Willem E. Fibbe

Isolation of Mesenchymal Stem Cells of Fetal or Maternal Origin from Human Placenta

Recently we reported that second‐trimester amniotic fluid (AF) is an abundant source of fetal mesenchymal stem cells (MSCs). In this study, we analyze the origin of these MSCs and the presence of MSCs in human‐term AF. In addition, different parts of the human placenta were studied for the presence of either fetal or maternal MSCs. We compared the phenotype and growth characteristics of MSCs derived from AF and placenta.

Mesenchymal Stem Cells Inhibit Generation and Function of Both CD34+-Derived and Monocyte-Derived Dendritic Cells

Mesenchymal stem cells (MSCs) are not only able to evade the immune system, but they have also been demonstrated to exert profound immunosuppressive properties on T cell proliferation. However, their effect on the initiators of the immune response, the dendritic cells (DCs), are relatively unknown. In the present study, the effects of human MSCs on the differentiation and function of both CD34+-derived DCs and monocyte-derived DCs were investigated. The presence of MSCs during differentiation blocked the differentiation of CD14+CD1a− precursors into dermal/interstitial DCs, without affecting the generation of CD1a+ Langerhans cells. In line with these observations, MSCs also completely prevented the generation of immature DCs from monocytes. The inhibitory effect of MSCs on DC differentiation was dose dependent and resulted in both phenotypical and functional modifications, as demonstrated by a reduced expression of costimulatory molecules and hampered capacity to stimulate naive T cell proliferation. The inhibitory effect of MSCs was mediated via soluble factors. Taken together, these data demonstrate that MSCs, next to the antiproliferative effect on T cells, have a profound inhibitory effect on the generation and function of both CD34+-derived and monocyte-derived DCs, indicating that MSCs are able to modulate immune responses at multiple levels.

Sarcoma derived from cultured mesenchymal stem cells

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole‐body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain‐specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon‐related genetic abnormality, and p53 was not overexpressed. Infusion of MSC‐derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

Human fetal lymphoid tissue–inducer cells are interleukin 17–producing precursors to RORC + CD127 + natural killer–like cells

The human body contains over 500 individual lymph nodes, yet the biology of their formation is poorly understood. Here we identify human lymphoid tissue–inducer cells (LTi cells) as lineage-negative RORC+ CD127+ cells with the functional ability to interact with mesenchymal cells through lymphotoxin and tumor necrosis factor. Human LTi cells were committed natural killer (NK) cell precursors that produced interleukin 17 (IL-17) and IL-22. In vitro, LTi cells gave rise to RORC+ CD127+ NK cells that retained the ability to produce IL-17 and IL-22. Postnatally, similar populations of LTi cell–like cells and RORC+ CD127+ NK cells were present in tonsils, and both secreted IL-17 and IL-22 but no interferon-γ. Our data indicate that lymph node organogenesis is controlled by an NK cell precursor population with adaptive immune features and demonstrate a previously unappreciated link between the innate and adaptive immune systems.

Mesenchymal Stromal Cells: Sensors and Switchers of Inflammation

In addition to their stem/progenitor properties, mesenchymal stromal cells (MSCs) possess broad immunoregulatory properties that are being investigated for potential clinical application in treating immune-based disorders. An informed view of the scope of this clinical potential will require a clear understanding of the dynamic interplay between MSCs and the innate and adaptive immune systems. In this Review, we outline current insights into the ways in which MSCs sense and control inflammation, highlighting the central role of macrophage polarization. We also draw attention to functional differences seen between vivo and in vitro contexts and between species. Finally, we discuss progress toward clinical application of MSCs, focusing on GvHD as a case study.

Mesenchymal stem cells promote engraftment of human umbilical cord blood–derived CD34+ cells in NOD/SCID mice

OBJECTIVE Mesenchymal stem cells (MSC) have been implicated as playing an important role in hematopoietic stem cell engraftment. We identified and characterized a new population of MSC derived from human fetal lung. In cotransplantation experiments, we examined the homing of MSC as well as the effect on engraftment of human umbilical cord blood (UCB)-derived CD34(+) cells in NOD/SCID mice. MATERIALS AND METHODS Culture-expanded fetal lung-derived CD34(+) cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. Irradiated (3.5 Gy) NOD/SCID mice (n = 51) were transplanted intravenously with 0.03 to 1.0 x 10(6) UCB CD34(+) cells in the presence or absence of 1 x 10(6) culture-expanded fetal lung-derived MSC, irradiated CD34(-) cells, B cells, or with cultured MSC only. RESULTS Culture-expanded fetal lung CD34(+) cells were identified as MSC based on phenotype (CD105(+), SH3(+), SH4(+), CD160(+)) and their multilineage potential. Cotransplantation of low doses of UCB CD34(+) cells and MSC resulted in a three-fold to four-fold increase in bone marrow engraftment after 6 weeks, whereas no such effect was observed after cotransplantation of irradiated CD34(-) or B cells. Homing experiments indicated the presence of MSC in the lung, but not in the bone marrow, of NOD/SCID mice. CONCLUSIONS We identified a population of MSC derived from human fetal lung. Upon cotransplantation, MSC, but not irradiated CD34(-) or B cells, promote engraftment of UCB CD34(+) cells in bone marrow, spleen, and blood by mechanisms that may not require homing of MSC to the bone marrow.

