (D), TGF-b (E), TNF-a (F), and INF-g (G) levels of BM-MSCT-recipients at 0, 1, 3, 6, and 12 months after the therapy. Data are expressed as mean – standard error of mean (SEM). Asterisks indicate statistically significant differences (P < 0.05) from the values at baseline. decompensated liver cirrhosis at the late stage of fibrosis [26], we enrolled PBC patients who were in early-to-moderate stages of liver diseases (histological stage IV was excluded) to accept the new therapy. First, we investigated the safety of this treatment. The vital signs of all the patients remained stable during and at 1, 6, 12, 24, 48, and 72 h after infusion. No adverse events were detected during the follow-up period. Routine blood and urine remained normal at 1, 3, 6, and 12 months after MSCT. Together, these data show that allogeneic MSCT was a safe approach. We next assessed the efficacy of BM-MSCT. PBC-40 form is a disease-specific health-related quality of life assessment that is developed and validated exclusively for PBC. It consists of six symptom criteria related to ‘‘fatigue,’’ ‘‘itch,’’ ‘‘cognition,’’ ‘‘symptoms,’’ ‘‘social’’ and ‘‘emotional,’’ and it has been used in PBC to assess severity of fatigue and response to treatment [27]. An improvement in the quality of life, including fatigue and pruritus, is an increasingly important therapeutic goal in the studies of patients with PBC. Although we could not exclude the possible influence of psychological factors, we observed that pruritus, fatigue, and emotional function of the patients significantly improved in this study. Serum biochemical markers such as ALP and g-GT are commonly elevated and defined, along with AMA, to di-
agnose PBC. Mildly elevated serum ALT and AST levels are usually observed in PBC but are not diagnostic. The increased serum level of bilirubin is a late phenomenon in PBC and is an independent predictor of survival. The complete remission of the biochemical markers indicates better prognosis and longer survival without liver transplantation [3]. The biochemical response to UDCA has been defined by numerous criteria: the French criteria [3], which are strongly recommended by AASLD and utilized in our trial, the Spanish criteria [2], and the Dutch criteria [28]. From them, we concluded that ALP, AST, and TBIL/DBIL are important biochemical markers for the evaluation of PBC during clinical trials. In this study, serum ALT and AST significantly diminished at 3 months after MSCT. The downregulation of ALT and AST was maintained at 6 and 12 months after MSCT, respectively. In addition, g-GT and DBIL were reduced 6 months after MSCT. No significant changes were observed in either serum TBIL or ALP. Altogether, these data suggest that MSCT improves liver function of the UDCA-resistant PBC patients. Although AMA and AMA-M2 are useful in diagnosing PBC and may be correlated with disease progression [29], most experts consider it is too rush to draw the conclusion that a high titer of these markers is related to disease severity or progressively worsening histology [30]. Moreover,
Table 3. Analysis of Histological Features by Two Pathology Experts (A&B) Fibrosis
Portal and periportal Lymphocytic periportal Intralobular Ductular inflammation piecemeal necrosis hepatocyte necrosis proliferation Ducto-penia
No. Months
A
B
A
B
A
B
A
B
A
B
A
B
1
1 2 1 1 1 1
1 1 1 1 0 1
2 2 2 2 2 3
1 1 1 1 1 2
2 2 0 0 0 1
1 0 1 0 0 1
0 0 1 1 0 0
0 1 0 1 0 0
0 0 0 0 0 0
0 0 0 0 0 1
1 1 1 0 0 0
0 1 1 1 0 0
3 4
0 12 0 12 0 12
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WANG ET AL.
FIG. 4. CD8 + T cells and TregsFoxP3 + cells immunohistochemistry staining in liver biopsy specimens. (A, B) are CD8 staining before and after MSCT, respectively. (C, D) are FoxP3 staining before and 12 months after MSCT, respectively. Positive cells have brown cytoplasm. Color images available online at www.liebertpub.com/scd
during the American Association for the Study of Liver Diseases endpoints conference, antibody detection was not recommended to be used as an endpoint [14]. Therefore, we did not use AMA/AMA-M2 titers as an endpoint. An important serology feature observed in PBC is the elevation of serum IgM. In this trial, we found that IgM levels were significantly reduced at 3 and 12 months after MSCT, further supporting the efficacy of MSCT. To investigate the role of MSCs in the recipients, we analyzed the lymphocyte phenotype of the PBMCs and several relevant serum cytokines. MSCT was associated with certain changes to the lymphocytes. Levels of CD8 + T cells decreased after MSCT, and were significantly less at month 3 and 6. Given that CD8 + T cells are the primary contributors to autoimmune cholangitis in the adoptive transfer models [31,32] and our observed reduction of CD8 + T cells in the PBMC, we suggest that MSCs might suppress the proliferation and hepatic infiltration of CD8 + T cells. The percentages of CD4 + T and CD19 + B cells did not significantly change during the follow up. CD4 + CD25 + Foxp3 + T cells are widespreadly identified as regulatory T cells (Treg cells) and crucial for the maintenance of immunologic self-tolerance and negative control of overactive autoimmune responses [33]. Lower levels of Treg cells have been observed in the peripheral blood of PBC patients and were attributed to the pathogenesis of the disease [34]. Roord et al. showed that MSCs restored immune homeostasis through activation of CD4 + CD25 + Foxp3 + T cells [35]. In our studies, the treatment of MSCT increased the percentage of CD4 + CD25 + Foxp3 + T cells after 3 months and maintained the levels at 6 months after MSCT, suggesting that upregulation of Treg cells by MSC was one of the mechanisms by which the disease was improved [25]. Previous reports indicate that PBC is a Th1-dominant disease [36]. We evaluated the change of serum cytokines of Th1 and Th2, including IFN-g, IL-4, IL-6, IL-10, and TGFb1 before and after MSCT. Among these cytokines, only IL-
