The clinical trial of allogenic mesenchymal stem cells (MSCs) transplantation for refractory SLE patients has shown significant safety and efficacy profiles. However, the optimum frequency of the MSCs transplantation (MSCT) is unknown. This study was undertaken to observe whether double transplantations of MSCs is superior to single transplantation. Fifty-eight refractory SLE patients were enrolled in this study, in which 30 were randomly given single MSCT, and the other 28 were given double MSCT. Patients were followed up for rates of survival, disease remission, and relapse, as well as transplantation-related adverse events. SLE disease activity index (SLEDAI) and serologic features were evaluated. Our results showed that no remarkable differences between single and double allogenic MSCT were found in terms of disease remission and relapse, amelioration of disease activity, and serum indexes in an SLE clinical trial with more than one year followup. This study demonstrated that single MSCs transplantation at the dose of one million MSCs per kilogram of body weight was sufficient to induce disease remission for refractory SLE patients.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiorgan involvement and loss of tolerance against self-antigens followed by antibody production. Current treatments of severe SLE flares consist of toxic immunosuppressive drugs, most commonly cyclophosphamide (CYC), mycophenolate mofetil, and leflunomide [
Mesenchymal stem cells (MSCs) have potent immunosuppressive capacity, which is demonstrated by the inhibition of T lymphocytes proliferation and proinflammatory cytokines production
As the first example of efficacy, clinical trials for prevention and treatment of graft-versus-host disease (GVHD) after HSC transplantation show that MSCs can modulate allogenic immune responses and effectively treat human disease. Now these multipotential cells have been applied in various physical and immune injuries including liver cirrhosis, multiple sclerosis, and Crohn’s disease [
From March 2007 through February 2010, 58 patients with SLE refractory to standard therapies were enrolled in allogenic MSCs transplantation (MSCT) trial at the Affiliated Drum Tower Hospital of Nanjing University Medical School after signing informed consent. The study was approved by the Ethics Committee at The Drum Tower Hospital of Nanjing University Medical School and registered at
Bone-marrow-derived MSCs (BMMSCs) were obtained from healthy family donors after signing informed consents. Bone marrow mononuclear cells were separated by density gradient centrifugation as previously described [
Criteria for release of MSCs for clinical use included presence of visible clumps, spindle-shape morphology, and absence of contamination by pathogens (as documented by aerobic and anaerobic cultures before release), as well as by virus for hepatitis B surface antigen, hepatitis B core antibody, hepatitis C virus antibody, human immunodeficiency virus antibodies I and II, cytomegalovirus IgM, and syphilis antibody (as determined by enzyme-linked immunosorbent assay [ELISA] before release), cell viability greater than 92% (as determined by trypan blue testing), and immune phenotyping proving expression of CD73, CD105, CD90, CD29 (
Randomization was conducted between once and double MSCT groups. The enrolled 58 refractory SLE patients were randomly assigned into once or double MSCT groups. Of all the patients, 30 were randomly given a single MSCs transplantation, and the other 28 patients received double allogenic MSCs transplantations, with an interval for 1 week. Before MSCT, all patients were administered CYC (10 mg per kilogram per day) intravenously on days 4, 3, and 2 to inhibit active lymphocytes. Patients received allogenic MSCs intravenously at the density of one million cells per kilogram of body weight in each transplantation.
After MSCT, all patients returned for scheduled followup at 1, 3, 6, and 12 months and then yearly thereafter. Medical history, physical examination, and serologic testing were performed. Complete remission was defined as SLEDAI score
After UC-MSCT, the dose of prednisone and immunosuppressive drugs was tapered when clinical efficacy was achieved for each patient. The withdrawal of prednisone and immunosuppressant was permitted if patient’s condition continued to improve. No other immunosuppressant was used unless disease relapsed. If the patient underwent disease relapse, he or she will withdraw from the study and will be given higher dose of prednisone or other immunosuppressants according to disease conditions. The patient’s clinical data after relapse and change of clinical regimens will not be included for analysis.
Descriptive statistics was used to summarize patient characteristics. Differences in patient demographics prior transplantation were analyzed by unpaired
Fifty-eight patients with refractory SLE enrolled in this trial, and all patients underwent allogenic MSCT and were followedup for more than 12 months. The mean followup was 27 months (range from 12 to 48 months) in single transplantation group and 26 months (range from 12 to 40 months) in double transplantation group. Patients’ demographics pretransplantation have been shown in Table
Patient demographics pretransplantation.
Variable | No. of patients | |
---|---|---|
Single MSCT | Double MSCT | |
( |
( |
|
Age in years | 30 (12–47) | 33 (16–54) |
Gender, |
25/5 | 26/2 |
Race, |
30/0 | 28/0 |
Disease duration ( |
62 (7–232) | 92 (12–264) |
MSCs source, |
||
Bone marrow (BM) | 12 | 9 |
Umbilical cord (UC) | 18 | 19 |
Medium followup for survivors ( |
27 (12–48) | 26 (12–40) |
MSCT, mesenchymal stem cells transplantation.
