The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs). Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages.

Mesenchymal stromal cells (MSCs), first found in bone marrow (BM), are the structural architects of all organs, participating in most biological functions. MSCs possess tissue-specific signatures that allow their discrimination according to their origin and location. Among their multiple functions, MSCs closely interact with immune cells, orchestrating their activity to maintain overall homeostasis. The phenotype of tissue MSCs residing in the bowel overlaps with myofibroblasts, lining the bottom walls of intestinal crypts (pericryptal) or interspersed within intestinal submucosa (intercryptal). In Crohn’s disease, intestinal MSCs are tightly stacked in a chronic inflammatory milieu, which causes their enforced expression of Class II major histocompatibility complex (MHC). The absence of Class II MHC is a hallmark for immune-modulator and tolerogenic properties of normal MSCs and, vice versa, the expression of HLA-DR is peculiar to antigen presenting cells, that is, immune-activator cells. Interferon gamma (IFNγ) is responsible for induction of Class II MHC expression on intestinal MSCs. The reversal of myofibroblasts/MSCs from an immune-modulator to an activator phenotype in Crohn’s disease results in the formation of a fibrotic tube subverting the intestinal structure. Epithelial metaplastic areas in this context can progress to dysplasia and cancer.

MiR-204 and 211 enforced expression in murine mesenchymal stromal cells (MSCs) has been shown to induce adipogenesis and impair osteogenesis, through RUNX2 down-modulation. This mechanism has been suggested to play a role in osteoporosis associated with obesity. However, two further fundamental MSC functions, chondrogenesis and hematopoietic supporting activity, have not yet been explored. To this end, we transduced, by a lenti-viral vector, miR-204 and 211 in a model primary human MSC line, opportunely chosen among our MSC collection for displaying all properties of canonical bone marrow MSCs, except adipogenesis. Enforced expression of miR-204&211 in these cells, rescued adipogenesis, and inhibited osteogenesis, as previously reported in murine MSCs, but, surprisingly, also damaged cartilage formation and hematopoietic supporting activity, which were never explored before. RUNX2 has been previously indicated as the target of miR-204&211, whose down modulation is responsible for the switch from osteogenesis to adipogenesis. However, the additional disruption of chondrogenesis and hematopoietic supporting activity, which we report here, might depend on diverse miR-204&211 targets. To investigate this hypothesis, permanent RUNX2 knock-down was performed. Sh-RUNX2 fully reproduced the phenotypes induced by miR-204&211, confirming that RUNX2 down modulation is the major event leading to the reported functional modification on our MSCs. It seems thus apparent that RUNX2, a recognized master gene for osteogenesis, might rule all four MSC commitment and differentiation processes. Hence, the formerly reported role of miR204&211 and RUNX2 in osteoporosis and obesity, coupled with our novel observation showing inhibition of cartilage differentiation and hematopoietic support, strikingly resemble the clinical traits of metabolic syndrome, where osteoarthritis, osteoporosis, anaemia and obesity occur together. Our observations, corroborating and extending previous observations, suggest that miR-204&211-RUNX2 axis in human MSCs is possibly involved in the pathogenesis of this rapidly growing disease in industrialized countries, for possible therapeutic intervention to regenerate former homeostasis.

Background Cancer cells, including colorectal cancer ones (CRC), release high amounts of nanovesicles (exosomes), delivering biochemical messages for paracrine or systemic crosstalk. Mesenchymal stromal cells (MSCs) have been shown to play contradicting roles in tumor progression. Results CRC exosomes induce in cMSCs: i) atypical morphology, higher proliferation, migration and invasion; ii) formation of spheroids; iii) an acidic extracellular environment associated with iv) a plasma membrane redistribution of vacuolar H+-ATPase and increased expression of CEA. Colon cancer derived MSCs, which were isolated from tumor masses, produce umbilicated spheroids, a future frequently observed in the inner core of rapidly growing tumors and recapitulate the changes observed in normal colonic MSCs exposed to CRC exosomes. Materials and Methods Tissue specific colonic (c)MSCs were exposed to primary or metastatic CRC exosomes and analysed by light and electron microscopy, proliferation in 2D and 3D cultures, migration and invasion assays, Western blot and confocal microscopy for vacuolar H+-ATPase expression. Conclusions CRC exosomes are able to induce morphological and functional changes in colonic MSCs, which may favour tumor growth and its malignant progression. Our results suggest that exosomes are actively involved in cancer progression and that inhibiting tumor exosome release may represent a way to interfere with cancer.

