Umbilical cord mesenchymal stem cell transplantation in active and refractory systemic lupus erythematosus: a multicenter clinical study

From December 2009 to August 2011, 40 SLE patients ranging in age from 17 to 54 years were enrolled into our trial. Informed consent was obtained from each patient and donor. All enrolled patients met at least four of the eleven American College of Rheumatology criteria for SLE. The eligibility criteria included treatment-refractory and active disease, as well as a Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score of more than 8 or at least one British Isles Lupus Assessment Group (BILAG) grade A or at least two BILAG grade B manifestations. Refractory to treatment was defined as lack of response to treatment with monthly intravenous pulse CYC (500 to 750 mg/m²) for at least 6 months [22, 23], or lack of response to treatment with oral MMF (≥ 1,000 mg/day) [24] or leflunomide (20 mg/day) for at least 3 months, or continued daily doses of at least 20 mg of prednisone (Pred) or its equivalent. Patients were excluded from the study if they had uncontrolled infection, New York Heart Association functional classification III or IV, failure of one of the vital organs or were pregnant or lactating. Active lupus nephritis (LN) was defined by meeting at least one of the following criteria: (1) laboratory tests documenting active LN three consecutive times: decrease in renal function (serum creatinine > 1.2 mg/dl), increase in proteinuria (> 1.0 g of protein excretion in a 24-hour urine specimen), deterioration in microscopic hematuria (> 10 red blood cells per high-power field) or the presence of cellular casts; or (2) renal biopsy documenting LN according to the International Society of Nephrology/Renal Pathology Society 2003 classification system criteria for active or active/chronic LN in renal biopsy class III, class IV-S or class IV-G, class V, class III + class V or class IV + class V [25]. The study was conducted in compliance with current good clinical practice (GCP) standards and in accordance with the principles set forth under the 1989 Declaration of Helsinki. The protocol was approved by the Ethics Committee at The Drum Tower Hospital of Nanjing University Medical School, The Affiliated Hospital of Jiangsu University, Jiangsu Provincial People’s Hospital and Subei People’s Hospital of Jiangsu Province.

UC MSCs were prepared by the Stem Cell Center of Jiangsu Province, which is the National Stem Cell Institute in China and a member of the International Society for Cellular Therapy. The Stem Cell Center was also certified by the American Association of Blood Banks. Fresh UCs were obtained from informed healthy mothers in a local maternity hospital after normal deliveries. The UCs were rinsed twice in phosphate-buffered saline in penicillin and streptomycin, and the cord blood was removed during this process. The washed UCs were cut into 1-mm² pieces and floated in low-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. The pieces of UC were subsequently incubated at 37°C in a humidified atmosphere consisting of 5% CO₂. Nonadherent cells were removed by washing. The medium was replaced every 3 days after the initial plating. When well-developed colonies of fibroblast-like cells appeared after about 10 days, the cells were trypsinized and passaged into a new flask for further expansion.

Cell viability was determined by trypan blue testing. The culture supernatant was analyzed for pathogenic microorganisms by direct cultivation analysis. Supernatant levels of alanine aminotransferase and endotoxins for each cell preparation were determined using an automatic biochemistry analyzer and by tachypleus amebocyte lysate analysis, respectively. In addition, supernatant virus indexes were determined by enzyme-linked immunosorbent assay. Cell surface labeling markers, including CD29, CD73, CD90, CD105, CD45, CD34, CD14, CD79 and human leukocyte antigen major histocompatibility complex class II molecule, DR haplotype (HLA-DR), as well as their isotype controls, were all purchased from eBioscience (San Diego, CA, USA), and cell phenotypes were studied by flow cytometric analysis (FCM). We used good manufacturing practice conditions and clinical grade reagents to prepare the cells, and the protocol was conducted in compliance with GCP standards. One million cells per kilogram of body weight were administered by intravenous infusion on days 0 and 7.