Intramyocardial Bone Marrow Cell Injection for Chronic Myocardial Ischemia: A Randomized Controlled Trial

CONTEXT Previous studies have suggested that bone marrow cell injection may improve myocardial perfusion and left ventricular (LV) function in patients with chronic myocardial ischemia. OBJECTIVE To investigate the effect of intramyocardial bone marrow cell injection on myocardial perfusion and LV function in patients with chronic myocardial ischemia. DESIGN, SETTING, AND PATIENTS Randomized, double-blind, placebo-controlled trial at a Netherlands university hospital, May 1, 2005-March 3, 2008 (6-month follow-up ended September 2008) of 50 patients with chronic myocardial ischemia (mean age [SD], 64 [8] years; 43 men). INCLUSION CRITERIA severe angina pectoris despite optimal medical therapy and myocardial ischemia. All patients were ineligible for conventional revascularization. INTERVENTIONS Intramyocardial injection of 100 x 10(6) autologous bone marrow-derived mononuclear cells or placebo solution. MAIN OUTCOME MEASURES Primarily, the summed stress score, a 17-segment score for stress myocardial perfusion assessed by Tc-99m tetrofosmin single-photon emission computed tomography (SPECT). Secondary included LV ejection fraction (LVEF), Canadian Cardiovascular Society (CCS) class, and Seattle Angina Questionnaire quality-of-life score (mean difference >5% considered clinically significant). RESULTS After 3-month follow-up, the summed stress score (mean [SD]) improved from 23.5 (4.7) to 20.1 (4.6) (P < .001) in the bone marrow cell group, compared with a decrease from 24.8 (5.5) to 23.7 (5.4) (P = .004) in the placebo group. In the bone marrow cell-treated patients who underwent magnetic resonance imaging (MRI), a 3% absolute increase in LVEF was observed at 3 months (95% CI, 0.5% to 4.7%; n = 18), but the placebo group showed no improvement. CCS angina score improved significantly in the bone marrow cell group (6-month absolute difference, -0.79; 95% CI, -1.10 to -0.48; P < .001) compared with no significant improvement in the placebo group. Quality-of-life score increased from 56% (9%) to 64% (12%) at 3 months and 69% (12%) at 6 months in bone marrow cell-treated patients, compared with a smaller increase in the placebo group from 57% (11%) to 61% (14%) to 64% (17%). The improvements in CCS class and quality of life score were significantly greater in bone marrow cell-treated patients than in placebo-treated patients (P = .03 and P = .04, respectively). CONCLUSIONS In this short-term study of patients with chronic myocardial ischemia refractory to medical treatment, intramyocardial bone marrow cell injection resulted in a statistically significant but modest improvement in myocardial perfusion compared with placebo. Further studies are required to assess long-term results and efficacy for mortality and morbidity. TRIAL REGISTRATIONS trialregister.nl Identifier: NTR400 and isrctn.org Identifier: ISRCTN58194927.

Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1

Mesenchymal stem cells are self-renewing cells with the ability to differentiate into osteocytes, chondrocytes and adipocytes. This article describes a subset of mesenchymal stem cells with distinct phenotypic and functional properties. Background Conventionally, mesenchymal stem cells are functionally isolated from primary tissue based on their capacity to adhere to a plastic surface. This isolation procedure is hampered by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during removal of undesired cells. To circumvent these limitations, several antibodies have been developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on natural killer cells. Design and Methods Bone marrow derived mesenchymal stem cells from healthy donors were analyzed and isolated by flow cytometry using a large panel of antibodies against surface antigens including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was monitored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mesenchymal stem cells into defined lineages was induced by culture in appropriate media and verified by immunostaining. Results Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were ~90-fold enriched in the MSCA-1+CD56− fraction and ~180-fold in the MSCA-1+CD56+ fraction. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 was restricted to the MSCA-1+CD56− mesenchymal stem cells subset and CD166 to MSCA-1+CD56± mesenchymal stem cells. Further differentiation of these subsets showed that chondrocytes and pancreatic-like islets were predominantly derived from MSCA-1+CD56± cells whereas adipocytes emerged exclusively from MSCA-1+CD56− cells. The culture of single sorted MSCA-1+CD56+ cells resulted in the appearance of phenotypically heterogeneous clones with distinct proliferation and differentiation capacities. Conclusions Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties were identified. Our data suggest that the MSCA-1+CD56+ subset is an attractive starting population for autologous chondrocyte transplantation.