Although the development of bone metastasis is a major detrimental event in prostate cancer, the molecular mechanisms responsible for bone homing and destruction remain largely unknown. Here we show that loss of miR-15 and miR-16 in cooperation with increased miR-21 expression promote prostate cancer spreading and bone lesions. This combination of microRNA endows bone-metastatic potential to prostate cancer cells. Concomitant loss of miR-15/miR-16 and gain of miR-21 aberrantly activate TGF-β and Hedgehog signaling, that mediate local invasion, distant bone marrow colonization and osteolysis by prostate cancer cells. These findings establish a new molecular circuitry for prostate cancer metastasis that was validated in patients' cohorts. Our data indicate a network of biomarkers and druggable pathways to improve patient treatment.

From a literature search (PubMed) on “SP1 transcription factor and osteoblasts”, SP1 is somehow interfering with the effect of Runx2, SP7 (Osx), FIAT (inhibitor of ATF4), ETS-like TFs, MZF1 (myeloid zinc finger), JunB, and also directly affecting the transcription of marker genes like Col1α1, Col5α1, Col5α3, Col11α2, Fibromodulin, Osteocalcin, MGP (matrix-gla protein), RANKL, Pit phosphate transporter, Integrin β5, and TGFβ-R1. Furthermore, a similar search on “FosB and osteoblasts” revealed that FosB is interfering with the effect of BMPs and TGFs on the expression of downstream signaling molecules like Smads, TCF/LEF, as well as c-myc, and fra-1, but also directly modulating the transcription of IL-11 (which suppresses DIKK1 & DIKK2, thus enhancing Wnt-signaling), stimulating Pref-mediated dedifferentiation of adipocytes, relaying stretch-mediated osteoblast differentiation, counteracting the negative effect of Notch1 (a decrease in the expression of Col1α1, Osteocalcin, and ALP) by obliterating its negative effect on the Wnt/β-catenin pathway. Finally, the recent literature describes the positioning of ETS1 in the differentiation of osteoblasts in this way: Leptin is a strong inducer of osteoblast differentiation working through Stat3 (ref), and it was shown that ETS1, along with Stat1, Stat3, and VDR were induced by Calcitriol (1,25(OH) 2 D 3 ) in UMR-106 osteoblast like cells, and that ETS1 is essential for connective tissue factor (CTGF/CCN2) induction by TGF-β1 in osteoblasts, synergizing with Smad3.

H osteoblastogenesis, as assessed by the expression of osteoblast specific genes, or by in vitro assays like ALP positive and mineralization surfaces are often attempted explained by the concerted action of developmental phase related appearance of transcription factors (TFs) like Msx2, Satb2, Runx2 and Osterix. The impact of other small protein subsets regulating β-catenin, being a key mediator of the canonical effect of Wnt signaling in osteoblasts, has also been emphasized. Over the past 6-7 years, differentiation of stem cells towards osteoblasts has been scrutinized in view of intrinsic levels of several microRNA species, like miR-133 and -135, miR-204 and -211, as well as miR-149, -328 and -339. Many excellent reviews describe a network of TF vs. microRNA interactions; however, few attempts to emulate such interactions for osteoblasts through bioinformatics have been made. In parallel, accumulating data on the impact of important epigenetic phenomena on osteoblastogenesis, via the concerted activity of various classes of histone deacetylases (HDACs), have been described.