Each patient returned for follow-up at 1, 3, 6, 9 and 12 months after MSCT. Evaluations performed at these follow-up visits included a physical examination, determination of SLEDAI score, BILAG analysis, serologic studies and evaluation of organ function. Adverse events and their severity were assessed and recorded throughout the study. Primary efficacy endpoints were major clinical response (MCR) and partial clinical response (PCR) assessed during the 12-month study period. A MCR was defined as achieving BILAG C scores or better in all organs at 6 months without experiencing a severe flare, which was defined, in turn, as one new domain with a BILAG A score or two new domains with BILAG B scores from MSC infusion and maintenance of this response throughout the 12-month study period. A PCR was defined as (1) BILAG C scores or better and maintenance of this response without a new BILAG A or B score within 3 months; and (2) having no more than one organ with a BILAG B score at 6 months without achieving at least one new BILAG A or B score throughout the 12-month study period [26]. No clinical response was defined as failure to meet the definition of a MCR or PCR. Clinical relapse was defined as development of at least one new domain with a BILAG A or B score after a previous MCR or PCR. Secondary efficacy endpoints included SLEDAI score, lupus serologic changes, systemic evaluations such as renal functional indexes, and hematological involvement. Transplantation-related mortality included all deaths associated with UC MSCT, except those related to recurrence of underlying disease. The investigators assessed and recorded adverse events and their severity throughout the study.

After UC MSCT, the doses of steroids as well as immunosuppressive drugs were tapered according to the amelioration of disease conditions. The dose of Pred was tapered by 5 to 10 mg every 2 weeks during the first month following transplantation for responders. If the clinical index was not improved or if disease activity had not declined, which was defined as nonresponse, the drug dose was not tapered or new drugs might be chosen. When relapse occurred, the dose of Pred or immunosuppressive drug would be added or new drugs would be given. This protocol is uniformly adhered to at each center, and the trial was monitored by the third party (The Stem Cell Center of Jiangsu Province).

Data were analyzed as of the last data collection in August 2011. Patients were censored at the time of death or last follow-up. We used Fisher’s exact test to compare the distribution of categorical variables. Pairwise comparisons of pre- and post-MSCT variables were analyzed by paired t-test analysis using SPSS version 13.0 statistical software (IBM SPSS, Chicago, IL, USA). The comparisons of clinical responses between patients with or without CYC treatment were analyzed by χ² test. The BILAG index for different organ systems was used to assess response, and scores were converted to numeric values (A = 9, B = 3, C = 1, D = 0 and E = 0) to enable evaluation [27, 28]. All P values were two-sided, and P < 0.05 was considered statistically significant.

Forty patients, including thirty-eight females and two males, were enrolled in this trial. Twenty-six patients were enrolled from The Department of Rheumatology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China, 6, 5 and 3 patients were enrolled from The Department of Rheumatology, the Affiliated Hospital of Jiangsu University, Zhenjiang, China, the Department of Rheumatology, Subei People’s Hospital of Jiangsu Province, Yangzhou, China, and the Department of Rheumatology, Jiangsu Provincial People’s Hospital, Nanjing, China, respectively. The mean disease duration was 90.9 months, ranging from 15 to 264 months. Baseline demographics and clinical manifestations for each patient are shown in Table 1. Thirty-nine patients (39/40, 97.5%) underwent two times of UC MSC infusions with an interval of one week, and one patient (1/40, 2.5%) was exempted from the second MSC infusion because of uncontrolled disease progression.

All the infused UC MSCs were derived from passages 2 to 4, with rigorous purification and quality control. The cell viability of purified MSCs was greater than 92%. The culture supernatant was negative for pathogenic microorganisms, including aerobic and anaerobic bacteria, as well as for hepatitis B surface antigen, hepatitis B core antibody, hepatitis C virus antibody, HIV antibodies I and II, cytomegalovirus immunoglobulin M and syphilis antibody. FCM analysis showed CD29, CD73, CD90 and CD105 expression greater than 95% in parallel with CD45, CD34, CD14, CD79 and HLA-DR expression less than 2%. In addition, levels of alanine aminotransferase and endotoxins in the supernatants of each cell preparation were strictly controlled within 40 IU/L and 5 endotoxin units, respectively. The capacity of MSCs to differentiate into adipogenic and osteogenic lineages was also assayed.

After 12 months, the overall survival rate was 92.5% (37 of 40 patients). Three patients died as a result of uncontrolled disease activity and organ failure. One patient had active lupus with malar rash, arthralgia, uncontrolled hypertension and rapid deterioration of renal function, hypoproteinemia and severe proteinuria. She died 7 days after the first MSC infusion as a result of uncontrolled progressive disease and acute heart failure. Another patient had a lupus relapse 8 months after MSC infusion, with pulmonary hypertension, and died as a result of right-sided heart failure 256 days after MSCT. The third patient also had disease relapse 6 months after MSCT, with steroid-resistant thrombocytopenia and uncontrolled septicemia, and ultimately died due to respiratory failure 192 days after MSC infusion. Two patients had moderate herpesvirus infection 291 and 135 days after MSC treatment, respectively, and one patient had tuberculosis infection at 326 days. All the infection adverse events were treated by conventional therapies. Adverse events were not considered to be possibly related to UC MSCT. All the adverse events are listed in Table 2.