With medium followup of over 24 months in both cohorts, one death was observed in double transplantation group. The survival rate was 100% for single and 96.4% for double transplantation group, respectively (log-rank = 1.071,
Rate of complete remission (a) and disease relapse (b) for patients with single and double MSCs transplantations, by Kaplan-Meier survival curve analysis.
We calculated the overall percentage of disease relapse that occurred in two groups (8/30, 26.7% in single MSCT group; 6/27, 22.2% in double MSCT group). Additionally, the exact rate of disease relapse was calculated by Kaplan-Meier survival curve (Figure
Disease activity shown by SLEDAI scores decreased significantly in both groups after allogenic MSCs transplantation by repeated measures ANOVA (
Comparisons of SLEDAI score (a), serum albumin (b), Complement 3 (C3, (c)), and anti-double-strand DNA antibody (dsDNA, (d)) for patients with single and double MSCs transplantations, by repeated measures ANOVA. Values are the mean ± SEM.
Twenty-six patients (26/30, 86.7%) in single MSCT group and 24 patients (24/27, 88.9%) in double MSCT group underwent renal involvement at baseline, shown by the presence of proteinuria, or hematuria, or renal disfunction. The 24 hour proteinuria significantly declined after allogenic MSCT within each group by repeated measures ANOVA (
Comparisons of proteinuria (a), serum creatinine (b), platelet count (c), and hemoglobin level (d) between patients given single or double MSCs transplantations, by repeated measures of ANOVA. Values are the mean ± SEM.
One patient in double transplantation group underwent uncontrolled disease recurrence 6 months after MSCT due to upper respiratory tract infection. She was not responsive to conventional treatments and finally died of acute heart failure. During 4 years followup, 7 patients in single transplantation group (23.3%) and 9 patients in double transplantation group (32.1%) suffered infection events, and no statistical difference was found between the two groups. Of 7 patients in single transplantation group, 3 had upper respiratory tract infection, 3 had intestinal infection, and one had oral fungi infection. Of 9 patients in double transplantation group, 4 had upper respiratory tract infection, 2 had intestinal infection, one had herpes zoster infection, one had pneumonia, and one had pulmonary tuberculosis. All the adverse events were not considered transplantation related.
Two patients in both single (2/30; 6.7%) and double transplantation groups (2/28; 7.1%) had discontinued immunosuppressive drugs in the last followup. Dose of prednisone was tapered to 5–10 mg/day for 24 patients (24/30, 80.0%) in single MSCT group and 22 patients (22/28, 78.6%) in double MSCT group, respectively. Maintenance therapy regimen was defined as the dose of prednisone was not more than 10 mg/day, combined with the dose of immunosuppressive drug was not more than 0.4–0.6 gm/3 months of CYC, 10 mg/day of leflunomide, or 0.5 gm/day of mycophenolate mofetil. Eleven and 7 patients in single (11/30; 36.7%) and double (7/28; 25.0%) transplantation groups achieved above-mentioned maintenance therapy in the last followup. Time to reach maintenance therapy was not different between single (11.8 months, 3–24 months) and double (10.0 months, 4–15 months) transplantation groups.
Systemic infusions of mesenchymal stem cells have been widely used in clinical applications. However, the appropriate dose of cells for each patient is still unknown. The dose of MSCs in current studies relied to a large extent on clinical experience and lack of rigorous standards. In a phase II clinical trial for MSCs transplantation in GVHD and followed up for 5 years [
This study for the first time represents a large single-institution series of refractory SLE patients receiving single or double MSCs transplantations. We found a considerable improvement in disease remission for patients transplanted single and double allogenic MSCs. In previous studies, we have proposed that single allogenic MSCs transplantation ameliorated disease phenotype in SLE mice and humans [
Although the routes of administration are different between diseases, such as intraportal injection for liver cirrhosis [
Most enrolled patients were unresponsive to CYC treatment before MSCT (for at least 6 months), the low dose of CYC given 4 days before MSCs infusion to each patient was used to inhibit active lymphocytes responses but not to treat disease. So we do not think the same pretreatment regimens before MSCT in both groups would influence the clinical response between once and double MSCT. Furthermore, the dose of CYC in the present study is much lower than that used in hematopoietic stem cells transplantation (total 30 mg/kg versus 200 mg/kg), and our previous animal studies had demonstrated that the addition of CYC before MSCT could not enhance clinical efficacy in MRL/
The role of transplanted MSCs
This study provides evidence that single transplantation at the dose of one million MSCs per kilogram of body weight is sufficient to induce disease remission in the treatment for refractory SLE patients, and double MSCT had no enhanced effect.
D. Wang and K. Akiyama contributed equally to this work.
The authors declare that they have no conflict of interests.
This work was supported by National Natural Science Foundation of China (no. 30972736), Major International (Regional) Joint Research Project (81120108021), Jiangsu Province Natural Science Foundation (BK2009034), Jiangsu Province Kejiao Xingwei Program (to L. Sun), and National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services (R01DE017449, R01 DE019932, and R01 DE019413 to S. Shi). This work was supported in part by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, National Institutes of Health (to W. Chen).