Introduction Due to their properties and characteristics, human mesenchymal stem cells (MSCs) appear to have great therapeutic potential, because they can contribute to the tissue regeneration. Many different populations of MSCs that differ for the embryonic origin and the anatomical localization have been described and to understand whether they have equivalent biological properties is a critical issue for their therapeutic application. Spine fusion is frequently used to treat traumatic, degenerative, and oncological spine diseases. Autologous bone graft has been considered the gold standard for spine fusion procedures because of its osteogenic, osteoinductive, and osteoconductive ability. However, its use is associated with significant disadvantages, including donor site pain, increased operative time, insufficient availability, and nonunion postlumbar fusion. Various bone substitutes have been developed to promote spinal fusion. Also, MSCs have been successfully tested for spinal fusion in several small and large animal models. Material and Methods To define the properties of MSCs for the use in clinical applications, we proposed to analyze the cellular and molecular characteristics of human adult MSCs derived from different body locations, such as bone marrow from the iliac crest (Ic-MSCs), sternum (St-MSCs), and vertebrae (vMSCs), as well as colon (Co-MSCs) and dental pulp (DPSCs), to identify the cell population showing better biological properties for spine fusion clinical application. Results MSCs from different sources presented very different growth kinetics. While iliac crest and sternum-derived MSCs could be maintained in culture for about 3 months, undergoing a limited expansion, mesenchymal cultures derived from dental pulp and colon grew much more rapidly, but could not be maintained longer. Strikingly, vMSCs continued to propagate at high rate even after 3.5 months ex vivo. MSCs from different sources were also induced to osteogenic, adipogenic, and chondrogenic differentiation following exposure to specific inducing agents. We observed that vMSCs generate mature cells of all mesenchymal lineages with greater efficiency, resulting into the best population both in terms of expansion and differentiation. Conclusion This finding could be very interesting and open new perspectives for the improvement of spine fusion, which is a mandatory step for the success of spinal surgical procedures. In the course of a surgical procedure for spinal fusion, vertebral bone marrow can be harvested simultaneously with the preparation of the site for pedicle screw insertion. Then multipotent MSCs can be isolated from vertebral bone marrow during the progress of the surgical procedure and reintroduced in the fusion site (using homologous or autologous bone tissue or biomaterials as scaffold), without any additional surgical time or any other donor site involvement.

Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co‐factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow‐derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell‐based therapeutical strategies for regeneration and repair of specific tissues. J. Cell. Physiol. 228: 879–889, 2013. © 2012 Wiley Periodicals, Inc.

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.

The coelomic cavity is part of the extraembryonic mesoderm, surrounding amniotic cavity, embryo, and yolk sac in the early gestation. It is now believed to represent an important transfer interface and a reservoir of nutrients for the embryo. Coelocentesis by ultrasound‐guided transvaginal puncture offers an easier access to the early human embryo, from 28 days post‐fertilization. However, despite some studies about its biochemical composition being reported, our knowledge about the presence of cellular elements and their quality in this compartment are still limited. Here we studied human coelomic fluids sampled from 6.6 (48 days) to 10 weeks of gestation, demonstrating the presence of functional embryonic erythroid precursors, that is, megaloblasts in the coelomic cavity. The ease of access of the coelomic cavity could allow the development of novel strategies for diagnostic or therapeutic purposes by ultrasound imaging and ultrasound‐guided puncture. J. Cell. Physiol. 225: 385–389, 2010. © 2010 Wiley‐Liss, Inc.

