Restrained Th17 response and myeloid cell infiltration into the central nervous system by human decidua-derived mesenchymal stem cells during experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis is a widespread inflammatory demyelinating disease. Several immunomodulatory therapies are available, including interferon-ß, glatiramer acetate, natalizumab, fingolimod, and mitoxantrone. Although useful to delay disease progression, they do not provide a definitive cure and are associated with some undesirable side-effects. Accordingly, the search for new therapeutic methods constitutes an active investigation field. The use of mesenchymal stem cells (MSCs) to modify the disease course is currently the subject of intense interest. Decidua-derived MSCs (DMSCs) are a cell population obtained from human placental extraembryonic membranes able to differentiate into the three germ layers. This study explores the therapeutic potential of DMSCs. METHODS: We used the experimental autoimmune encephalomyelitis (EAE) animal model to evaluate the effect of DMSCs on clinical signs of the disease and on the presence of inflammatory infiltrates in the central nervous system. We also compared the inflammatory profile of spleen T cells from DMSC-treated mice with that of EAE control animals, and the influence of DMSCs on the in vitro definition of the Th17 phenotype. Furthermore, we analyzed the effects on the presence of some critical cell types in central nervous system infiltrates. RESULTS: Preventive intraperitoneal injection of DMSCs resulted in a significant delay of external signs of EAE. In addition, treatment of animals already presenting with moderate symptoms resulted in mild EAE with reduced disease scores. Besides decreased inflammatory infiltration, diminished percentages of CD4(+)IL17(+), CD11b(+)Ly6G(+) and CD11b(+)Ly6C(+) cells were found in infiltrates of treated animals. Early immune response was mitigated, with spleen cells of DMSC-treated mice displaying low proliferative response to antigen, decreased production of interleukin (IL)-17, and increased production of the anti-inflammatory cytokines IL-4 and IL-10. Moreover, lower RORγT and higher GATA-3 expression levels were detected in DMSC-treated mice. DMSCs also showed a detrimental influence on the in vitro definition of the Th17 phenotype. CONCLUSIONS: DMSCs modulated the clinical course of EAE, modified the frequency and cell composition of the central nervous system infiltrates during the disease, and mediated an impairment of Th17 phenotype establishment in favor of the Th2 subtype. These results suggest that DMSCs might provide a new cell-based therapy for the control of multiple sclerosis.

Human decidua-derived mesenchymal stromal cells differentiate into hepatic-like cells and form functional three-dimensional structures.

BACKGROUND AIMS: Previously, we have shown that human decidua-derived mesenchymal stromal cells (DMSC) are mesenchymal stromal cells (MSC) with a clonal differentiation capacity for the three embryonic layers. The endodermal capacity of DMSC was revealed by differentiation into pulmonary cells. In this study, we examined the hepatic differentiation of DMSC. METHODS: DMSC were cultured in hepatic differentiation media or co-cultured with murine liver homogenate and analyzed with phenotypic, molecular and functional tests. RESULTS AND CONCLUSIONS: DMSC in hepatic differentiation media changed their fibroblast morphology to a hepatocyte-like morphology and later formed a 3-dimensional (3-D) structure or hepatosphere. Moreover, the hepatocyte-like cells and the hepatospheres expressed liver-specific markers such as synthesis of albumin (ALB), hepatocyte growth factor receptor (HGFR), α-fetoprotein (AFP) and cytokeratin-18 (CK-18), and exhibited hepatic functions including glycogen storage capacity and indocyanine green (ICG) uptake/secretion. Human DMSC co-cultured with murine liver tissue homogenate in a non-contact in vitro system showed hepatic differentiation, as evidenced by expression of AFP and ALB genes. The switch in the expression of these two genes resembled liver development. Indeed, the decrease in AFP and increase in ALB expression throughout the co-culture were consistent with the expression pattern observed during normal liver organogenesis in the embryo. Interestingly, AFP and ALB expression was significantly higher when DMSC were co-cultured with injured liver tissue, indicating that DMSC respond differently under normal and pathologic micro-environmental conditions. In conclusion, DMSC-derived hepatospheres and DMSC co-cultured with liver homogenate could be suitable in vitro models for toxicologic, developmental and pre-clinical hepatic regeneration studies.

Isolation and characterization of true mesenchymal stem cells derived from human term decidua capable of multilineage differentiation into all 3 embryonic layers.

OBJECTIVE: The objective of the study was to isolate and characterize a population of mesenchymal stem cells (MSCs) from human term placental membranes. STUDY DESIGN: We isolated an adherent cell population from extraembryonic membranes. Morphology, phenotype, growth characteristics, karyotype, and immunological and differentiation properties were analyzed. RESULTS: The isolated placental MSCs were from maternal origin and named as decidua-derived mesenchymal stem cells (DMSCs). DMSCs differentiated into derivatives of all germ layers. It is the first report about placental MSC differentiation into alveolar type II cells. Clonally expanded DMSCs differentiated into all embryonic layers, including pulmonary cells. DMSCs showed higher life span than placental cells from fetal origin and proliferated without genomic instability. CONCLUSION: The data suggest that DMSCs are true multipotent MSCs, distinguishing them from other placental MSCs. DMSCs could be safely used in the mother as a potential source of MSCs for pelvic floor dysfunctions and immunological diseases. Additionally, frozen DMSCs can be stored for both autologous and allogeneic tissue regeneration.

CSL-MAML-dependent Notch1 signaling controls T lineage-specific IL-7R{alpha} gene expression in early human thymopoiesis and leukemia.