Phase I/II trial of autologous stem cell transplantation in systemic sclerosis : procedure related mortality and impact on skin disease

BACKGROUND Systemic sclerosis (SSc, scleroderma) in either its diffuse or limited skin forms has a high mortality when vital organs are affected. No treatment has been shown to influence the outcome or significantly affect the skin score, though many forms of immunosuppression have been tried. Recent developments in haemopoietic stem cell transplantation (HSCT) have allowed the application of profound immunosuppression followed by HSCT, or rescue, to autoimmune diseases such as SSc. METHODS Results for 41 patients included in continuing multicentre open phase I/II studies using HSCT in the treatment of poor prognosis SSc are reported. Thirty seven patients had a predominantly diffuse skin form of the disease and four the limited form, with some clinical overlap. Median age was 41 years with a 5:1 female to male ratio. The skin score was >50% of maximum in 20/33 (61%) patients, with some lung disease attributable to SSc in 28/37 (76%), the forced vital capacity being <70% of the predicted value in 18/36 (50%). Pulmonary hypertension was described in 7/37 (19%) patients and renal disease in 5/37 (14%). The Scl-70 antibody was positive in 18/32 (56%) and the anticentromere antibody in 10% of evaluable patients. Peripheral blood stem cell mobilisation was performed with cyclophosphamide or granulocyte colony stimulating factor, alone or in combination. Thirty eight patients had ex vivo CD34 stem cell selection, with additional T cell depletion in seven. Seven conditioning regimens were used, but six of these used haemoimmunoablative doses of cyclophosphamide +/- anti-thymocyte globulin +/- total body irradiation. The median duration of follow up was 12 months (3–55). RESULTS An improvement in skin score of >25% after transplantation occurred in 20/29 (69%) evaluable patients, and deterioration in 2/29 (7%). Lung function did not change significantly after transplantation. One of five renal cases deteriorated but with no new occurrences of renal disease after HSCT, and the pulmonary hypertension did not progress in the evaluable cases. Disease progression was seen in 7/37 (19%) patients after HSCT with a median period of 67 (range 49–255) days. Eleven (27%) patients had died at census and seven (17%) deaths were considered to be related to the procedure (direct organ toxicity in four, haemorrhage in two, and infection/neutropenic fever in one). The cumulative probability of survival at one year was 73% (95% CI 58 to 88) by Kaplan-Meier analysis. CONCLUSION Despite a higher procedure related mortality rate from HSCT in SSc compared with patients with breast cancer and non-Hodgkins lymphoma, the marked impact on skin score, a surrogate marker of mortality, the trend towards stabilisation of lung involvement, and lack of other treatment alternatives justify further carefully designed studies. If future trials incorporate inclusion and exclusion criteria based on this preliminary experience, the predicted procedure related mortality should be around 10%.

Autologous Hematopoietic Stem Cell Transplantation vs Intravenous Pulse Cyclophosphamide in Diffuse Cutaneous Systemic Sclerosis: A Randomized Clinical Trial

IMPORTANCE High-dose immunosuppressive therapy and autologous hematopoietic stem cell transplantation (HSCT) have shown efficacy in systemic sclerosis in phase 1 and small phase 2 trials. OBJECTIVE To compare efficacy and safety of HSCT vs 12 successive monthly intravenous pulses of cyclophosphamide. DESIGN, SETTING, AND PARTICIPANTS The Autologous Stem Cell Transplantation International Scleroderma (ASTIS) trial, a phase 3, multicenter, randomized (1:1), open-label, parallel-group, clinical trial conducted in 10 countries at 29 centers with access to a European Group for Blood and Marrow Transplantation-registered transplant facility. From March 2001 to October 2009, 156 patients with early diffuse cutaneous systemic sclerosis were recruited and followed up until October 31, 2013. INTERVENTIONS HSCT vs intravenous pulse cyclophosphamide. MAIN OUTCOMES AND MEASURES The primary end point was event-free survival, defined as time from randomization until the occurrence of death or persistent major organ failure. RESULTS A total of 156 patients were randomly assigned to receive HSCT (n = 79) or cyclophosphamide (n = 77). During a median follow-up of 5.8 years, 53 events occurred: 22 in the HSCT group (19 deaths and 3 irreversible organ failures) and 31 in the control group (23 deaths and 8 irreversible organ failures). During the first year, there were more events in the HSCT group (13 events [16.5%], including 8 treatment-related deaths) than in the control group (8 events [10.4%], with no treatment-related deaths). At 2 years, 14 events (17.7%) had occurred cumulatively in the HSCT group vs 14 events (18.2%) in the control group; at 4 years, 15 events (19%) had occurred cumulatively in the HSCT group vs 20 events (26%) in the control group. Time-varying hazard ratios (modeled with treatment × time interaction) for event-free survival were 0.35 (95% CI, 0.16-0.74) at 2 years and 0.34 (95% CI, 0.16-0.74) at 4 years. CONCLUSIONS AND RELEVANCE Among patients with early diffuse cutaneous systemic sclerosis, HSCT was associated with increased treatment-related mortality in the first year after treatment. However, HCST conferred a significant long-term event-free survival benefit. TRIAL REGISTRATION isrctn.org Identifier: ISRCTN54371254.