Abstract Abstract 5086 Background two fluid compartments, amniotic and coelomic cavity, surrounded the developing embryo during the first trimester of pregnancy. This opens the possibility of functional studies of both fluid compartments by ultrasound imaging and ultrasound-guided puncture. The composition of amniotic fluid has been studied extensively since the advent of second-trimester amniocentesis. Instead, while some studies about biochemical composition of coelomic cavity have been reported and reviewed by Santolaya-Forgas (2006), our knowledge about cellular composition of this compartment is still limited. Aim of the study to study cellular pattern of human coelomic fluids sampled from 6.6 to 10 weeks of gestation. Patients and Methods women seeking for voluntary abortion were advised to be included in this study. Coelomic fluid was sampled by ultrasound guided transvaginal puncture. The first sample (0.2ml) was discarded. Then aspirated 1-2ml of coelomic fluid were used for analysis. Coelomic fluid cells were pelletted and divided into two aliquots. One aliquot was underwent to CD45 selection and observed at the inverted microscope. Then coelomic cells were May Grunwald-Giemsa stained and re-analyzed by inverted microscope. Cells from the same sample were also analyzed by quantitative fluorescent-PCR (QF-PCR) and ε- γ- and β globin chains cDNA specific primers RT-PCR, to verify the presence of foetal cells only. Cells from second aliquot were incubated with specific mAbs to detect CD71+ and CD45+ cell lines. Flow cytometry analysis was carried out using forward scatter/ side scatter gating. The study complied with the Declaration of Helsinki. All patients gave written informed consent to the protocol. The institutional review board approved this study. Results coelomic fluid cells May Grunwald-Giemsa staining analysis showed red cells and few cells with eccentric nuclei and vacuoles. These were very similar to megaloblasts (Fig1). QF-PCR analysis showed that all selected cells were foetal cells, and RT-PCR showed the expression of ε and γ globin mRNAs according to the pregnancy weeks. Flow cytometry analysis, performed by CD71 and CD45 mAbs, showed a cellular clone CD71+/CD45- (Fig2), consistent with the presence of megaloblasts (red cells are CD71 negative). Conclusions presence of erythroid precursors in the coelomic fluid could indicate a cellular traffic between the yolk sac and the coelomic cavity. Thus, the coelomic cavity potentially could be a new way to access the foetus for possible diagnostic and therapeutical interventions. Acknowledgments. We would like to thank all women who agreed to participate to this study. This work was supported,partially, by “Stem Cell Project” of Fondazione Roma, Rome (Italy) and by Foundation Franco and Piera Cutino, Palermo (Italy). Disclosures No relevant conflicts of interest to declare.

The rarity of hematopoietic stem and progenitor cells (HSCs, HPCs) has hampered the analysis of cellular and molecular mechanisms underlying early hematopoiesis. Methodology for HPC purification has partially offset this limitation. A further hurdle has been represented by the heterogeneity of the analyzed HPC/precursor populations: recently, development of unilineage HPC differentiation cultures has provided homogeneous populations of hematopoietic cells, particularly in the early differentiation state, i.e., populations pertaining to a single lineage and a restricted stage of differentiation/maturation, but sufficiently large for cellular/molecular analysis. This report focuses on the development and characterization of the unilineage HPC differentiation culture systems. A section is devoted to selected cellular and molecular mechanisms underlying hematopoiesis, which have been investigated by the HPC unilineage culture approach. Finally, recent advances in the development of HPC unilineage cultures at single cell level are discussed.

Mesenchymal stromal cells (MSCs) represent a bone marrow (BM) population, classically defined by five functional properties: extensive proliferation, ability to differentiate into osteoblasts, chondrocytes, adipocytes, and stromal cells -supporting hematopoiesis. However, research progress in this area has been hampered by lack of suitable markers and standardized procedures for MSC isolation. We have isolated a CD146(+) multipotent MSC population from 20 human BM donors displaying the phenotype of self-renewing osteoprogenitors; an extensive 12-week proliferation; and the ability to differentiate in osteoblasts, chondrocytes, adipocytes, and stromal cells supporting hematopoiesis. Furthermore, the CD146+ MSCs secrete a complex combination of growth factors (GFs) controlling hematopoietic stem cells (HSCs) function, while providing a > 2-log increase in the long-term culture (LTC) colony output in 8-week LTC over conventional assays. The hematopoietic stromal function exhibited by the MSCs was further characterized by manipulating LTCs with the chemical inhibitors Imatinib or SU-5416, targeting two GF receptors (GFRs), KIT or VEGFR2/1, respectively. Both treatments similarly impaired LTC colony output, indicating key roles for these two GF/GFR interactions to support LTC-initiating cell activity. CD146+ MSCs may thus represent a tool to explore the NISC-HSC cross-talk in an in vitro surrogate model for HSC "niches," and for regenerative therapy studies. In addition, the MSC microRNA (miRNA) expression profile was analyzed by microarrays in both basic conditions and chondrogenic differentiation. Our analysis revealed that several miRNAs are modulated during chondrogenesis, and many of their putative targets are genes involved in chondrogenic differentiation. (c) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Adult mesenchymal stromal cells (MSCs) are undifferentiated multi-potent cells predominantly residing in the bone marrow (BM), but also present with similar but not identical features in many other tissues such as blood, placenta, dental pulp, and adipose tissue. MSCs have the potential to differentiate into multiple skeletal phenotypes like osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and possibly tendons. MSCs differentiation potential, ex vivo expansion capacity, nurturing and immunomodulatory proficiencies oriented these versatile cells in several areas of ongoing clinical applications. However, the absence of MSC-specific markers for isolation and characterization together with the lack of a comprehensive view of the molecular pathways governing their particular biological properties, remains a primary obstacle to their research and application. In this review we discuss some areas of growing interest in MSCs biology: their contribution to the hematopoietic stem cell (HSC) niche, to regenerative medicine, their role in cancer and in therapy as delivery tools and their micro-RNA (miRNA) signatures. Despite rapid progress in the MSC field, it is generally thought that only a fraction of their full potential has been realized thus far.

