Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors.

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.

Effect of miR-204&211 and RUNX2 control on the fate of human mesenchymal stromal cells.

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.

Gut Mesenchymal Stromal Cells in Immunity.

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.

Exosomes from human colorectal cancer induce a tumor-like behavior in colonic mesenchymal stromal cells.

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.

Mesenchymal stem cells derived from vertebrae (vMSCs) show best biological properties.

PURPOSE: Due to their properties and characteristics human mesenchymal stem cells (MSCs) appear to have great therapeutic potential. Many different populations of MSCs have been described and to understand whether they have equivalent biological properties is a critical issue for their therapeutic application. METHODS: We proposed to analyze the in vitro growth kinetics of MSCs derived from different body sites (iliac crest bone marrow, vertebrae bone marrow, colon mucosa, dental pulp). RESULTS: Mesenchymal stem cells derived from vertebrae can be maintained in culture for a greater number of steps and they also generate mature cells of all mesenchymal lineages with greater efficiency, when induced into osteogenic, adipogenic and chondrogenic differentiation. CONCLUSIONS: The ability of vertebrae-derived MSCs in terms of expansion and differentiation is very interesting at the light of a clinical application for bone fusion in spine surgery.

HOX and TALE signatures specify human stromal stem cell populations from different sources.

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.

Identity and ranking of colonic mesenchymal stromal cells.

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.

Embryo-fetal erythroid megaloblasts in the human coelomic cavity.

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.

The mesenchymal stromal cell contribution to homeostasis.

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.

Isolation and characterization of CD146+ multipotent mesenchymal stromal cells.

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 MSC-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.

MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression primarily through translational repression. In erythropoietic (E) culture of cord blood CD34+ progenitor cells, the level of miR 221 and 222 is gradually and sharply down-modulated. Hypothetically, this decline could promote erythropoiesis by unblocking expression of key functional proteins. Indeed, (i) bioinformatic analysis suggested that miR 221 and 222 target the 3' UTR of kit mRNA; (ii) the luciferase assay confirmed that both miRs directly interact with the kit mRNA target site; and (iii) in E culture undergoing exponential cell growth, miR down-modulation is inversely related to increasing kit protein expression, whereas the kit mRNA level is relatively stable. Functional studies show that treatment of CD34+ progenitors with miR 221 and 222, via oligonucleotide transfection or lentiviral vector infection, causes impaired proliferation and accelerated differentiation of E cells, coupled with down-modulation of kit protein: this phenomenon, observed in E culture releasing endogenous kit ligand, is magnified in E culture supplemented with kit ligand. Furthermore, transplantation experiments in NOD-SCID mice reveal that miR 221 and 222 treatment of CD34+ cells impairs their engraftment capacity and stem cell activity. Finally, miR 221 and 222 gene transfer impairs proliferation of the kit+ TF-1 erythroleukemic cell line. Altogether, our studies indicate that the decline of miR 221 and 222 during exponential E growth unblocks kit protein production at mRNA level, thus leading to expansion of early erythroblasts. Furthermore, the results on kit+ erythroleukemic cells suggest a potential role of these miRs in cancer therapy.

The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein.

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.

Lung cancer susceptibility in Fhit-deficient mice is increased by Vhl haploinsufficiency.

The FHIT gene plays important roles in cancer development, including lung cancers, in which the Fhit protein is frequently lost. To determine if Fhit-deficient mice exhibit increased susceptibility to carcinogen-induced lung cancer, mice were treated with the pulmonary carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone. Wild-type and Fhit-deficient animals did not exhibit significantly different frequencies of lung lesions, but Fhit-/- mice showed significantly increased average tumor volume (1.62 mm3) and multiplicity in tumor-bearing mice, compared with wild-type mice (0.70 mm3). Tumors of Fhit-/- mice were all carcinomas, whereas Fhit+/+ mice did not develop carcinomas. To determine if Fhit absence, in combination with deficiency of an additional 3p tumor suppressor, would affect the frequency of tumor induction, we examined the spontaneous and dimethylnitrosamine-induced tumor phenotype of Fhit-/-Vhl+/- mice. Whereas no spontaneous lung tumors were observed in Fhit-/- or Vhl+/- mice, 44% of Fhit-/-Vhl+/- mice developed adenocarcinomas by 2 years of age. Dimethylnitrosamine (6 mg/kg body weight) induced lung tumors (adenomas and carcinomas) in 100% of Fhit-/-Vhl+/- mice and adenomas in 40% of Fhit-/- mice by 20 months of age. Thus, double deficiency in murine homologues of 3p suppressor genes, including haploinsufficiency of Vhl, predisposes to spontaneous and induced lung cancers, showing that Fhit-deficient mice will be useful, in combination with other 3p tumor suppressors, in recapitulating a pattern of lung cancer development similar to the human pattern; such double- or triple-deficient mice will be excellent lung cancer prevention and therapy models.

Identification of the hemangioblast in postnatal life.

Postnatal CD34(+) cells expressing vascular endothelial growth factor receptor 2 (KDR) generate hematopoietic or endothelial progeny in different in vitro and in vivo assays. Hypothetically, CD34(+)KDR(+) cells may comprise hemangioblasts bipotent for both lineages. This hypothesis is consistent with 2 series of experiments. In the first series, in clonogenic culture permissive for hematopoietic and endothelial cell growth, CD34(+)KDR(+) cells generate large hemato-endothelial (Hem-End) colonies (5% of seeded cells), whereas CD34(+)KDR(-) cells do not. Limiting-dilution analysis indicates that Hem-End colonies are clonally generated by single hemangioblasts. Sibling cells generated by a hemangioblast, replated in unicellular culture, produce either hematopoietic or Hem-End colonies, depending on the specific culture conditions. Identification of endothelial cells was based on the expression of VE-cadherin and endothelial markers and with lack of CD45 and hematopoietic molecules, as evaluated by immunofluorescence, immunocytochemistry, and reverse transcription-polymerase chain reaction. Furthermore, endothelial cells were functionally identified using low-density lipoprotein (LDL) uptake and tube-formation assays. In the second series, to evaluate the self-renewal capacity of hemangioblasts, single CD34(+)KDR(+) cells were grown in 3-month extended long-term culture (ELTC) through 3 serial culture rounds-that is, blast cells generated in unicellular ELTC were reseeded for a subsequent round of unicellular ELTC. After 9 months, 10% blasts from tertiary ELTC functioned as hemangioblasts and generated macroscopic Hem-End colonies in clonogenic culture. These studies identified postnatal hemangioblasts in a CD34(+)KDR(+) cell subset, endowed with long-term proliferative potential and bilineage differentiation capacity. Although exceedingly rare, hemangioblasts may represent the lifetime source/reservoir for primitive hematopoietic and endothelial progenitors.