Notch1 activation is essential for T-lineage specification of lymphomyeloid progenitors seeding the thymus. Progression along the T cell lineage further requires cooperative signaling provided by the interleukin 7 receptor (IL-7R), but the molecular mechanisms responsible for the dynamic and lineage-specific regulation of IL-7R during thymopoiesis are unknown. We show that active Notch1 binds to a conserved CSL-binding site in the human IL7R gene promoter and critically regulates IL7R transcription and IL-7R alpha chain (IL-7Ralpha) expression via the CSL-MAML complex. Defective Notch1 signaling selectively impaired IL-7Ralpha expression in T-lineage cells, but not B-lineage cells, and resulted in a compromised expansion of early human developing thymocytes, which was rescued upon ectopic IL-7Ralpha expression. The pathological implications of these findings are demonstrated by the regulation of IL-7Ralpha expression downstream of Notch1 in T cell leukemias. Thus, Notch1 controls early T cell development, in part by regulating the stage- and lineage-specific expression of IL-7Ralpha.

Consistency of the initial cell acquisition procedure is critical to the standardization of CD34+ cell enumeration by flow cytometry: results of a pairwise analysis of umbilical cord blood units and cryopreserved aliquots.

BACKGROUND: The CD34+ cell content is a predictive factor for engraftment and survival after umbilical cord blood (UCB) transplantation. The high variability in the CD34 assay results in different recommended cell doses for infusion across transplant centers and also limits the clinical utility of the CD34+ cell counts provided by cord blood banks (CBBs). This bi-institutional study was intended to understand the sources of this variability. STUDY DESIGN AND METHODS: The level of CD34 agreement between the University of Minnesota (UM) and the Madrid CBB (MCBB) was evaluated on 50 UCB units before and after cryopreservation. Two cryopreserved vials per unit were thawed and processed at both laboratories. Dual-platform ISHAGE-based flow cytometry was used for CD34 enumeration. RESULTS: Postthaw nucleated cell recoveries were similar. However, whereas CD34+ cell enumeration before freezing was 0.35 +/- 0.22 percent, the results after thawing were 0.98 +/- 0.65 and 0.57 +/- 0.39 percent at UM and MCBB, respectively. Bland-Altman plots analysis ruled out the interchangeability of MCBB and UM CD34 values. Differences in the initial cell acquisition settings accounted for most of the CD34 discrepancy, which was no longer present after normalization of the forward scatter threshold for cell acquisition. CONCLUSIONS: The standardization of CD34+ cell enumeration by flow cytometry is strongly reliant on a consistent initial cell acquisition procedure. The interlaboratory variation can be minimized by using frozen cell aliquots as reference samples. Both requisites should be considered for CD34 testing and UCB unit selection by regulatory institutions involved with cord blood banking and transplantation.

A modified cord blood collection method achieves sufficient cell levels for transplantation in most adult patients.

Umbilical cord blood transplantation (UCBT) has been used increasingly in both pediatric and adult patients. The total nucleated cell (NC) dose infused is the most critical factor in determining speed of engraftment and survival. Using standard collection techniques, the mean NC content of UCB units is about 10 x 10(8) and only 25% of these units reach the target cell dose of 2 x 10(7)/kg in UCBT patients weighing 50-70 kg. We have designed a modified placental/umbilical two-step collection method in which a standard blood fraction obtained by umbilical venipuncture is combined with a second fraction harvested after placental perfusion with 50 ml heparinized 0.9% saline. This second fraction contributed 32% volume and 15% NCs to the whole UCB unit (123.7 +/- 50.1 ml and 1.26 +/- 0.52 x 10(9) NC). The proportion of progenitor cells in both fractions was not significantly different, indicating that the hematopoietic potential of these larger units is 20% (range, 2%-100%) higher than UCB units collected by standard methods. In addition, the bacterial contamination rate associated with this novel collection method (2.78%) compares favorably. Since 1998 we have further enriched our units by processing only UCB units over 0.8 x 10(9) NCs, resulting in a 36% cell increment (1.46 +/- 0.52 x 10(9) NCs). Thus, 84% and 54% of the Madrid UCB Bank inventory would fulfill the target cell dose of 2 x 10(7)/kg in patients weighing 50 and 65 kg, respectively. This significant UCB banking improvement gives larger pediatric and adult patients a greater chance of finding adequate grafts in order to achieve better clinical outcomes after UCBT.

A novel E2 box-GATA element modulates Cdc6 transcription during human cells polyploidization.

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements.

Inconclusive presence of adenovirus sequences in human leukemias and lymphomas.

Adenoviruses are ubiquitous viruses related to human mild upper tract respiratory infections. Murine cells are semi-permissive to adenovirus replication, and persistent or abortive infections have been associated with tumorigenic potential. Given that only human lymphoid cells are semi-permissive and abortive infections have been described, we hypothesized that adenovirus could be related to the transformation of human haematopoietic cells. We studied 30 lymphomas, 46 leukemias, 10 reactive lymphadenopathies and 40 normal human spleens. The presence of adenovirus sequences and proteins were studied using PCR, southern-blot, slot-blot, in situ hybridization, immunohistochemistry, and western-blot techniques. By using nested PCR, adenovirus sequences were detectable in about 30% of lymphomas, but in less than 10% of leukemias, reactive lymphadenopathies and normal spleens. In no case were we able to demonstrate adenovirus products by in situ hybridization, immunohistochemistry or western-blot. These results indicate that adenovirus sequences are present in a significant number of human lymphomas, but that the number of positive cells is extremely low and no protein expression could be detected. Therefore, we are unable to conclude that persistent infections of human lymphoid cells by adenovirus is related to a higher risk of developing malignant lymphomas or leukemias.