A tight control of kinase and phosphatase activity is fundamental for normal cell growth, survival and differentiation. The deregulated kinase activity of the BCR/ABL oncoprotein is responsible for the emergence and maintenance of chronic myelogenous leukemia (CML). By contrast, PP2A, a serine-threonine phosphatase involved in the regulation of many cellular functions, was found genetically inactivated in many types of cancer. We show here that, in BCR/ABL-transformed cells and CD34+ CML blast crisis progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the physiological PP2A-inhibitor SET whose expression is enhanced by BCR/ABL and increased in blast crisis CML. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and in CD34+ CML blast crisis cells, molecular and/or pharmacological activation of PP2A leads to dephosphorylation of important regulators of proliferation and survival of CML progenitors, suppresses BCR/ABL kinase activity and promotes BCR/ABL proteasome degradation via a mechanism that requires the SHP-1 tyrosine phosphatase activity. Furthermore, PP2A activation achieved by shRNA-mediated SET knock-down or PP2Ac overexpression or treatment with the PP2A activator forskolin results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential and decreased in vivo leukemogenesis of wild type and T315I BCR/ABL-transformed myeloid cells. Thus, functional inactivation of PP2A phosphatase activity is essential for BCR/ABL leukemogenesis and, perhaps, required for transition of CML into blast crisis.

The oncogenic BCR/ABL kinase activity induces and maintains chronic myelogenous leukemia (CML). We show here that, in BCR/ABL-transformed cells and CML blast crisis (CML-BC) progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the BCR/ABL-induced expression of the PP2A inhibitor SET. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and CML-BC progenitors, molecular and/or pharmacological activation of PP2A promotes dephosphorylation of key regulators of cell proliferation and survival, suppresses BCR/ABL activity, and induces BCR/ABL degradation. Furthermore, PP2A activation results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cells. Thus, functional inactivation of PP2A is essential for BCR/ABL leukemogenesis and, perhaps, required for blastic transformation.

1098The Fragile Histidine Triad (FHIT) gene is a tumor suppressor gene that plays a role in many human epithelial cancers, including cervical carcinomas. The gene is located at chromosome region 3p14.2, and encompasses a large chromosomal region which includes the common FRA3B fragile site. Common fragile sites have been shown to be highly recombinogenic and to participate in deletions and translocations. Accordingly, a papillomavirus integration site and cancer specific translocations have been mapped within the FRA3B fragile site in the Fhit gene. We have generated a Fhit knockout mouse for in vivo studies of Fhit function in tumorigenesis. Although the number of tumor-affected animals among Fhit deficient mice was higher than in wild type littermates, cervical carcinomas were not found in the spectrum of spontaneous tumors. Fhit deficient mice were exquisitely sensitive to carcinogen induction …