10 levels increased after infusion, while the other cytokine levels did not change. IL-10 is known to have potent immunosuppressive properties [37–39]. It inhibits the growth and differentiation of B cells, T cells, NK cells, and other cells that influence inflammatory responses and stimulates naı¨ve T cells to become Treg cells, which can again produce IL-10 to generate positive feedback [40]. Therefore, upregulation of the IL-10 pathway may be due to MSCT and result in the augmentation of Treg cells, which is the underlying therapeutic pathway for PBC patients. In addition, MSCs may also secrete TGF-b1 to promote the generation of Treg cells [41] and obstruct the differentiation of Th17 [42], thus mediating immune-regulation. However, we did not detect increased serum TGF-b1 levels in this trial in contrast to a previously reported animal experiment [25]. The examination of serum IL-17A levels revealed a slight (but not significant) reduction after MSCT, which may be helpful to PBC patients. No significant alterations of serum IL-4, IL-6, TNF-a, or IFN-g were detected during the follow-up period. Histological evaluation focused on the lesions, and fibrosis has been identified as a predictor of survival [43]. Improvement in liver histology is a desirable outcome in clinical trials of the treatment of PBC [14]. Twelve months after BM-MSCT, all but three patients were reluctant to have two liver biopsies because of surgical discomfort. Interestingly, no histological progression was observed in the three re-biopsied cases, although we did not find any improvement in ductopenia, extent of periportal cell necrosis, interface hepatitis, or fibrosis. The frequency of CD8 + T cells and FoxP3 + Tregs cells was also analyzed in these liver biopsy specimens, and no significant difference was observed. The percentage of CD8 + T cells and Treg-FoxP3 + cells in peripheral blood was also analyzed pretreatment and at 1, 3, 6, and 12 months post-treatment. The percentage of CD8 + T cells was significantly decreased at 3 and 6 months, and that of Treg-FoxP3 + cells increased at 6 months, though these favorable changes did not continue at 12 months
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BM-MSCT TREATMENT IN UDCA-RESISTANT PBC PATIENTS
after stem cell therapy. These results may advise that, for further studies, the second liver biopsy and the analysis of CD8 + T cells and FoxP3 + Treg cells be performed at an earlier posttreatment stage. All patients enrolled in our study had an incomplete response to UDCA, and were destined to have a poor prognosis and more rapid histological progression [1]. During 1 year follow up, no patient progressed rapidly to decompensated liver disease in the trial group. Therefore, these results demonstrate a relatively satisfying outcome because of the minimal histological and clinical deterioration. Our results need confirmation in a prospective randomized trial to confirm efficacy. In summary, this study provides evidence of the safety and efficacy of allogeneic BM-MSCT for the treatment of UDCAresistant PBC patients. BM-MSCT improved the life quality of the patients as demonstrated by responses to the well-established PBC-40 questionnaire. Serum levels of ALT, AST, g-GT, DBIL, and IgM significantly decreased. The immunoregulatory role of BM-MSC administration in PBC patients is likely attributable to an increase of CD4 + CD25 + Foxp3 + T cells, an increase in IL-10 levels, and a reduction of CD8 + T cells. PBC patients undergoing BM-MSCT had no histological liver deterioration such as fibrosis. The optimal therapeutic effect was observed at 3 to 6 months after MSCT and could be maintained for 12 months. Further studies are required to determine the optimal frequency of infusions.
Acknowledgments The authors thank Cui Quan-cai, Lu Zhao-hui (Department of Pathology, PUMCH) for the reading of liver biopsy and analyzing the changes of histological features. This study is supported by the Research Special Fund for Public Welfare Industry of Health (201202004), National Major Scientific and Technological Special Project for ‘‘Significant New Drugs Development’’ (2012ZX09303006-002), National High Technology Research and Development Program of China (2011AA020111), ‘‘863 Projects’’ of Ministry of Science and Technology of PRChina (no. 2011AA020100), the National Key Scientific Program of China (no. 2011CB964901), and Program for Changjiang Scholars and Innovative Research Team in University-PCSIRT (no. IRT0909).
Author Disclosure Statement The authors declare that there is no conflict of interest.
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Address correspondence to: Feng-chun Zhang, MD Department of Rheumatology and Clinical Immunology Peking Union Medical College Hospital (PUMCH) Chinese Academy of Medical Science Beijing 100730 China E-mail: [email protected] Robert Chun-hua Zhao, MD, PhD Institute of Basic Medical Sciences Chinese Academy of Medical Sciences School of Basic Medicine Peking Union Medical College Peking Union Medical College Hospital Center of Excellence in Tissue Engineering Chinese Academy of Medical Sciences Beijing 100005 China E-mail: [email protected] Received for publication October 13, 2013 Accepted after revision May 15, 2014 Prepublished on Liebert Instant Online XXXX XX, XXXX