Lupus disease activity, as defined by SLEDAI score, significantly decreased after MSCT (mean ± SD values = 10.83 ± 4.63 at baseline, 8.55 ± 3.99 at 1 month, 7.43 ± 3.93 at 3 months, 6.30 ± 3.63 at 6 months, 6.40 ± 3.84 at 9 months and 6.48 ± 3.52 at 12 months; all P < 0.01 versus baseline levels) (Figure 1A). Total BILAG score was markedly ameliorated after UC MSC infusion (mean ± SD values = 10.78 ± 6.09 at baseline, 5.35 ± 4.48 at 1 month, 5.28 ± 4.71 at 3 months, 4.23 ± 4.43 at 6 months, 3.85 ± 4.73 at 9 months and 3.55 ± 4.33 at 12 months; all P < 0.001 versus baseline levels) (Figure 1B).

Serum albumin levels improved shortly after UC MSC infusions, were normal at the 1-month follow-up visit and remained normal during the succeeding 9 months until the 12-month follow-up visit, when they declined (mean ± SD values = 3.17 ± 0.75 g/dl at baseline, 3.70 ± 0.58 g/dl at 1 month, 3.80 ± 0.67 g/dl at 3 months, 3.84 ± 0.63 g/dl at 6 months, 3.89 ± 0.64 g/dl at 9 months and 3.67 ± 0.78 g/dl at 12 months; all P < 0.05 versus baseline levels) (Figure 2A). Serum complement 3 improved with statistical significance found at 6 months (Figure 2B). Serum complement 4 levels showed no obvious changes after MSC treatment in those patients. We observed that serum anti-double-stranded DNA antibody levels decreased after MSCT with statistically significant differences found at the 6- and 12-month follow-up visits (mean ± SD values = 710.83 ± 814.05 U/ml at baseline, 526.78 ± 666.7 U/ml at 1 month, 590.41 ± 702.99 U/ml at 3 months, 492.67 ± 615.15 U/ml at 6 months, 513.58 ± 378.6 U/ml at 9 months and 212.62 ± 244.77 U/ml at 12 months; n = 16), along with decreased serum antinuclear antibody (mean ± SD values = 5.77 ± 2.32 at baseline, 5.40 ± 2.08 at 1 month, 5.24 ± 2.66 at 3 months, 4.85 ± 2.83 at 6 months, 4.46 ± 2.21 at 9 months and 4.73 ± 2.36 at 12 months) (Figures 2C and D).

Thirty-eight (95%) of forty patients had active LN (renal BILAG A or B score) at baseline, but their renal BILAG scores decreased significantly after two UC MSC infusions (Figure 3A). Twenty-four-hour proteinuria levels decreased significantly after UC MSC treatment (mean ± SD values = 2.24 ± 1.43 g at baseline, 2.13 ± 1.35 g at 1 month, 1.91 ± 1.20 g at 3 months, 1.65 ± 1.11 g at 6 months, 1.24 ± 1.09 g at 9 months and 1.41 ± 1.33 g at 12 months; P < 0.05 at 9- and 12-month follow-up visits) (Figure 3B). Renal function index, as assessed by serum creatinine and blood urea nitrogen levels, also decreased, and both showed statistically significant differences at the 6-month follow-up visit (Figures 3C and D), but they increased at the 12-month follow-up visit. Twenty-five (62.5%) and twenty-eight (70%) of the forty patients had hematopoietic and cutaneous system involvement, respectively, at baseline. The BILAG score for the two systems also ameliorated after MSC treatment (Figures 3E and F).

The dose of Pred was tapered from 5 to 10 mg every 2 weeks during the first month following transplantation, according to clinical status and laboratory indicators of disease amelioration. During the 12-month follow-up visits, 30 (81.08%) of 37 patients underwent steroid tapering, and, though 19 (54.29%) of 35 patients underwent immunosuppressant tapering after MSCT, two patients were excluded because they had not been taking immunosuppressive drugs at baseline (Table 3).